Synthesis of novel 17-triazolyl-androst-5-en-3-ol epimers via Cu(I)-catalyzed azide-alkyne cycloaddition and their inhibitory effect on 17α-hydroxylase/C17,20-lyase

Article abstract of DOI:10.1016/j.steroids.2018.03.006

The regioselective Cu(I)-catalyzed 1,3-dipolar cycloaddition of 17α- and 17β-azidoandrost-5-en-3β-ol epimers (3b and 5b) with different terminal alkynes afforded novel 1,4-substituted triazolyl derivatives (8a–k and 9a–k). For the preparation of 5′-iodo-1

Full text of DOI:10.1016/j.steroids.2018.03.006

Accepted Manuscript
Synthesis of novel 17-triazolyl-androst-5-en-3-ol epimers via Cu(I)-catalyzed
azide-alkyne cycloaddition and their inhibitory effect on 17α-hydroxylase/
C17,20-lyase
Anita Kiss, Bianka Edina Herman, Tamás Görbe, Erzsébet Mernyák, Barnabás
Molnár, János Wölfling, Mihály Szécsi, Gyula Schneider
PII:
S0039-128X(18)30059-X
https://doi.org/10.1016/j.steroids.2018.03.006
STE 8243
DOI:
Reference:
Steroids
To appear in:
Received Date:
Accepted Date:
26 February 2018
15 March 2018
Please cite this article as: Kiss, A., Herman, B.E., Görbe, T., Mernyák, E., Molnár, B., Wölfling, J., Szécsi, M.,
Schneider, G., Synthesis of novel 17-triazolyl-androst-5-en-3-ol epimers via Cu(I)-catalyzed azide-alkyne
cycloaddition and their inhibitory effect on 17α-hydroxylase/C17,20-lyase, Steroids (2018), doi: https://doi.org/
10.1016/j.steroids.2018.03.006
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Synthesis of novel 17-triazolyl-androst-5-en-3-ol epimers via Cu(I)-
catalyzed azide-alkyne cycloaddition and their inhibitory effect on 17α-
hydroxylase/C17,20-lyase
Anita Kissa, Bianka Edina Hermanb, Tamás Görbec, Erzsébet Mernyáka, Barnabás Molnára,
János Wölflinga, Mihály Szécsib*, Gyula Schneidera*
a Department of Organic Chemistry, University of Szeged, Dóm tér 8., H-6720 Szeged, Hungary
b 1st Department of Medicine, University of Szeged, Korányi fasor 8-10., H-6720 Szeged, Hungary
c Organic Chemistry, Arrhenius Laboratory, Stockholm University, 106 91 Stockholm, Sweden
ABSTRACT
The regioselective Cu(I)-catalyzed 1,3-dipolar cycloaddition of 17α- and 17β-azidoandrost-5-
en-3β-ol epimers (3b and 5b) with different terminal alkynes afforded novel 1,4-substituted
triazolyl derivatives (8ak and 9ak). For the preparation of 5’-iodo-1’,2’,3’-triazoles (8mn
and 9mn), an improved method was developed, directly from steroidal azides and terminal
alkynes, in reaction mediated by CuI and ICl as iodinating agents. Acetolysis and subsequent
hydrolysis of 8n and 9n yielded 5’-hydroxy-1’,2’,3’-triazoles 8o and 9o. The inhibitory effect
of 8ao, 9ao, 3, and 5 on rat testicular C17,20-lyase was investigated by means of an in vitro
radioincubation technique. The results revealed that the C-17 epimers of steroidal triazoles
influence the C17,20-lyase effect. Inhibitors were found only in the 17α-triazolyl series (8ao),
whereas in the C-17 azide pair the 17β compound (5b) was more potent.
Keywords: Azide-alkyne cycloaddition, 17- and 17-azido-androst-5-ene, Inhibitory effect,
Chemoselectivity.
1. Introduction
Prostate cancer is an age-related disease and a major cause of cancer-related mortality
worldwide. It is androgen dependent in the majority of cases and can be treated with androgen
1

ablation. Steroidal precursors are transformed to preandrogens by the 17α-hydroxylase-C17,20
-
lyase enzyme (P45017α), which is highly active in both testes and adrenals [1]. Inhibitors of
this enzyme can block androgen synthesis in its early steps, and may be suitable for
pharmacological treatment of androgen dependent disorders, among them the prostate cancer
[24]. In the past decades a large number of compounds were developed as P45017α inhibitors
[5,6]. The most promising compounds have been designed by the ligand-base approach. These
are steroidal derivatives possessing heterocycle on the C-17 position. Heterocycles containing
sp2 hybrid N were demonstrated to be the most effective coordinating groups to the heme iron
of the enzyme active center. The Δ16,17 double bond can significantly increase inhibitory
potency [7]. Abiraterone [17-(3-pyridyl)androsta-5,16-dien-3β-ol] and Galeterone [17-(1H-
benzimidazole-1-yl)androsta-5,16-dien-3β-ol] have become the most successful drug
candidates of this group of compounds [8, 9].
We previously described the synthesis of series of C-17β heterocyclic steroids, and
investigated their inhibitory activity againts C17,20-lyase in vitro [1015]. As a continuation of
our research program we set out to prepare 1,4-substituted-1,2,3-triazolyl derivatives in the
androstane series, in which the heterocycle is attached to the steroid C-17 position.
The Huisgen 1,3-dipolar cycloaddition of organic azides and terminal alkynes has been of
considerable interest in recent years following the independent introduction of Cu(I) catalysis
in 2002 by the research groups of Sharpless [16] and Meldal [17]. The catalyst dramatically
improves both the rate and the regioselectivity of the reaction leading exclusively to 1,4-
disubstituted 1,2,3-triazoles.
The synthetic routes involve the introduction of an azide group into the C-17 position
of the sterane framework and the subsequent ring closure of the steroidal azid with a terminal
acetylene in the presence of a Cu(I) source. The copper(I)-catalyzed azide–alkyne
cycloaddition (CuAAC) is convenient for the regioselective construction of 1,4-disubstituted
triazoles in which the heterocycle is attached directly to the steroid nucleus through a nitrogen
atom.
The position and steric orientation of the azide group are determined by the synthetic
method applied. The C-17 azide group is generally in the α position, because the nucleophilic
exchange reaction of 17β-tosylate or mesylate by sodium azide takes place with Walden
inversion [18]. Here we decided to synthetize not only the 17α-azides, but a novel series of
17β-azide epimers, as starting materials for CuAAC in order to obtain novel 1,4-disubstituted
triazolyl androstane derivatives with varied terminal alkynes as reagents.
2

We set out to obtain answers to the following questions: (1) how the CuAAC process
influenced by the steric structure of the steroidal azides, and (2) how the inhibitory activity
against C17,20-lyase in the C-17 epimer series differ.
2. Experimental
2.1. General
Melting points (mp) were determined on a Kofler block and are uncorrected. Specific
○
rotations were measured in CHCl3 (c 1) at 20 C with a POLAMAT-A (Zeiss-Jena)
polarimeter and are given in units of 10–1 deg cm2 g–1. Elementary analysis data were
determined with a Perkin-Elmer CHN analyzer model 2400. The reactions were monitored by
TLC on Kieselgel-G (Merck Si 254 F) layers (0.25 mm thick); solvent systems (ss): (A)
isopropyl ether, (B) acetone/toluene/hexane (30:35:35, v/v). The spots were detected by
spraying with 5% phosphomolybdic acid in 50% aqueous phosphoric acid. The Rf values were
determined for the spots observed by illumination at 254 and 365 nm. Flash chromatography:
silica gel 60, 40–63 m. All solvents were distilled prior to use. NMR spectra were recorded
on a Bruker DRX 500 instrument at 500 (1H NMR) or 125 MHz (13C NMR). Chemical shifts
are reported in ppm (δ scale), and coupling constants (J) in Hertz. For the determination of
multiplicities, the J-MOD pulse sequence was used.
2.2. 3β-Acetoxy-androst-5-ene-17β-p-toluenesulfonate (2)
3β-Acetoxy-androst-5-en-17β-ol (1, 16.6 g, 50 mmol) was dissolved in pyridine (100 m), and
a solution of p-toluenesulfonyl chloride (14.25 g, 75 mmol) in anhydrous pyridine (50 ml)
was added during cooling with ice. The reaction mixture was allowed to stand for 24 h and
then poured onto a mixture of ice (500 g) and concentrated H2SO4 (50 ml). The crystalline
precipitate separating out was filtered off, washed thoroughly with water, and recrystallized
о
from a mixture of acetone and water to give pure 2 (21.55 g, 88%). Mp: 165166 C, Rf =
0.80 (ss A); [α]D20 = 70 (c 1 in CHCl3) (Ref. [19] 162164 оC, [α]D20 = – 72, c 1 in ethanol).
1
(Found C, 68.95; H, 7.94. C28H38O5S requires C, 69.10; H, 7.87%). H NMR (δ, ppm,
CDCl3): 0.80 and 0.99 (s, 3H, 18-H3, and s, 3H, 19-H3), 2.01 (s, 3H, Ac-CH3 ), 2.44 (s, 3H,
Ts-CH3), 4.24 (t, 1H, J = 8.0 Hz, 17-H), 4.57 (m, 1H, 3-H), 5.33 (d, 1H, J = 4.5 Hz, 6-H),
13
7.32 (d, 2H, J = 8.0 Hz, 3’- and 5’ -H), 7.77 (d, 2H, J =8.0 Hz, 2’- and 6’-H). C NMR (δ,
ppm, CDCl3): 11.6 (C-18), 19.3 (C-19), 20.3, 21.4, 21.6, 23.3, 27.6, 31.2, 31.6, 35.9, 36.6(C-
3

3), 36.9, 38.0, 42.8 (C-13), 49.8, 50.2, 73.7 (Ac-CH3), 89.9 (C-17), 121.9 (C-6), 127.8 (C-2’
and C-6’), 129.6 (C-3’and C-5’), 134.2 (C-4’), 139.8, 144.3 (C-1’), 170.4 (C=O).
2.3. 3β-Acetoxy-androst-5-en-17α-azide (3a)
Compound 2 (9.73 g, 20 mmol) was dissolved in N,N-dimethylformamide (150 ml), and
о
NaN3 (5.2 g, 80 mmol) was added. The mixture was stirred for 48 h at 100 C, then poured
into water (600 ml). The precipitate separating out was filtered off and washed with water.
The residue obtained was dissolved in CH2Cl2 and chromatographed on silica gel with
CH2Cl2/hexane (1:1, v/v) to give 3a (5.6 g, 78%) as a white solid. Mp: 153155 оC, Rf = 0.85
20
(ss A); [α]D = – 113 (c 1, in CHCl3). (Found: C, 70.76; H, 8.62; N, 11.93. C21H31N3O2
1
requires C, 70.55; H, 8.74; N, 11.75%.) H NMR (δ, ppm, CDCl3): 0.75 and 1.01 (s, 3H, 18-
H3 and s, 3H, 19-H3), 2.02 (s, 3H, Ac-H3), 3.52 (d, 1H, J = 6.5 Hz, 17-H), 4.60 (m, 1H, 3-H),
13
5.37 (d, 1H J = 4.5 Hz, 6-H). C NMR (δ, ppm, CDCl3): 17.4 (Ac-CH3, 19.3 (C-18), 20.5,
21.4 (C-19), 24.7, 27.7, 28.6, 32.0, 32.1 (C-8), 32.4, 36.6, 37.0, 38.0, 45.7 (C-13), 49.6 (C-9),
49.8 (C-14), 71.4 (C-17), 73.8 (C-3), 122.3 (C-6), 139.6 (C-5), 170.5 (C=O).
2.4. 17α-Azido-androst-5-en-3β-ol (3b)
Compound 3a (3.57 g, 10 mmol) was dissolved in methanol (100 ml) containing NaOCH3
(108 mg, 2 mmol), and the solution was allowed to stand for 24 h. It was then diluted with
water and the white precipitate separating out was separated by filtration and recrystallized
from a mixture of acetone/water to give 3b (3.15 g, 92%). Mp: 132134 оC, Rf = 0.65 (ss B);
[α]D20 = − 118 (c 1, in CHCl3). (Found: C, 72.47; H, 9.05; N, 13.51. C19H29N3O requires C,
72.34; H, 9.27; N, 13.32%.) 1H NMR (δ, ppm, CDCl3): 0.77 and 1.01 (s, 3H, 18-H3 and s, 3H,
19-H3), 3.51 (m, 2H, 3-H and –OH), 5.35 (t, 1H, J = 2.5 Hz, 6-H). 13C NMR (δ, ppm, CDCl3):
17.4 (C-18), 19.4 (C-19), 20.7, 24.8, 28.7, 31.6, 31.7, 32.1, 32.3, 32.6, 36.6, 37.3, 42.3, 45.8,
49.9, 50.0, 71.6 (C-17), 71.7 (C-3), 121.4 (C-6), 140.8 (C-5).
2.5. 3β-Acetoxy-androst-5-en-17α-iodide (4)
Compound 1 (5 g, 15 mmol), Ph3P (15.78 g, 60.15 mmol), and imidazol (4.10 g, 60.15 mmol)
were dissolved in toluene (250 ml) and I2 (15.26 g, 60.15 mmol) was added in two portions.
The reaction mixture was stirred at 80 оC for 2 h and allowed to cool to room temperature. A
saturated aqueous Na2SO3 solution (150 ml) was then added and the resulting mixture was
stirred until all solids dissolved. EtOAC (100 ml) was added, and the organic phase was
washed with saturated aqueous NaHCO3 (2 × 100 ml) and brine (100 ml), dried over Na2SO4
4

and evaporated in vacuo. The residue was subjected to column chromatography on silica gel
о
in CH2Cl2/hexane (1:3, v/v) to yield 4 (5.27 g, 79%) as a white solid. Mp: 162164 C, (Ref.
о
20
[7] 164166 C), Rf = 0.80 (ss A); [α]D = – 138 (c 1 in CHCl3). (Found: C, 56.93; H, 7.27.
1
C21H31IO2 requires C, 57.02; H, 7.06%). H NMR (δ, ppm, CDCl3): 0.84 and 1.02 (s, 3H, 18-
H3 and s, 3H, 19-H3), 2.30 (s, 3H, Ac-CH3), 2.79 (m, 1H, 6-H), 4.36 (d, 1H, J = 6.5 Hz, 1=
H), 4.60 (m, 1H, 3-H), 5.39 (s, 1H, 6-H). 13C NMR (δ, ppm, CDCl3):15.5 (C-18), 19.4 (C-19),
21.4 (Ac-CH3), 21.8, 25.2, 27.7, 31.9, 32.5, 36.6, 36.8, 37.0, 38.1, 40.6, 45.1, 47.7, 49.4 (C-
2), 73.8 (C-3), 122.3 (C-6), 139.6 (C-5), 170.4 (Ac-C=O).
2.6. 3β-Acetoxy-androst-5-en-17β-azide (5a) and 3β-acetoxy-androst-5,16-diene (6)
Compound 4 (4.42 g, 10 mmol) was dissolved in N,N-dimethylformamide (150 ml), and
о
NaN3 (5 g, 75 mmol) was added. The mixture was stirred for 32 h at 60 C, then poured onto
ice (500 g). The resulting emulsion was extracted with CH2Cl2 (3 × 150 ml). The CH2Cl2
phase was washed with water, dried over Na2SO4, evaporated in vacuo and subjected to
chromatographic separation on silica gel in CH2Cl2/hexane (1:3 v/v) to yield 6 (1.32 g, 42%)
о
20
as a slowly-crystallizing colourless oil. Mp: 4546 C, Rf = 0.85 (ss A); [α]D = – 92 (c 1 in
CHCl3). (Found: C, 80.37; H, 9.46. C21H30O2 requires: C, 80.21; H, 9.62%). 1H NMR (δ ppm,
CDCl3): 0.78 and 1.06 (s, 3H, 18-H3 and s, 3H, 19-H3), 2.03 (s, 3H, Ac-CH3), 2.33 (m, 2H,
15-H2), 4.61 (m, 1H, 3-H), 5.39 (d, 1H, J = 5.0 Hz, 6-H), 5.70 (m, 1H, 16-H), 5.84 (dd, 1H, J
=5.0 Hz, J = 1.0 Hz, 17-H). 13C NMR (δ, ppm CDCl3): 16.8 (Ac-CH3), 19.2 (C-18), 21.4 (C-
19), 27.7, 30.4 (C-8), 31.7, 32.1, 35.8, 36.9, 26.9, 38.1, 45.3 (C-13), 50.8 (C-9), 56.1 (C-14),
73.9 (C-3), 122.5 (C-6), 129.3 (C-16), 139.9 (C-5), 143.8 (C-17), 170.5 (C=O). Continued
20
elution resulted in 5a (1.64 g, 46%) as a white solid. Mp: 143145 оC, Rf = 0.75 (ss A); [α]D
= 68 ( c 1 in CHCl3). (Found: C, 70.76; H, 8.55; N, 11.94. C21H31N3O2 requires C, 70.55; H,
1
8.74; N, 11.75%). H NMR (δ, ppm, CDCl3): 0.76 and 1.02 (s, 3H, 18-H3 and s, 3H, 19-H3),
3.32 (t, 1H, J = 9.0 Hz, 17-H), 4.59 (m, 1H, 3-H), 5.36 (d, 1H, J = 4.5 Hz, 6-H).
13C NMR (δ, ppm, CDCl3): 12.1 (Ac-CH3), 19.3 (C-18), 20.5, 21.4 (C-19), 23.6, 26.9, 27.7,
31.5, 31.9 (C-8), 36.6, 37.0, 38.0, 44.2 (C-13), 49.9 (C-9), 52.3 (C-14), 71.2 (C-17), 73.8 (C-
3), 122.1 (C-6), 139.7 (C-5), 170.5 (C=O).
2.7. 17β-Azido-androst-5-en-3β-ol (5b)
Compound 5a (7.15 g, 20 mmol) was dissolved in methanol (200 ml) containing NaOCH3
(270 mg, 5 mmol), and the solution was allowed to stand for 24 h. It was then diluted with
5

water, the white precipitate separating out was filtered off, recrystallized from a mixture of
о
acetone/hexane to give 5a (5.45 g, 86%) as a white solid. Mp: 7880 C, Rf = 0.60 (ss B);
20
[α]D = − 67 (c 1 in CHCl3). (Found: C, 72.55; H, 9.42; N, 13.65. C19H29N3O requires C,
72.34; H, 9.27; N, 13.32%). 1H NMR (δ, ppm, CDCl3): 0.76 and 1.00 (s, 3H, 18-H3 and s, 3H,
13
19-H3), 3.31 (t, 1H, J = 8.5 Hz, 17-H), 3.50 (m, 1H, 3-H) 5.34 (s, 1H, 6-H). C NMR (δ,
ppm, CDCl3): 12.1 (C-18), 19.4 (C-19), 20.6, 23.6, 26.9, 31.5, 31.6, 32.0, 37.1, 37.2, 42.1,
42.2, 44.2, 50.0, 52.4, 71.2 (C-17), 71.6 (C-3), 121.1 (C-6), 140.9 (C-5).
2.8. General procedure for the synthesis of triazoles (8a–k and 9a–k)
17α-Azido-androst-5-en-3β-ol (3b) (315.45 mg, 1 mmol) or 17β-azido-androst-5-en (5b)
(315.45 mg, 1 mmol) was dissolved in CH2Cl2 (20 ml), then CuI (19 mg, 0.10 mmol), Et3N
(0.2 ml, 2 mmol), and the appropriate terminal alkyne (7a-k, 2 mmol) were added. The
mixture was stirred under reflux for 24 h, then diluted with water (30 ml) and extracted with
CH2Cl2 (2 × 30 ml). The combined organic phases were dried over Na2SO4 and evaporated in
vacuo. The crude product was purified by flash chromatography using ethyl acetate/CH2Cl2 in
different concentrations.
2.8.1. 17α-(4’-Phenyl-1’H-1’,2’,3’-triazol-1’-yl)androst-5-en-3β-ol (8a)
Compound 3b (315.45 mg, 1 mmol) and phenylacetylene (7a, 0.22 ml) were used for the
synthesis as described in Section 2.8. The crude product was chromatographed on silica gel
with ethyl acetate/CH2Cl2 (1:99 v/v) to yield pure 8a (234 mg, 56%) as a white solid. Mp:
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20
234236 C; Rf= 0.45 (ss B); [α]D = − 73 (c 1 in CHCl3). (Found: C, 77.52; H, 8.26; N,
9.87. C27H35N3O requires C, 77.66; H, 8.45; N, 10.06%). 1H NMR (δ, ppm, CDCl3): 0.97 and
0.98 (s, 3H, 18-H3 and s, 3H, 19-H3), 3.47 (m, 1H, 3-H), 4.63 (d, 1H, J = 7.5 Hz, 17-H), 5.33
(s, 1H, 6-H), 7.32 (t, 1H, J = 7.4 Hz, 4’’-H), 7.41 (t, 1H, J = 7.4 Hz, 3’’- and 5’’-H), 7.68 (s,
1H, 5’-H), 7.83 (d, 2H, J = 7.4 Hz, 2’’-H and 6’’-H). 13C NMR (δ, ppm, CDCl3): 18.4 (C-18),
19.3 (C-19), 20.4, 25.3, 28.6, 31.5, 31.9, 32.2, 32.4, 36,4, 37.1, 42.1, 46.1, 49.4, 50.2, 70.4 (C-
17), 71.5 (C-3), 119.8 (C-5’), 121.1 (C-6), 125.6 and 128.8 (4C, C-2’’, C-3’’, C-5’’ and C-
6’’), 128.0 (C-4’’), 130.6 (C-1’’), 140.7 (C-5), 146.8 (C-4’).
2.8.2. 17α-[4’-(4’’-Tolyl)-1’H-1’,2’,3’-triazol-1’-yl]androst-5-en-3β-ol (8b)
Compound 3b (315.45 mg, 1 mmol) and 4-tolylacetylene (7b, 0.22 ml) were used for the
synthesis as described in Section 2.8. The crude product was chromatographed on silica gel
with ethyl acetate/CH2Cl2 (1:99 v/v) to yield pure 8b (193 mg, 45%) as a colorless crystalline
6

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20
solid. Mp: 258261 C; Rf = 0.45 (ss B); [α]D = − 75 (c 1 in CHCl3). (Found: C, 78.05; H,
1
8.42; N, 9.87. C28H37N3O requires C, 77.92; H, 8.64; N, 9.74%). H NMR (δ, pm, CDCl3):
0.99 and 1.03 (s, 3H, 18-H3 and s, 3H, 19-H3), 2.40 (s, 3H, 4’’-CH3), 3.49 (m, 1H, 3-H), 4.74
(d, 1H, J = 6.5 Hz, 17-H), 5.35 (s, 1H, 6-H), 7.27 and 7.84 (d, 2H, J = 7.0 Hz, 3’’- and 5’’-H,
and d, 2H, J = 7.0 Hz, 2’’-H and 6’’-H). 13C NMR (δ, ppm, CDCl3): 18.4 (C-18), 19.3 (C-19),
20.5, 21.3 (4’’-CH3), 25.6, 29.1, 31.5, 31.8, 32.1, 32.5, 36.4, 37.1, 42.1, 46.8, 49,2, 49.7, 69.8
(C-17), 71.5 (C-3), 78.9 (C-4’), 121.5 (C-6), 127.5 and 129.1 (4C, C-2’’, C-3’’, C-5’’ and C-
6’’), 127.5 (C-1’’), 138.3 (C-4’’), 140.5 (C-5), 187.7 (C-5’’).
2.8.3. 17α-[4’-(4’’-Methoxyphenyl)-1’H-1’,2’,3’-triazol-1’-yl]androst-5-en-3β-ol ( 8c)
Compound 3b (315.45 mg, 1 mmol) and 4-methoxyphenyl acetylene (7b, 0.22 ml) were used
for the synthesis as described in Section 2.8. The crude product was chromatographed on
silica gel with ethyl acetate/CH2Cl2 (5:95 v/v) to yield pure 8c (170 mg, 38%) as a white
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20
solid. Mp: 246248 C ; Rf = 0.50 (ss B); [α]D = – 74 (c 1 in CHCl3). (Found: C, 75.34; H,
1
8.17; N, 9.55. C28H37N3O2 requires C, 75.13; H, 8.33; N, 9.39%). H NMR (δ, ppm, CDCl3):
0.97 and 0.99 (s, 3H, 18-H3 and s, 3H, 19-H3), 3.47 (m, 1H, 3-H), 3.83 (s, 3H, 4’’-OCH3),
4.61 (d, 1H, J = 8.5 Hz, 17-H), 5.34 (d, 1H, J = 2.3 Hz, 6-H), 6.95 (d, 2H, 8.2 Hz, 3’’-5’’-H2),
7.58 (s, 1H, 5’-H), 7.76 (d, 2H, J = 8.2 Hz, 2’’-H and 6’’-H). 13C NMR (δ, ppm, CDCl3): 18.5
(C-18), 19.5 (C-19), 20.5, 25.4, 28.8, 31.7, 32.1, 32.3 (4’-OCH3), 32.6, 36.6, 37.3, 42.3, 46.3,
49.6, 50.3, 55.5, 70.4 (C-17), 114.3 (C-2” and -6”), 119.0 (C-5’), 121.3 (C-6), 123.6 (C-1”),
127.05 (C-3” and -5”), 140.8 (C-5), 146.9 (C-4”), 159.6 (C-4’).
2.8.4. 17α-(4’-Cyclopropyl-1’H-1’,2’,3’-triazol-1’-yl)androst-5-en-3β-ol (8d)
Compound 3b (315.45 mg, 1 mmol) and cyclopropylacetylene (7d, 0.22 ml) were used for the
synthesis as described in Section 2.8. The crude product was chromatographed on silica gel
о
with ethyl acetate/CH2Cl2 (5:95 v/v) to yield pure 8d (360 mg, 96%). Mp: 197200 C; Rf =
20
0.55 (ss B); [α]D = −70 (c 1 in CHCl3). (Found: C, 75.81; H, 9.08; N, 10.94. C24H35N3O
requires C, 75.55; H, 9.25; N, 11.01%). 1H NMR (δ, ppm, CDCl3): 0.98 and 0.99 (s, 3H, 18-
H3 and s, 3H, 19-H3), 3.49 (m, 1H, 3-H), 4.57 (t, 1H, J = 8.1, 2.5 Hz, 17-H), 5.33 (d, 1H, J =
13
2.3 Hz, 6-H). C NMR (δ, ppm, CDCl3): 7.3, 7.4, 7.8, 18.5 (C-18), 19.5 (C-19), 20.6, 22.0,
25.7, 29.1, 29.8, 31.7, 32.0, 32.2, 32.6, 36.6, 37.3, 42.4, 46.9, 49.3, 49.8, 69.7 (C-17), 71.7 (C-
3), 121.7 (C-6), 140.6 (C-5).
7

2.8.5. 17α-(4’-Cyclopentyl-1’H-1’,2’,3’-triazol-1’-yl)androst-5-en-3β-ol (8e)
Compound 3b (315.45 mg, 1 mmol) and cyclopentylacetylene (7e, 0.22 ml) were used for the
synthesis as described in Section 2.8. The crude product was chromatographed on silica gel
о
with ethyl acetate/CH2Cl2 (5:95 v/v) to yield pure 8e (300 mg, 45%). Mp: 108110 C; Rf =
0.53 (ss B); [α]D20 = – 68 (c 1 in CHCl3). (Found: C, 76.38, H, 9.47; N, 10.03. C26H39N3O
1
requires C, 76.24; H, 9.60; N, 10.26%). H NMR (δ, ppm, CDCl3): 0.93 and 0.98 (s, 3H, 18-
H3 and s, 3H, 19-H3), 3.15 (t, 1H, J = 8.2 Hz, 1’-H), 3.48 (m, 1H, 3-H), 4.52 (t, 1H, J = 8.6
13
Hz, 17-H), 5.34-5.33 (m, 1H, 6-H),7.13 (s, 1H, 5’-H). C NMR (δ, ppm, CDCl3): 18.5 (C-
18), 19.5 (C-19), 20.6, 22.8, 25.3, 25.5, 28.8, 31.7, 32.1, 32.4, 32.5, 33.4, 33.4, 36.6, 36.9,
37.3, 42.3, 46.2, 49.6, 50.3, 70.1 (C-17), 71.7 (C-3), 120.0 (C-5’), 121.3 (C-6), 140.9 (C-5),
151.9 (C-4’).
2.8.6. 17α-(4’-Cyclohexyl-1’H-1’,2’,3’-triazol-1’-yl)androst-5-en-3β-ol (8f)
Compound 3b (315.45 mg, 1 mmol) and cyclohexylacetylene (7f, 0.22 ml) were used for the
synthesis as described in Section 2.8. The crude product was chromatographed on silica gel
with ethyl acetate/CH2Cl2 (5:95 v/v) to yield pure 8f (240 mg, 71%) as white crystalline
product. Mp: 127129 оC; Rf = 0.50 (ss B); [α]D20 = – 65 (c 1 in CHCl3). (Found C, 76.73; H,
1
9.55; N, 10.06. C27H41N3O requires C, 76.55; H, 9.76; N, 9.92%). H NMR (δ, ppm, CDCl3):
0.92 and 0.98 (s, 3H, 18-H3 and s, 3H, 19-H3), 2.73 (m, 1H, 1’’-H), 3.48 (m, 1H, 3-H), 4.52
(d, 1H, J = 8.5 Hz, 17-H), 5.33 (t, 1H, J = 2.3 Hz, 5-H), 7.12 (s, 1H, 5’-H). 13C NMR (δ, ppm,
CDCl3): 18.5 (C-18), 19.5 (C-19), 20.5, 25.5, 26.2, 26.2, 26.3, 28.7, 31.7, 32.1, 32.3, 32.5,
33.2, 33.2, 35.4, 36.6, 37.3, 42.3, 46.2, 49.6, 50.3, 70.1 (C-17), 71.7 (C-3), 119.6 (C-5’),
121.3 (C-6), 140.9 (C-5), 152.9 (C-4’).
2.8.7. 17α-[4’-(2’’-Pyridyl)-1’H-1’,2’,3’-triazol-1’-yl]androst-5-en-3β-ol (8g)
Compound 3b (315.45 mg, 1 mmol) and 2-ethynylpyridine (7g, 0.22 ml) were used for the
synthesis as described in Section 2.8. The crude product was chromatographed on silica gel
with ethyl acetate/CH2Cl2 (10:90 v/v) to yield pure 8g (270 mg, 64%) as a white solid. Mp:
о
20
211213 C; Rf = 0.30 (ss B); [α]D = – 88 (c 1 in CHCl3). (Found C, 74.48; H, 8.35; N,
1
13.12. C26H34N4O requires C, 74.61; H, 8.19; N, 13.39%). H NMR (δ, ppm, CDCl3): 0.97 (s,
6H, 18-19-H6), 3.46 (m, 1H, 3-H), 4.714.70 (m, 1H, 17-H), 5.325-31 (m, 1H, 6-H),
7.227.20 (m, 1H, 5’’-H), 7.76 (m, 1H, 4’’-H), 8.09 (s, 1H, 5’-H), 8.19 (d, 1H, J = 7.9 Hz,
13
3’’-H), 8.56-8.55 (m, 1H, 6’’-H). C NMR (δ, ppm, CDCl3): 18.0 (C-18), 19.0 (C-19), 20.0,
8

24.9, 28.4, 31.1, 31.49, 31.8, 32.1, 36.1, 36.7, 41.8, 45.7, 49.0, 49.9, 70.0 (C-17), 71.1 (C-3),
120.0, 120.8 (C-6), 121.3 (C-1’), 122.4, 136.6, 140.3 (C-5), 147.1 (C-1”), 148.9 (C-3”), 150.1
(C-4’).
2.8.8. 17α-(4’-Phenoxymethyl-1’,2’,3’-triazol-1’-yl)androst-5-en-3β-ol (8h)
Compound 3b (315.45 mg, 1 mmol) and phenyl propargyl ether (7h, 0.22 ml) were used for
the synthesis as described in Section 2.8. The crude product was chromatographed on silica
gel with ethyl acetate/CH2Cl2 (15:85 v/v) to yield pure 8h (332 mg, 76%) as white crystals.
Mp: 173175 оC; Rf = 0.50 (ss B); [α]D20 = – 54 (c 1 in CHCl3). (Found C, 75.05; H, 8.51; N,
1
9.952. N28H37N3O2 requires C, 75.13; H, 8.33; N, 9.39%). H NMR (δ, ppm, CDCl3): 0.91
and 0.95 (s, 3H, 18-H3 and s, 3H, 19-H3), 3.46 (m, 1H, 3-H), 4.55 (d, 1H, 17-H), 5.18 (s, 2H,
4’-CH2-O-), 5.31 (t, 1H, 6-H), 6.93 (m, 3H, 2’’-H, 4’’-H and 6’’-H), 7.24 (t, 2H, 3’’-, 5’’-H),
7.50 (s, 1H, 5’-H). 13C NMR (δ, ppm, CDCl3): 18.3 (C-18), 19.3 (C-19), 20.4, 25.3, 28.6,
31.5, 32.2, 32.4, 36.4, 37.1, 42.2, 46.1, 49.3, 50.0, 62.2, 70.3 (C-17), 71.5 (C-3), 114.9 (C-2”
and -6”), 121.2 (C-6 and C-4”), 122.8 (C-5’), 129.4 (C-3” and -5”), 140.7 (C-5), 143.4 (C-4’),
158.1 (C-1”).
2.8.9. 17α-(4’-Benzoyloxymethyl-1’H-1’,2’,3’-triazol-1’-yl)androst-5-en-3β-ol (8i)
Compound 3b (315.45 mg, 1 mmol) and propargyl benzoate (7i, 0.20 ml) were used for the
synthesis as described in Section 2.8. The crude product was chromatographed on silica gel
with ethyl acetate/CH2Cl2 (10:90 v/v) to yield pure 8i (385 mg, 81%) as a white solid. Mp:
222223 оC; Rf = 0.45 (ss B); [α]D20 = − 56 (c 1 in CHCl3). (Found C, 73.48; H, 7.66; N, 8.68.
C29H37N3O3 requires C, 73.23; H, 7.84; N, 8.83%). 1H NMR (δ, ppm, DMSO): 0.90 and 0.92
(s, 3H, 18-H3 and s, 3H, 19-H3), 3.21 (m, 1H, 3-H), 4.61 (d, 1H, J = 4.5 Hz, -OH ), 4.62 (d,
1H, J = 7.5 Hz, 17-H), 5.26 (s, 1H, 6-H), 5.39 ( s, 2H, -CH2-O-), 7.51 (t, 2H, J = 7.5 Hz, 3’’-
and 5’’ -H), 7.66 (t, 1H, J = 7.5 Hz, 4’’-H), 7.95 (d, 2H, J = 7.5 Hz, 2’’-H and 6’’-H), 8.24 (s,
13
1H, 5’-H). C NMR (δ, ppm, DMSO): 17.7 (C-18), 19.1 (C-19), 19.9, 24.9, 28.0, 31.3, 31.5,
31.6, 31.9, 36.0, 36.8, 42.1, 45.4, 49.3, 49.6, 58.0 (4’-CH2-), 69.0 (C-17), 69.9 (C-3), 120.2
(C-6), 125.2 (C-5’), 128.7 (C-2” and -6”), 129.1 (C-3” and -5”), 129.3 (C-1”), 133.4 (C-4”),
141.1 (C-4’), 141.1 (C-5), 165.4 (C=O).
2.8.10. 17α-[4’-(4’’-Toluoyloxymethyl)-1’H-1’,2’,3’-triazol-1’-yl]androst-5-en-3β-ol (8j)
Compound 3b (315.45 mg, 1 mmol) and propargyl 4-methylbenzoate (7j, 0.20 ml) were used
for the synthesis as described in Section 2.8. The crude product was chromatographed on
9

silica gel with ethyl acetate/CH2Cl2 (10:90 v/v) to yield pure 8j (430 mg, 88%) as a white
о
20
crystalline solid. Mp: 250252 C; Rf = 0.48 (ss B); [α]D = – 55 (c 1 in CHCl3). (Found C,
73.72; H, 7.95; N, 8.44. C30H39N3O3 requires C, 73.59; H, 8.03; N, 8.58%). 1H NMR (δ, ppm,
DMSO): 0.90 and 0.92 (s, 3H, 18-H3 and s, 3H, 19-H3), 2.37 (s, 3H, 4’’-H3), 4.60 (d, 1H, J =
4.5 Hz, 3-H), 4.69 (d, 1H, J = 8.0 Hz, 17-H), 5.27 (d, 1H, J = 3.0 Hz, 6-H), 5.36 (s, 2H, 4’-
H2), 7.32 (d, 2H, J = 8.0 Hz, 3’’- and 5’’-H), 7.84 (d, 2H, J = 8.0 Hz, 2’’- and 6’’-H), 8.22 (s,
1H, 5’-H). 13C NMR (δ, ppm, DMSO): 17.7 (C-18), 19.1 (C-19), 19.9, 24.9, 28.0, 31.3, 31.5,
31.6, 31.9, 36.0, 36.8, 42.1, 45.4, 49.3, 49.6, 58.0, 69.0, (C-17), 69.9 (C-3), 120.1 (C-6), 125.2
(C-5’), 128.7 and 129.1 (C-4, C-2’’, C-3’’, C-5’’ and C-6’’), 133.5 (C-1’’), 141.0 (C-5), 141.1
(C-4’), 165.4 (C=O).
2.8.11. 17α-[4’-(4’’-Nitrobenzoyloxymethyl)-1’H-1’,2’,3’-triazol-1’-yl]androst-5-en-3β-ol
(8k)
Compound 3b (315.45 mg, 1 mmol) and propargyl 4-nitrobenzoate (7k, 411 mg) were used
for the synthesis as described in Section 2.8. The crude product was chromatographed on
silica gel with ethyl acetate/CH2Cl2 (10:90 v/v) to yield pure 8k (360 mg, 62%) as pale yellow
о
crystals. Mp: 201202 C; Rf = 0.45 (ss B); [α]D20 = – 63 (c 1 in CHCl3). (Found C, 67.02; H,
1
6.75; N, 10.85. C29H36N4O5 requires C, 66.90; H, 6.97; N, 10.76%). H NMR (δ, ppm,
CDCl3): 0.95 and 0.97 (s, 3H, 18-H3 and s, 3H, 19-H3), 3.47 (m, 1H, 3-H), 4.59 (d, 1H, J =
7.5 Hz, 6-H), 5.49 (s, 2H, 4’-H2), 7.61 (s, 1H, 5’-H), 8.20 (d, 2H, J = 9.0 Hz, 3’’- and 5’’-H),
8.26 (d, 2H, J = 9.0 Hz, 2’’- and 6’’-H). 13C-NMR (δ, ppm, CDCl3): 18.4 (C-18), 19.3 (C-19),
20.3, 25.3, 28.7, 31.5, 31.9, 32.1 (C-8), 32.4, 36.4, 37.1, 42.1, 46.1 (C-13), 49.4 (C-9), 50.1
(C-14), 58.9 (4’-CH2-), 70.4, (C-17), 71.5 (C-3), 121.1 (C-6), 123.5 (C-2’’ and C-6’’), 124.4
(C-5’), 130.9 (C-3’’ and C-5’’), 135.1 (C-1’), 140.7 (C-5), 141.2 (C-4’), 150.6 (C-4’’), 164.6
(C=O).
2.8.12. 17α-(4’-Hydroxymethyl-1’H-1’,2’,3’-triazol-1’-yl)androst-5-en-3β-ol (8l)
Compound 8k (520 mg, 1 mmol) was dissolved in methanol (10 ml) containing NaOCH3 (27
mg, 0.5 mmol), and the solution was allowed to stand for 24 h. It was then diluted with water,
and the white precipitate separating out was filtered off, dried and recrystallized from a
mixture of ethyl acetate/hexane to afford 8l (350 mg, 94%) as a white solid. Mp: 217219 оC;
20
Rf = 0.20 (ss B); [α]D = – 38 (c 1 in ethanol). (Found C, 71.25; H, 9.03; N, 11.16.
C22H33N3O2 requires C, 71.12; H, 8.95; N, 11.31%). 1H NMR (δ, ppm, DMSO): 0.89 and 0.93
10

(s, 3H, 18-H3 and s, 3H, 19-H3), 4.49 (d, 2H, J = 5.0 Hz, 4’-CH2-), 5.14 (d, 1H, J = 5.0 Hz,
13
17-H), 5.27 (s, 1H, 6-H), 7.92 (s, 1H, 5’-H). C NMR (δ, ppm, DMSO): 17.8 (C-18), 19.1
(C-19), 19.9, 24.9, 28.0, 31.3, 31.5, 31.6 (C-8), 31.9, 36.0, 36.8, 42.1, 45.3, 49.3 (C-9), 49.6
(C-14), 55.0 (4’-CH2-), 68.8 (C-17), 69.9 (C-3), 120.2 (C-6), 123.0 (C-5’), 141.1 (C-4’), 147.1
(C-5).
2.9. General procedure for the synthesis of 5’-iodotriazoles (8mk and 9mk)
Compound 3b or 5b (315.45 mg, 1 mmol) was dissolved in CH2Cl2 (20 ml), then Et3N (0.2
ml, 2 mmol), substituted acetylene derivative (7mk, 2 mmol), ICl (1.5 mmol) and, finally,
CuI (190 mg, 10 mol) were added. The heterogeneous reaction mixture was stirred under N2
for 24 h, then diluted with 1% N2S2O3 solution (30 ml), and extracted with CH2Cl2 (2 × 30
ml). The combined organic phases were dried over Na2SO4 and evaporated in vacuo. The
crude product was purified by flash chromatography using ethyl acetate/CH2Cl2 (5:95 v/v) to
obtain the steroidal 5’-iodotriazoles (8mn and 9mn).
2.9.1. 17α-[4’-(4’’-Nitrobenzoyloxymethyl)-5’-iodo-1’H-1’,2’,3’-triazol-1’-yl]androst-5-en-
3β-ol (8m)
Compound 3b (315.45 mg, 1 mmol) and propargyl 4-nitrobenzoate (7k, 204 mg) were used
for the synthesis as described in Section 2.9. The crude product was chromatographed on
silica gel with ethyl acetate/CH2Cl2 (5:95 v/v) to yield a pale yellow crystalline material (320
о
mg, 49%). Mp: 278281 C; Rf = 0.60 (ss B); [α]D20 = + 81 (c 1 in CHCl3). (Found C, 54.02;
1
H, 5.37; N, 8.55. C29H35IN4O5 requires C, 53.87; H, 5.46; N, 8.67%). H NMR (δ, ppm,
CDCl3): 0.99 and 1.02 (s, 3H, 18-H3 and s, 3H, 19-H3), 3.50 (m, 1H, 3-H), 4.64 (dd, 1H, J =
9.0 Hz, J = 4.5 Hz, 17-H), 5.35 (d, 1H, J = 6.5 Hz, 6-H), 5.47 (d, 2H, J = 5.5 Hz, 4’-H2), 8.22
13
(d, 2H, J = 11.0 Hz, 3’’-H and 5’’-H), 8.27 (d, 2H, J = 11.0 Hz, 2’’-H and 6’’-H). C NMR
(δ, ppm, CDCl3): 18.4 (C-18), 19.4 (C-19), 20.5, 25.6, 29.1, 31.5, 31.8, 32.1, 32.5, 36.4, 37.1,
42.2 (C-13), 46.9, 49.2 (C-9), 49.7 (C-14), 59.1 (4’-CH2-), 70.3 (C-17), 71.6 (C-3), 121.4 (C-
5’), 121.5 (C-6), 123.5 (C-2’’ and C-6’’), 11.0 (C-3’’ and C-5’’), 135.1 (C-1’), 140.5 (C-5),
145.1 (C-4’), 150.6 (C-4’’), 164.3 (C=O).
2.9.2. 17α-(4’-Cyclopropyl-5’-iodo-1’H-1’,2’,3’-triazol-1’-yl)androst-5-en-3β-ol (8n)
Compound 3b (315.45 mg, 1 mmol) and cyclopropylacetylene (7d, 0.22 ml) were used for the
synthesis as described in Section 2.9. The crude product was chromatographed on silica gel
11

with ethyl acetate/CH2Cl2 (5:95 v/v) to yield 8n (285 mg, 57%) as white crystals. Mp:
176179 оC; Rf = 0.55 (ss B); [α]D20 = + 43 (c 1 in CHCl3). (Found C, 56.63; H, 6.89; N, 8.42.
1
C24H34IN3O requires C, 56.80; H, 6.78; N, 8.28%). H NMR (δ, ppm, CDCl3): 0.92 and 0.98
(s, 3H, 18-H3 and s, 3H, 19-H3), 3.50 (m, 1H, 3-H), 4.51 (d, 1H, J = 5.5 Hz, 17-H), 5.33 (d,
1H, J = 4.5 Hz, 6-H). 13C NMR (δ, ppm, CDCl3): 7.2 and 7.7 (C-2’’ and C-3’’), 18.4 (C-18),
19.3 (C-19), 20.4, 25.3, 28.6, 31.5, 31.9, 32.1, 32.3, 36,5, 37.1, 42.2, 46.0 (C-13), 49.4 (C-14),
50.1, 70.0 (C-17), 71.5 (C-3), 121.5 (C-6), 140.4 (C-5), 149.2 (C-4’), 180.5 (C-5’).
2.10. General procedure for the synthesis of 5’-hydroxytriazoles (8o, 9o)
Compound 8m or 9m (127 mg, 0.25 mmol) was dissolved in Ac2O (3 ml) containing KOAc
(196 mg, 2 mmol), and the mixture was treated at reflux temperature for 4 h. The progress of
the reaction was monitored by TLC. After completion of the transformation, the reaction
mixture was diluted with water, then extracted with CH2Cl2 (2 × 30 ml). The CH2Cl2 solution
was washed with NaHCO3 solution and then with water, dried and evaporated in vacuo. The
residual yellow oil was dissolved in methanol (15 ml) containing KOH (56 mg, 1 mmol) and
the solution was treated at reflux temperature for 2 h. The precipitate separating out was
filtered off and recrystallized from a mixture of acetone/water.
2.10.1. 17α-(4’-Cyclopropyl-5’-hydroxy-1’H-1’,2’,3’-triazol-1’-yl)androst-5-en-3β-ol (8o)
Compound 8n (127 mg, 0.25 mmol) were used for the synthesis described in Section 2.10.
8o (68 mg, 69%). Mp: 193195 оC; Rf = 0.15 (ss B); [α]D20 = – 40 (c 1 in ethanol). (Found C,
1
72.68; H, 8.45; N, 10.72. C24H35N3O2 requires C, 72.51; H, 8.87; N, 10.57%). H NMR (δ,
ppm, DMSO): 0.87 and 0.92 (s, 3H, 18-H3 and s, 3H, 19-H3), 2.33 (m, 1H, 3-H), 4.04 (d, 1H,
J = 8.5 Hz, 17-H), 4.28 (d, 1H, J = 8.0 Hz, 6-H), 5.27 (s, 1H, 5’-OH). 13C NMR (δ, ppm,
DMSO): 5.0 (C-18), 6.2, 6.6, 10.0 (C-19), 17.9, 19.1 (C-8), 19.9, 25.1, 27.1, 31.3, 31.6 (C-9),
36.0, 36.8, 42.1 (C-13), 45.3 (C-5’), 49.3 (C-14), 49.4 (C-1’’), 50.8, 61.6, 63.6 (C-17), 69.9
(C-3), 120.2 (C-6), 141.1 (C-4’), 172.5 (C-5).
2.11.1. 17β-(4’-Phenyl-1’H-1’,2’,3’-triazol-1’-yl)androst-5-en-3β-ol (9a)
Compound 5b (315.45 mg, 1 mmol) and phenylacetylene (7a, 0.22 ml) were used for the
synthesis as described in Section 2.8. The crude product was chromatographed on silica gel
with ethyl acetate/CH2Cl2 (1:99 v/v) to yield pure 9a (283 mg, 68%) as a white crystalline
product. Mp: 280283 оC; Rf = 0.40 (ss B); [α]D20 = – 40 (c 1 in CHCl3). (Found: C, 77.48; H,
12

1
8.31; N, 10.27. C27H35N3O requires C, 77.66; H, 8.45; N, 10.6%). H NMR (δ, ppm, DMSO):
0.95 and 1.24 (s, 3H, 18-H3 and 19-H3), 4.52 (t, 1H, J = 9.5 Hz, 17-H), 4.61 (d, 1H, J = 4.0
Hz, 3-H), 5.31 (dd, 1H, J = 4.0 Hz, J = 1.5 Hz, 6-H), 7.32 (t, 1H, J = 7.5 Hz, 4’’-H), 7.44 (t,
2H, J = 7.5 Hz, 3’’-H and 5’’-H). 13C NMR (δ, ppm, DMSO): 12.5 (C-18), 19.6 (C-19), 21.0,
23.8, 26.1, 31.4, 31.7, 32.4, 36.9, 37.2, 37.6, 42.1 (C-13), 44.7, 50.4, (C-9), 53.5 (C-14), 71.2
(C-17), 71.4 (C-3), 120.0 (C-6), 121.2 (C-5’), 126.0 (C-2’’ and C-6’’), 128.6, 129.2 (C-3’’ and
C-5’’), 130.6, 141.4, (C-5), 147.4 (C-4’).
2.11.2. 17β-[4’(4’’-Tolyl)-1’H-1’,2’,3’-triazol-1’-yl]androst-5-en-3β-ol (9b)
Compound 5b (315.45 mg, 1 mmol) and 4-tolylacetylene (7b, 0.22 ml) were used for the
synthesis as described in Section 2.8. The crude product was chromatographed on silica gel
with ethyl acetate/CH2Cl2 (99:1 v/v) to yield pure 9b (210 mg, 77%) as a white crystalline
material. Mp: 293295 оC; Rf = 0.45 (ss B); [α]D20 = – 42 (c 1 in CHCl3). (Found C, 77.78; H,
1
8.83; N, 9.54. C28H37N3O requires C, 77.92; H, 8.64, N, 9.79%). H NMR (δ, ppm, DMSO):
0.51 and 0.94 (s, 3H, 18-H3 and s, 3H, 19-H3), 4.49 (s, 1H, 17-H), 4.53 (d, 1H, J = 2.0 Hz, 3-
H), 5.30 (d, 2H, J = 2.0 Hz, 6-H2), 7.24 (d, 2H, J = 1.5 Hz, 3’’- H and 5’’- H), 7.75 (d, 2H, J
= 1.5 Hz, 2’’- H and 6’’- H), 8.59 (s, 1H, 5’- H).
2.11.3. 17β-[4’(4’’-Methoxyphenyl)-1’H-1’,2’,3’-triazol-1’-yl]androst-5-en-3β-ol (9c)
Compound 5b (315.45 mg, 1 mmol) and 4-methoxyphenyl acetylene (7c, 0.22 ml)) were used
for the synthesis as described in Section 2.8. The crude product was chromatographed on
silica gel with ethyl acetate/CH2Cl2 (5:95 v/v) to yield pure 9c (143 mg, 67%) as a white
о
20
solid. Mp: 260262 C; Rf = 0.40 (ss B); [α]D = – 30 (c 1 in CHCl3). (Found C, 75.27; H,
1
8.22; N, 9.46. C28H37N3O2 requires C, 75.13; H, 8.33; N, 9.39%). H NMR (δ, ppm, CDCl3):
0.62 and 1.01 (s, 3H, 18-H3 and s, 3H, 19-H3) 3.53 (m, 1H, 3-H), 3.84 (-OCH3), 4.43 (t, 1H, J
= 9.5 Hz, 17-H), 5.38 (s, 1H, 6-H), 6.95 (d, 2H, J = 8.5 Hz, 2’’- and 6’’-H), 7.67 (s, 1H, 5’-
13
H), 7.76 (d, 2H, J = 8.5 Hz, 3’’-H and 5’’-H). C NMR (δ, ppm, CDCl3): 12.0 (C-18), 19.4
(C-19), 20.7, 23.5, 25.9, 31.5, 31.6, 32.0, 36.9, 37.2, 42.2, 44.3, 50.1, 53.2, 55.3, 63.3, 65.2,
70.2, 71.6 (C-3), 114.2 (2C, C-2’’ and C-6’’), 118.3 (C-5’) 121.0 (C-6), 126.9 (2C, C-3’’ and
C-5’’), 140.9 (C-5), 149.0, 159.6.
2.11.4. 17β-(4’-Cyclopropyl-1’H-1’,2’,3’-triazol-1’-yl)androst-5-en-3β-ol (9d)
Compound 5b (315.45 mg, 1 mmol) and cyclopropylacetylene (7d, 0.22 ml) were used for the
synthesis as described in Section 2.8. The crude product was chromatographed on silica gel
13

о
with ethyl acetate/CH2Cl2 (1:99 v/v) to yield pure 9d (170 mg, 45%). Mp: 242244 C; Rf =
20
0.48 (ss B); [α]D =− 36 (c 1 in CHCl3). (Found C, 75.62; H, 9.03; N, 11.25. C24H35N3O
requires C, 75.55; H, 9.25; N, 11.01%). 1H NMR (δ, ppm, CDCl3): 0.55 and 1.00 (s, 3H, 18-
H3 and s, 3H, 19-H3), 3.52 (m, 1H, 3-H), 4.32 (t, 1H, J = 9.5 Hz, 17-H), 5.35 (d, 1H, J = 5.0
13
Hz, 6-H), 7.23 (s, 1H, 5’-H). C NMR (δ, ppm, CDCl3): 6.6 (C-18), 7.8 (C-2’’ and C-3’’),
11.4 (C-19), 19.4 (C-8), 20.6, 23.4, 25.8, 29.7, 31.5, 31.6, 32.0 (C-9), 36.3, 36.9, 37.2, 42.2,
44.2 (C-13), 50.0 (C-14), 53.1 (C-1’’), 70.5 (C-17), 71.6 (C-3), 119.3 (C-6), 121.0 (C-4’),
141.0 (C-5), 149.2 (C-5’).
2.11.5. 17β-(4’-Cyclopentyl-1’H-1’,2’,3’-triazol-1’-yl)androst-5-en-3β-ol (9e)
Compound 5b (315.45 mg, 1 mmol) and cyclopentylacetylene (7e, 0.22 ml) were used for the
synthesis as described in Section 2.8. The crude product was chromatographed on silica gel
with ethyl acetate/CH2Cl2 (5:95 v/v) to yield 9e (290 mg, 71%) as a white crystalline product.
Mp: 275277 оC; Rf = 0.45 (ss B); [α]D20 = − 48 (c 1 in CHCl3). (Found C, 76.38; H, 9.83; N,
1
10.12. C26H39N3O requires C, 76.24; H, 9.60; N, 10.26%). H NMR (δ, ppm, CDCl3): 0.53
and 0.98 (s, 3H, 18-H3 and s, 3H, 19-H3), 3.50 (m, 1H, 3-H), 4.33 (t, 1H, J = 9.5 Hz, 17-H),
5.34 (s, 1H, 6-H), 7.21 (d, 2H, J = 8.0 Hz, 2’’- and 6’’-H), 7.67 (s, 1H, 5’-H), 7.92 (d, 2H, J =
8.0 Hz, 3’’- and 5’’-H). 13C NMR (δ, ppm, CDCl3): 11.9 (C-18), 19.3 (C-19), 20.6, 23.4, 25.1,
25.8, 31.4 (C-13), 32.0, 33.1, 33.2, 36.5, 36.7, 36.8, 37.2, 42.0, 44.1, 50.0, 53.1, 70.4 (C-17),
71.4 (C-3), 119.1 (C-5’), 121.0 (C-6), 141.0 (C-5), 151.8 (C-4’).
2.11.6. 17β-(4’-Cyclohexyl-1’H-1’,2’,3’-triazol-1’-yl)androst-5-en-3β-ol (9f)
Compound 5b (315 mg, 1 mmol) and cyclohexyacetylene (7f, 0.22 ml) were used for the
synthesis as described in Section 2.8. The crude product was chromatographed on silica gel
with ethyl acetate/CH2Cl2 (5:95 v/v) to yield 9f (250 mg, 59%) as a white solid. Mp: 296297
20
оC; Rf = 0.40 (ss B); [α]D = − 49 (c 1 in CHCl3). (Found C, 76.73; H, 9.55; N, 9.76.
C27H41N3O requires C, 76.55; H, 9.76; N, 9.92%). 1H NMR (δ, ppm, CDCl3): 0.55 (s, 3H, 18-
H3), 1.01 (s, 3H, 19-H3), 1.84 (m, 8H, 2”-, 3”-, 5”- and 6”-H2), 3.40 (m, 1H, 3-H), 4.45 (t, 1H,
J = 9.5 Hz, 17-H), 5.34 (dd, 1H, J = 3.5 Hz, J = 2.5 Hz, 6-H), 7.71 (s, 1H, 4’-H).
2.11.7. 17β-[4’(2’’-Pyridyl)-1’H-1’,2’,3’-triazol-1’-yl)]androst-5-en-3β-ol (9g)
Compound 5b (315 mg, 1 mmol) and 2-ethynylpiridine (7g, 0.22 ml) were used for the
synthesis as described in Section 2.8. The crude product was chromatographed on silica gel
14

with ethyl acetate/CH2Cl2 (10:90 v/v) to yield 9g (330 mg, 79%) as a white crystalline
material. Mp: 254256 оC; Rf = 0.35 (ss B); [α]D20 = – 34 (c 1 in CHCl3). (Found C, 74.38; H,
8.25; N, 13.55. C26H34N4O requires C, 74.61; H, 8.19; N, 13.39%). 1H NMR (δ, ppm, CDCl3):
1.00 and 1.24 (s, 3H, 18-H3 and s, 3H 19-H3), 3.53 (m, 1H, 3-H), 4.46 (t, 1H, J = 9.5 Hz, 17-
H), 5.36 (s, 1H, 6-H), 7.21 (t, 1H, J = 6.1 Hz, 5’’-H), 7.77 (m, 1H, 4’’-H), 8.18 (s, 1H, 5’-H),
13
8.20 (s, 1H, 3’’-H), 8.57 (d, 1H J = 4.1 Hz, 5’’-H). C NMR (δ, ppm, CDCl3): 12.0 (C-18),
19.4 (C-19), 20.6, 23.5, 25.9, 29.7, 31.5, 31.6, 32.0, 36.9, 37.2, 42.2, 44.3 (C-13), 50.0, 53.2,
70.7 (C-17), 71.6 (C-3), 120.2, 121.0 (C-6), 121.4 (C-5’), 122.7, 131.9, 141.0 (C-5), 147.4 (C-
1”), 149.3 (C-3”), 150.5 (C-4’).
2.11.8. 17β-(4’-Phenoxymethyl-1’H-1’,2’,3’-triazol-1’-yl)androst-5-en-3β-ol (9h)
Compound 5b (315 mg, 1 mmol) and phenyl propargyl ether (7h, 0.22 ml) were used for the
synthesis as described in Section 2.8. The crude product was chromatographed on silica gel
with ethyl acetate/CH2Cl2 (5:95 v/v) to yield 9h (356 mg, 79%) as white crystals. Mp:
198201 оC; Rf = 0.48 (ss B); [α]D20 = – 46 (c 1 in CHCl3). (Found C, 75.36; H, 8.22; N, 9.51.
1
C28H37N3O2 requires C, 75.13; H, 8.33; N, 9.39%). H NMR (δ, ppm, CDCl3): 0.56 and 1.00
(s, 1H, 18-H3 and s, 1H, 19-H3, and q, J = 11.0 Hz, 1H), 3.52 (m, 1H, 3-H), 4.39 (t, 1H, J =
9.5 Hz, 17-H), 5.21 (s, 1H, 4’-CH2-), 3.53 (d, 1H, J = 2.5 Hz, 6-H), 5.45 (s, 1H, 4’-CH2-) 6.97
(overlapping multiplets, 3H, 3’’-,4’’- and 5’’-H), 7.29 (t 2H, J = 8.3 Hz, 2’’-H and 6’’-H),
7.60 (s, 1H, 5’-H). 13C (δ, ppm, CDCl3): 12.0 (C-18), 19.4 (C-19), 20.6, 23.5, 25.9, 31.4, 31.5,
32.0, 36.5, 36.8, 37.2, 42.2, 44.2, 50.0, 53.1, 62.2 (4’-CH2-), 70.6, 71.6 (C-3), 114.8 (2C, C-
2’’ and C-6’’) 121.0 (C-6), 121.2, 122.1, 1129.5 (2C, C-3’’ and C-5’’), 141.0 (C-5), 143.4,
158.2.
2.11.9. 17β-(4’-Benzoyloxymethyl-1’H-1’,2’,3’-triazol-1’-yl)androst-5-en-3β-ol (9i)
Compound 5b (315 mg, 1 mmol) and propargyl benzoate (7i, 0.22 ml) were used for the
synthesis as described in Section 2.8. The crude product was chromatographed on silica gel
with ethyl acetate/CH2Cl2 (10:90 v/v) to yield 9i (230 mg, 48%) as a white solid. Mp 203-206
20
оC; Rf = 0.40 (ss B); [α]D = – 50 (c 1 in CHCl3). (Found C, 73.42; H, 8.02; N, 8.67.
1
C29H37N3O3 requires C, 73.23; H, 7.84; N, 8.83%). H NMR (δ, ppm, CDCl3) 0.55 and 0.99
(s, 3H, 18-H3 and s, 3H, 19-H3), 3.52 (m, 1H, 3-H), 4.40 (t, 1H, J = 9.5 Hz, 17-H), 5.35 (s,
1H, 6-H), 5.47 (s, 2H, -CH2-O-), 7.42 (t, 2H, J = 7.5 Hz, 3’’-H and 5’’-H), 7.55 (t, 1H, J = 7.5
13
Hz, 4’’-H), 7.69 (s, 1H, 5’-H), 8.03 (d, 2H, J = 7.5 Hz, 2’’-H and 6’’-H). C NMR (δ, ppm,
CDCl3) 12.0 (C-18), 19.4 (C-19), 20.6, 23.4, 25.9, 29.7, 31.4, 31.5, 32.0, 36.5, 36.8, 37.2,
15

42.1, 44.2, 50.0, 53.1, 58.2, 70.6 (C-17), 71.5 (C-3), 121.0 (C-6), 123.5 (C-5’), 128.3 and
129.7 (4C, C-2’’, C-3’’, C-5’’ and C-6’’), 133.1 (C-4’’), 140.9, 142.0, 166.5 (C=O).
2.11.10. 17β-[4’-(4’’-Toluoyloxymethyl)-1’H-1’,2’,3’-triazol-1’-yl]androst-5-en-3β-ol (9j)
Compound 5b (315 mg, 1 mmol) and propargyl 4-methylbenzoate (7j, 0.22 ml) were used for
the synthesis as described in Section 2.8. The crude product was chromatographed on silica
gel with ethyl acetate/CH2Cl2 (10:90 v/v) to yield 9j (250 mg, 51%) as a white solid. Mp:
212215 оC; Rf = 0.45 (ss B); [α]D20 = – 48 (c 1 in CHCl3). (Found C, 73.70; H, 9.87, N, 8.75.
1
C30H39N3O3 requires C, 73.59, H, 8.03; N, 8.58%). H NMR (δ, ppm, CDCl3): 0.55 and 0.99
(s, 3H, 18-H3 and s, 3H, 19-H3), 2.39 (s, 3H, 4’’-H3), 2.51 (m, 1H), 3.53 (m, 1H, 3-H), 4.39 (t,
1H, J = 9.5 Hz, 17-H), 5.35 (d, 1H, J = 2.5 Hz, 6-H), 5.45 (s, 2H, 4’-CH2-), 7.21 and 7.92 (d,
2H, J = 8.0 Hz, 3’’-H and 5’’-H and d, 2H, J = 8.0 Hz, 2’’- and 6’’-H), 7.67 (s, 1H, 5’-H). 13C
NMR (δ, ppm, CDCl3): 12.0 (C-18), 19.4 (C-19), 20.6, 21.6 (4’’-CH3), 23.4, 25.9, 31.4, 31.5,
32.0, 36.5, 36.8, 37.2, 42.2, 44.2, 50.0, 53.1, 58.0 (4’-CH2-), 70.6 (C-17), 71.6 (C-3), 121.0
(C-6), 123.4 (C-5’), 127.1 (C-1”), 129.0 and 129.7 (4C, C-2’’, C-3’’, C-5’’ and C-6’’), 140.1
(C-5’), 141.2 (C-4’), 143.8 (C-1”), 166.5 (C=O).
2.11.11. 17β-[4’-(4’’-Nitrobenzoyloxymethyl)-1’H-1’,2’,3’-triazol-1’-yl]androst-5-en-3β-ol
(9k)
Compound 5b (315 mg, 1 mmol) and propargyl 4-nitrobenzoate (7k, 411 mg) were used for
the synthesis as described in Section 2.8. The crude product was chromatographed on silica
gel with ethyl acetate/CH2Cl2 (10:90 v/v) to yield 9k (375 mg, 72%) as a pale yellow solid.
Mp: 198199 оC; Rf = 0.42 (ss B); [α]D20 = − 43 (c 1 in CHCl3). (Found C, 66.82; H, 7.12; N,
1
10.64. C29H36N4O5 requires C, 66.90; H, 6.97; N, 10.76%). H NMR (δ, ppm, CDCl3): 0.56
and 0.99 (s, 3H, 18-H3 and s, 3H, 19-H3), 3.52 (m, 1H, 3-H), 4.41 (t, 1H, J = 9.5 Hz, 17-H),
5.35 (d, 1H, J = 5.0 Hz, 6-H), 5.51 (s, 2H, 4’-H2), 7.71 (s, 1H, 5’-H), 8.20 (d, 2H, J = 9.0 Hz,
3’’-H and 5’’-H), 8.26 (d, 2H, J = 9.0 Hz, 2’’-H and 6’’-H). 13C NMR (δ, ppm, CDCl3): 12.0
(C-18), 19.4 (C-18), 20.6, 23.4, 25.9, 29.6, 31.4, 31.5, 32.0 (C-8), 36.8, 37.2, 42.2, 44.3 (C-
13), 50.0 (C-9), 53.1 (C-14), 58.8 (4’-CH2-), 70.7 (C-17), 71.5 (C-3), 120.9 (C-6), 123.5 (C-
2’’ and C-6’’), 123.7 (C-5’), 130.9 (C-3’’ and C-5’’), 135.2 (C-1’’), 141.0 (C-5), 141.2 (C-4’),
150.6 (C-4’’), 164.6 (C=O).
2.11.12. 17β-(4’-Hydroxymethyl-1’H-1’,2’,3’ -triazol-1’-yl)androst-5-en-3β-ol (9l)
16

Compound 9k (520 mg, 1 mmol) was dissolved in methanol (10 ml) containing NaOCH3 (27
mg, 0.5 mmol), and the solution was allowed to stand for 24 h. It was then diluted with water,
and the precipitate separating out was filtered off, and recrystallized from a mixture of ethyl
acetate/hexane to afford 9l (320 mg, 86%) as a white crystalline product. Mp: 244245 оC; Rf
= 0.18 (ss B); [α]D20 = − 18 (c 1 in ethanol). (Found C, 70.98; H, 9.11; N, 11.46. C22H33N3O2
requires C, 71.12; H, 8.95; N, 11.31%). 1H NMR (δ, pp, DMSO): 0.47 and 0.94 (s, 3H, 18-H3
and s, 3H, 19-H3), 4.51 (s, 2H, 4’-CH2-), 4.61 (s, 1H, 3-H), 5.14 (s, 1H, 17-H), 5.29 (s, 1H,
6H), 7.98 (s, 1H, 5’-H). 13C NMR (δ, ppm, DMSO): 11.7 (C-18), 19.1 (C-19), 20.1, 23.0,
25.1, 30.9, 31.3, 31.5 (C-8), 36.1, 36.2, 36.9, 42.1, 43.5, 49.6 (C-9), 52.2 (C-14), 55.0 (4’-
CH2- ), 69.3 (C-17), 69.9 (C-3), 120.1 (C-6), 122.1 (C-5’), 141.3 (C-4’), 147.1 (C-5).
2.12.1. 17β-[4’-(4’’-Nitrobenzoyloxymethyl)-5’-iodo-1’H-1’,2’,3’-triazol-1’-yl]androst-5-en-
3β-ol (9m)
Compound 5b (315.45 mg, 1 mmol) and propargyl 4-nitrobenzoate (7k, 204 mg) were used
for the synthesis as described in Section 2.9. The crude product was chromatographed on
silica gel with ethyl acetate/CH2Cl2 (5:95 v/v) to yield 9m (375 mg, 72%) as a white solid.
Mp: 198200 оC; Rf = 0.55 (ss B); [α]D20 = + 89 (c 1 in CHCl3). (Found C, 53.62; H, 5.68; N,
8.72. C29H35IN4O5 requires C, 53.87; H, 5.46; N, 8.67%). 1H NMR (δ, ppm, CDCl3): 0.77 and
1.01 (s, 3H, 18-H3 and 19-H3), 3.53 (m, 1H, 3-H), 4.49 (t, 1H, J = 8.5 Hz, 17-H), 5.38 (d, 1H,
J = 5.0 Hz, 6-H), 5.49 (dd, 1H, J = 20.0 Hz, J = 13.0 Hz, 4’-CH2-), 8.24 (t, 2H, J = 7.0 Hz,
3’’-H and 5’’-H), 8.28 (t, 2H, J = 7.0 Hz, 2’’- and 6’’-H). 13C NMR (δ, ppm, CDCl3): 14.1 (C-
18), 19.4 (C-19), 20.8, 22.7, 23.6, 27.1, 29.4 (C-8), 31.5, 31.6, 31.9 , 32.0, 36.6 (C-9), 37.2,
38.2, 42.2, 45.6, (C-13), 50.0 (C-14), 53.7 (C-17), 59.1 (4’-CH2-), 71.6 (C-3), 121.1 (C-6),
123.5 (C-2’’ and C-6’’), 131.0 (C-3’’and C-5’’), 135.1 (C-1’), 140.9 (C-5), 164.4 (C=O).
2.12.2. 17β-(4’-Cyclopropyl-5’-iodo-1’H-1’,2’,3’-triazol-1’-yl)androst-5-en-3β-ol (9n)
Compound 5 (315.45 mg, 1 mmol) and cyclopropylacetylene (7d, 0.22 ml) were used for the
synthesis as described in Section 2.9. The crude product was chromatographed on silica gel
with ethyl acetate/CH2Cl2 (5:95 v/v) to yield 9n (260 mg, 53%) as a white crystalline
material. Mp: 210213 оC; Rf = 0.52 (ss B); [α]D20 = + 63 (c 1 in CHCl3). (Found C, 56.63; H,
1
6.91; N, 8.42. C24H34IN3O requires C, 6.80; H, 6.75, N, 8.28%). H NMR (δ, ppm, CDCl3):
0.70 and 0.98 (s, 3H, 18-H3 and s, 3H, 19-H3), 3.51 (m, 1H, 3-H), 4.40 (t, 1H, J = 9.5 Hz, 17-
13
H), 5.34 (d, 1H, J = 4.5 Hz, 6-H). C NMR (δ, ppm, CDCl3): 7.24, 7.26, 7.63 7.63, 12.7 (C-
17

18), 19.4 (C-19), 20.8, 23.5, 26.9, 29.7, 31.5, 32.0 (C-9), 37.2, 38.2, 42.2, 45.4 (C-13), 50.0
(C-14), 53.7 (C-1’’), 70.0 (C-17), 71.6 (C-3), 114.0 (C-5’), 121.1 (C-6), 140.9 (C-4’), 151.7
(C-5).
2.13.1. 17β-(4’-Cyclopropyl-5’-hydroxy-1’H-1’2’,3’-triazol-1’-yl)androst-5-en-3β-ol (9o)
Compound 9n (127 mg, 0.25 mmol) were used for the synthesis described in Section 2.10. 9r
о
20
(76 mg, 76%). Mp: 178180 C; Rf = 0.12 (ss B); [α]D = − 31 (c 1 in ethanol). (Found C,
72.67; H, 8.55, N, 10.68. C24H35N3O2 requires C, 72.51; H, 8.87; N, 10.57%).1H NMR (δ,
ppm, DMSO): 0.56 and 0.97 (s, 3H, 18-H3 and s, 3H, 19-H3), 1.98 (s, 4H, 2’’-H and 3’’-H),
2.62 (dd, 1H, J = 21.5 Hz, J = 10.5 Hz, 3-H), 4.18 (t, 1H, J = 9.5 Hz, 17-H), 4.45 (m, 1H, 6-
13
H), 5.37 (s, 1H, 5’-OH). C NMR (δ, ppm, DMSO): 5.0 (C-18), 6.1 and 6.5 (C-2’’ and C-
3’’), 12.2 (C-19), 18.9 (C-8), 20.1, 23.0, 24.3, 27.2, 30.9, 31.4 (C-9), 31.6, 36.4, 36.7, 37.6,
43.8, 49.3 (C-14), 52.4 (C-1’’), 61.5 (C-5’), 64.7 (C-17), 73.0 (C-3), 121.8 (C-6), 139.5 (C-
4’), 169.6 (C-5).
2.14. Determination of the C17,20-lyase inhibition
The inhibitory effect on the rat testicular C17,20-lyase enzyme exerted by the new compounds
were determined via an in vitro radiosubstrate incubation method [20]. In brief, adult Wistar
rat testicular tissue was homogenized with an Ultra-Turrax in 0.1 M HEPES buffer (pH = 7.3)
containing 1 mM EDTA and 1 mM dithiotreitol. Aliquots of this homogenate were incubated
in 200 μl final volume at 37 оC for 20 min in the presence of 0.1 mM NADPH. 1 μM [3H]-17-
hydroxyprogesterone was added to the incubate in 20 μl of a 25% (v/v) propylene glycol
solution. Test compounds were applied at 50 μM and introduced in 10 μl of DMSO. (Such
minor quantities of organic solvents used here did not reduce the enzyme activity
substantially). Control incubates without test substances, and incubates with the reference
compound ketoconazole and abirateron were also prepared in every series. Following
incubation, the androst-4-ene-3,17-dione formed and the 17-hydroxyprogesterone remaining
were isolated through extraction and TLC. C17,20-lyase activity was calculated from the
radioactivity of the androst-4-ene-3,17-dione obtained. Two experiments were performed with
each test compound and the standard deviations of the mean enzyme activity results (S.D.)
were within ± 15%. IC50 values were determined for the more active inhibitors. In this case,
the conversion was measured at 5 or 6 different concentrations of the test compound. IC50
results were calculated using an un-weighted iterative least squares method for four-parameter
18

logistic curve fitting. IC50 of unlabelled 17-hydroyprogesterone substrate, as well as
abiraterone, and another clinically relevant, nonsteroidal imidazole P45017α inhibitor,
ketoconazole were measured as reference. Reference IC50 values were determined for
unlabelled 17-hydroxyprogesterone substrate (1.6 μM) as well as for abiraterone (0.0125 μM)
and ketoconazole (0.32 μM).
2.14.1. Time dependence of inhibitory effect
Time-dependent change in the inhibitory effect was investigated by preincubation with the
testicular homogenate. Testicular homogenate in incubation mixture was preincubated with
IC50 concentration in the presence or the absence of NADPH cofactor for 40 minutes at 37 оC,
then the inhibition test was started by the addition of the substrate.
2.14.2. Investigation of binding mechanism
Reversibility of test compounds was investigated via preincubation and charcoal adsorption
method. Testicular homogenate in incubation mixture was preincubated with high
concentration (10 μM and 50 μM) of the tested compounds, in the presence of NADPH
о
cofactor for 10 and 40 minutes at 37 C. After the addition of charcoal suspension in 0.6%
final concentration, the preincubated samples were kept on ice for 15 minutes and centrifuged
at 4 оC. Supernatants were drained and used for inhibition tests.
3. Results and discussion
3.1. Synthetic studies
To prepare novel steroid triazoles via 1,3-dipolar cycloaddition, 17α- and 17β-azidoandrost-5-
en-3β-ol (3b and 5b) were selected as starting compounds. The synthetic strategy for the
preparation of the starting azides is illustrated in Scheme 1 and the synthesis of steroidal
1,2,3-triazoles by CuAAC is outlined in Scheme 1 and 2.
(Scheme 1.)
(Scheme 2.)
3β-Acetoxy-androst-5-en-17β-ol (1) was esterified with p-toluenesulfonyl chloride to
give tosylate 2, which then underwent facile SN2 substitution with NaN3 in N,N-
dimethylformamide to furnish the corresponding 17α-azido compound 3a. Deacetylation of
19

3a by the Zemplén method produced 17α-azido-androst-5-en-3β-ol 3b. Iodination of 1 by the
Appel reaction proceeded with Walden inversion to yield 3-acetoxy-androst-5-en-17α-iodide
4 [7]. Further nucleophilic exchange reaction with NaN3 in N,N-dimethylformamide furnished
17β-azido compound 5a in moderate yield. The exchange reaction was accompanied by
elimination to give 3-acetoxy-androst-5,16-diene (6) too.
The 17-azido derivatives (3b or 5b) were subjected to azide–alkyne CuAAC reactions
with substituted phenylacetylenes (7ac), small cycloalkyl acetylenes (7df), 2-
piridylacetylene (7g), phenyl propargyl ether (7h), and propargyl phenyl esters (7ik). The
CuAAC of 3b or 5b with substituted acetylenes (7ak) were carried out in refluxing CH2Cl2
with CuI as catalyst in the presence of Et3N (0.2 equivalent) to obtain the required 1,4-
disubstituted triazoles (8ak and 9ak) in moderate yields after 24 h.
Alkaline hydrolysis of 4’-benzoyloxymethyl triazoles 8ik and 9ik yielded polar 4’-
hydroxymethyltriazolyl derivatives 8l and 9l.
We found earlier that the CuAAC reaction in the presence of Et3N is accompanied by
building of 5’-iodo-1,4-triazoles in small quantities [21]. When 1.5 equivalent of ICl was used
as electrophile in the presence of 10 equivalents of CuI and Et3N, the quantity of 5’-iodo-
triazoles increased to 4972%. With this method we prepared 4’-p-nitrobenzoyloxymethyl-5’-
iodo- and 4’-cyclopropyl-5’-iodo triazoles 8mn and 9mn. Acetolysis of 8n and 9n in acetic
anhydride in the presence of KOAc followed by alkaline hydrolysis yielded 4’-cyclopropyl-5’
hydroxy triazolyl derivatives 8o and 9o.
3.2. C17,20-lyase inhibition studies
The P45017α catalyzed biotransformation of C21 steroids consists of hydroxylation and a
subsequent side-chain cleavage at the C-17 carbon. This second C17,20-lyase process is the
rate-limiting step in the synthesis of the C19 preandrogens. Inhibition of the C17,20-lyase
activity is, therefore, the primary evaluation criteria of compounds with presumed androgen-
withdrawing effect achieved by blockade of the P45017α [1, 5, 22]. In this study we
investigated inhibition against C17,20-lyase activity of P45017α exerted by the new androst-5-
en-3β-ol derivatives possessing diverse C-substituted triazolyl substituents or the azide group
in 17α or 17β position.
20

3.2.1 Inhibition effect of the17-heterocyclic compounds
Several related 5-membered N-containing imidazole, pyrazole, triazole, and tetrazole isomers
of androstane compounds were studied as potential C17,20-lyase inhibitors [5, 6, 23–25]. We
have previously observed with C-17β and C-17β phenyl-substituted pyrazolinylandrostenes
that the extended 17β heterocyclic side-chain with various substituted aromatic rings may
have substantial inhibitory effect [11]. Structural investigations proposed the existence of a
second hydrophobic binding pocket beside the heme structure, which also supports the
inhibitor development strategy involving an adjacent phenyl group on the C-17 substituent
[26]. As a further example, related studies with galeterone also indicate that compounds with
more spacious aromatic side chains may improve affinity and inhibitory effect towards
P45017α, and the extension of the side chain may be useful in modification of the activity
profile of a pharmacon candidate [27, 28].
In this report we investigated inhibition against C17,20-lyase activity of P45017α exerted
by the new androst-5-en-3β-ol derivatives (8 and 9) possessing diverse C-substituted triazolyl
substituents in the 17α and 17β position (Table 1 and Table 2).
(Table 1)
(Table 2)
Compounds bearing 17β-triazolyl substituents on the androst-5-en-3β-hydroxy
skeleton (9) exerted weak inhibition with the relative conversions more than 70% even in the
relatively high 50 μM test concentration (Table 1). In the 17α-triazolyl series two cyclopropyl
derivatives (8d, 8o) and three phenyl ester derivatives (8i, 8k, 8l) exerted more pronounced
inhibition (Table 1). Compounds 8o and 8i were found to be the best inhibitors among them
with IC50 values 3.1 μM and 3.5 μM, respectively (Table 1, Fig 1). Compounds 8g in the 17α-
triazolyl series and 9g in the 17β series containing heteroaryl substituents at the C-4’ carbon
were also weak inhibitors. In the series of the cycloalkyl-substituted 17α-triazolyl compounds
(8d-f) a decreasing inhibition could be observed with increasing carbon numbers in the cycle.
An additional iodo substituent on the triazole ring (8-m,n, 9m,n) was disadvantageous for
inhibition, whereas a hydroxyl group at the same position (8o ) improved the effect.
These results demonstrate substantial inhibitory effect of the C-4’-hydroxymethyl
benzoate 8i and p-nitrobenzoate 8k to the enzyme. Their IC50 values indicate affinities to the
enzyme similar to that of the substrate. The presence as well as the flexible and appropriate
21

positioning of phenyl group seems to be essential for the good binding such as the precursor
alcohol 8l. The C-4’ coupled phenyl group (8a), and the C4’-phenoxymethyl ether (8h)
derivatives were weak inhibitors. The p-nitro function on the phenyl group (8k) decreased the
inhibition in a lesser, whereas the p-methyl group (8j) decreased in a higher extent. Our
results reveal that stereoisomerism at the C-17 of the new androst-5-en-3β-ol derivatives
influences the C17,20-lyase effect. Inhibitors were found only in the 17α-triazolyl series,
whereas the 17β counterparts (9) were found to be almost inactive in the C17,20-lyase test.
3.2.2. Inhibition effect of the 17-azido compounds
Only a few steroid azides similar to those investigated here can be found in the literature. The
synthesis and antituberculosis activity of 17α-azido-5α-androstan-3β-ol was investigated by
Merlani et al. [29]. The neuroactive 17α- and 17β-azido-5β-androstan-3α-ols were studied by
Vidnra et al. [30]. 17-Azidomethyl-androst-5,16-dien-3β-ol was synthesized by the group of
Njar and Brodie during their P45017α inhibitor development studies [31]. P45017α or C17,20
-
lyase inhibitory potential of these related compounds, however, was not reported in either of
the corresponding publications.
Wright et al. investigated the inhibition of 19-azido-4-androstene-3,17-dione against
the aromatase, another cytochrome P450 dependent enzyme in the steroid hormone
metabolism [32]. They have found reversible inhibitor binding and hypothesized that the first
nitrogen atom in the azide group (attached directly to the C-19 carbon) is most likely to be
involved in coordination to the heme iron of aromatase.
Other, non-steroidal primary azides were found to be converted to the corresponding
aldehydes via biocatalysis of myoglobin, another heme-dependent metalloprotein similar to
cytochrome P450 [33]. The proposed binding and reaction mechanisms of the myoglobin-
catalyzed azide reaction also indicate that the first-nitrogen is coordinated to the heme iron of
the active center.
In this report we investigated C17,20-lyase inhibition of the two androst-5-en-3β-ol
compounds possessing azide group in the 17α or 17β position. Our results revealed that the
17-azido androstene compounds (3b, 5b) tested here were able to inhibit the C17,20-lyase
activity. Stereoisomerism at the C-17 atom influenced the effect in the pair of the 3β-hydroxy-
5-androstene compounds (3b, 5b). 17β-Azido derivative 5b was found to be more effective
(IC50 = 0.60 μM) than 17α-azide 3b (IC50 = 21 μM) (Table 2, Fig 1). The IC50 parameter of 5b
indicated a 2.5-fold stronger binding compared to the substrate, and its inhibitory potential
was found to be close to that of the nonsteroidal imidazole reference ketoconazole.
22

Potent inhibition of the 17β-azido 5b is an interesting finding; therefore, we further
investigated inhibitory properties of this compound.
(Fig 1)
3.2.2.1. Time dependence of inhibitory effect of 5b
The possible time-dependent change of inhibitory effect of 5b was investigated in
experiments in which the compound was applied in its IC50 concentration (0.60 μM) and
preincubated with the testicular homogenate before the usual inhibition test. No difference
was found between the normal and the preincubated samples either in case of test compound
5b or the reference abiraterone and ketoconazole (Fig. 2). The presence of cofactor during the
preincubation had no influence either on the measured inhibitory potentials. This observation
indicates that conversion of the tested compound, which could modify the inhibitory potential
of 17β-azido-androst-5-en-3β-ol 5b, does not occur under the conditions of incubation
procedures applied.
(Fig 2)
3.2.2.2. Reversibility of inhibition of 5b
Reversibility of the inhibition was studied via preincubation with high concentration of the
test compounds followed by the removal of unbound and reversibly bound inhibitor
molecules by charcoal adsorption. Samples pretreated with the known reversible inhibitor
compound ketoconazole as reference [34] displayed almost non-inhibited C17,20-lyase activity
[34], whereas irreversible reference inhibitor compound abiraterone [7] blocked enzyme
activity (Fig. 3). Incubations pretreated with compound 5b resulted in conversions close to
those in the inhibited control inhibition tests with high inhibitor concentration. These results
indicate that 5b molecules cannot be removed by charcoal treatment from the binding, that is
binding to the enzyme takes place via an irreversible mechanism. Note that in the case of
abiraterone and several other C-17 heterocyclic P45017α inhibitors, the Δ16,17 double bond not
only increases the inhibitory potency but also switches the binding mode from reversible to
irreversible [7]. Nevertheless, the irreversible binding of 17β-azido-androst-5-en-3β-ol (5b)
occurs in the absence of Δ16,17 double bond.
(Fig 3)
23

3.2.2.3. Aromatase inhibitory effect of 5b
Taking into account of the aromatase inhibitory effect of 19-azido-4-androstene-3,17-dione
[32] and certain P45017α inhibitor abiraterone analogs [35], we performed tests whether 5b is
capable of inhibiting this enzyme. The in vitro radiosubstrate incubations were based on
similar incubation methodology as described here for the C17,20-lyase. Incubations with high
concentrations (10 µM and 50 µM) resulted in relative conversions similar to the non-
inhibited controls (results are not shown). These results indicate that 17β-azido-androst-5-en-
3β-ol 5b does not inhibit aromatase activity substantially.
SUMMARY
P45017α is a pivotal enzyme of steroid biosynthesis. Inhibition of its C17,20-lyase activity leads
to suppressed androgen production, which can be applied for antiandrogen therapies. Results
on C17,20-lyase inhibitory effect of our new aryl-substituted 17-triazolyl androstenes revealed
that compounds bearing a 17α-heterocyclic side chain may also have substantial affinity to
this enzyme. Our results verified that extension of the C-17 side chain may improve inhibitory
potential. In addition, an azido group on the steroidal C-17 atom is a suitable pharmacophore
in the inhibition of the C17,20-lyase activity. The potent inhibition of the azido compound is an
interesting finding worth further investigations.
Acknowledgements
The work of Anita Kiss was supported by a PhD Fellowship of the Talentum Fund of Richter
Gedeon Plc. (Budapest). Financial support from the Economic Development and Innovation
Operative Programme of Hungary (GINOP-2.3.2-15-2016-00038) is gratefully
acknowledged. This research was supported by the Hungarian Scientific Research Fund
(OTKA K113150).
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27

Legends for Schemes and Figures
Scheme 1. Reagents and conditions: (i) KBH4, MeOH; (ii) TsCl, pyridine, rt, 24 h; (iii) NaN3,
DMF, 100 °C, 24 h; (iv) Ph3P, imidazole, I2, toluene, 80 °C, 2 h; (v) NaN3, DMF, 60 °C, 24h.
Fig. 1. Concentration-dependent inhibitory effects of test compounds 5b, 8o and 8i on the in
vitro activity of C17,20-lyase. [I]: inhibitor concentration in μM.
Fig. 2. Inhibitory potential of the IC50 concentration of 5b and reference compounds
(abiraterone and ketoconazole) with or without 40 min preincubations with the rat testicular
homogenate. Following the preincubation period, the usual incubation procedure was applied.
Fig. 3. Investigation of inhibition reversibility of 5b and reference compounds (abiraterone
and ketoconazole). Compounds were preincubated with rat testicular homogenate. Following
a charcoal separation step, the usual incubation procedure was applied.
28

Scheme 1.
OTs
OH
N3
H
H
H
H
H
H
H
H
H
AcO
AcO
RO
2
1
3
a
b
R
Ac
H
N3
H
H
H
I
RO
5
a
b
R
Ac
H
H
H
H
AcO
H
4
H
H
AcO
6
29