Synthesis of ferrocene-labeled steroids via copper-catalyzed azide-alkyne cycloaddition. Reactivity difference between 2β-, 6β- and 16β-azido-androstanes

Article abstract of DOI:10.1016/j.steroids.2012.04.005

Copper-catalyzed cycloaddition of steroidal azides and ferrocenyl-alkynes were found to be an efficient methodology for the synthesis of ferrocene-labeled steroids. At the same time, a great difference between the reactivity of 2β- or 16β-azido-androstane

Full text of DOI:10.1016/j.steroids.2012.04.005

Steroids 77 (2012) 738–744
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis of ferrocene-labeled steroids via copper-catalyzed azide–alkyne
cycloaddition. Reactivity difference between 2b-, 6b- and 16b-azido-androstanes
Klaudia Fehér a, János Balogh a, Zsolt Csók b, Tamás Kégl b, László Kollár b, Rita Skoda-Földes a,
⇑
a University of Veszprém, Institute of Chemistry, Department of Organic Chemistry, Egyetem u. 10, P.O. Box 158, H-8200 Veszprém, Hungary
b University of Pécs, Department of Inorganic Chemistry, Ifjúság u. 6, P.O. Box 266, H-7624 Pécs, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
Copper-catalyzed cycloaddition of steroidal azides and ferrocenyl-alkynes were found to be an efficient
methodology for the synthesis of ferrocene-labeled steroids. At the same time, a great difference between
the reactivity of 2b- or 16b-azido-androstanes and a sterically hindered 6b-azido steroid toward both
ferrocenyl-alkynes and simple alkynes, such as phenylacetylene, 1-octyne, propargyl acetate and methyl
propiolate, was observed.
Received 31 January 2012
Received in revised form 7 March 2012
Accepted 4 April 2012
Available online 13 April 2012
Ó 2012 Elsevier Inc. All rights reserved.
Keywords:
Copper
Cycloaddition
Triazole
Ferrocene
Azidosteroid
1. Introduction
Cholesterol [12] and pregnane [13] derivatives with an azido group
in the 17-alkyl side chain were converted to the corresponding tri-
High stability, accessibility of a great variety of derivatives and
favorable electrochemical properties make ferrocene and its deriv-
atives very suitable for biological applications and for conjugation
with biomolecules [1]. Combination of a ferrocenyl moiety with
structures of pharmacological importance may increase their bio-
logical activity. Low toxicity towards mammals make ferrocene
derivatives ideal candidates for drug design [2]. The incorporation
of the redox-active ferrocenyl moiety into a biomolecule (e.g. into a
steroid), makes its electrochemical detection possible [3]. The cop-
per-catalyzed 1,3-dipolar cycloaddition of azides and terminal al-
kynes (CuAAC, copper-catalyzed azide–alkyne cycloaddition), one
of the so-called ‘click reactions’ (Scheme 1) [4] is an efficient meth-
odology to attach a ferrocene label to compounds of diverse struc-
ture [5].
azoles in the presence of various terminal alkynes. Recently, some
new steroid–ferrocene conjugates were synthesised in our group
via the ‘click reaction’ of ferrocenyl azides and steroids with an
ethynyl group in the side chain [14].
These steroidal triazoles were obtained either in the reaction of
ethynyl/alkynyl steroids with azides of various structure, [7–
11,14,15] or via the cycloaddition of simple alkynes with steroids
bearing an azido group in the side chain [12,13,16]. However, there
are only a few examples for the ‘click reaction’ of steroids with an
azido group attached directly to the steroid core, at the easily
accessible 3b-[17,18] or 17a-positions [19].
Although CuAAC is reported to be unaffected by steric factors,
[7] we decided to explore if this methodology can be used for the
attachment of a ferrocene-containing label to different positions
of the steroidal skeleton. As a result, a great difference between
the reactivity of 2b- or 16b-azido–androstanes and a sterically hin-
dered 6b-azido steroid toward ferrocenyl-alkynes was observed. In
order to clarify if this difference is restricted to the reaction of ferr-
ocenyl alkynes, the ‘click reaction’ was also carried out with al-
kynes of different structures.
The CuAAC reaction has also been shown to serve as invaluable
tool for the selective labeling of biological molecules with different
moieties [6]. At the same time, fewer applications of this method-
ology for the synthesis of steroidal derivatives, compared to other
biomolecules, have been reported. The conversion of 17a-ethyny-
lestradiol to the corresponding 4-substituted 1,2,3-triazole was
already mentioned by Sharpless [7]. A similar reaction was used
for the synthesis of estradiol–cyclodextrin,[8] estradiol–peptide,
[9] bile acid–peptide [10] or bile acid–b-lactam [11] conjugates.
2. Experimental
1H and 13C NMR spectra were recorded in CDCl3 on a Varian Ino-
va 400 spectrometer at 400.13 MHz and 100.62 MHz, respectively.
Chemical shifts are reported in ppm relative to CHCl3 (7.26 and
⇑
Corresponding author.
E-mail address: skodane@almos.uni-pannon.hu (R. Skoda-Földes).
0039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2012.04.005

K. Fehér et al. / Steroids 77 (2012) 738–744
739
66; 185/2; 121/38; 56/21. IR (KBr,
m
(cmꢀ1)): 2110, 1643. Analysis
calculated for C14H13FeN (251.11): C, 66.96; H, 5.22; N, 5.58.
Found: C, 66.89; H, 5.31; N, 5.49. Yield: 78%.
Ferrocenylimine 12 (0.6 mmol, 150.6 mg), ethyl diazoacetate
(11) (0.6 mmol, 64 ll), Co2(CO)8 (0.03 mmol, 10 mg) and CH2Cl2
Scheme 1. Copper-catalyzed azide–alkyne cycloaddition (CuAAC).
(4 ml) were transferred under an inert atmosphere into a stainless
steel autoclave. It was charged with carbon monoxide (60 bar at
room temperature) and stirred at room temperature for 24 h. (Cau-
tion! Cylinders containing CO should be used according to the
safety instructions. The high pressure reactor should have the
appropriate capacity and pressure rating. The reactor should be lo-
cated in a laboratory hood during the reaction and should be
vented carefully and slowly in the hood.) After evaporation of the
solvent, the product was purified by column chromatography (sil-
ica, n-hexane/EtOAc = 3/1) to yield 7b. 1H NMR(d, CDCl3): 7.60 (s,
1H,=CH); 6.16 (t, J = 3.8 Hz, 1H, NH); 4.58–4.60 (m, 2H, NCH2);
4.46 (t, J = 1.8 Hz, 2H, 2,5Cp); 4.15–4.21 (m, 9H, OCH2, 3,4Cp,
unsubstituted Cp); 2.24 (t, J = 2.5 Hz, 1H, CCH); 1.31 (t, J = 7.1 Hz,
3H, CH3). 13C NMR(d, CDCl3): 164.3; 163.4; 143.3; 120.7; 77.2;
73.6; 70.2; 70.1; 69.3; 68.1; 59.4; 27.5; 12.4. MS (m/z/rel. int.):
365 (M+)/100; 320/4; 300/23; 254/12; 216/14; 173/20; 121/17;
77.00 ppm for 1H and 13C, respectively). Mass spectra were ob-
tained on an Autoflex II TOF/TOF (Bruker Daltonics, Bremen, Ger-
many) spectrometer in 2,5-dihydroxybenzoic acid matrix. Mass
spectrum of 7f was recorded on a HP-5971A MSD connected to a
HP-5890/II gas chromatograph. IR spectra were made using a Ther-
mo Nicolet Avatar 330 FT–IR instrument. Samples were prepared
as KBr pellets. Elemental analyses were measured on a 1108 Carlo
Erba apparatus.
2.1. Synthesis of steroidal azides 4–6
To a mixture of the epoxide (1–3) (6 mmol), sodium azide
(42 mmol, 2.73 g) and DMSO (45 ml), glacial acetic acid (9 ml)
was added. The mixture was heated with stirring at 110 °C for
4 h. The mixture was poured on ice, filtered, washed with 0.1 M
NaOH, water and diethyl ether and dried.
56/9. IR (KBr,
m
(cmꢀ1)): 3311; 1693; 1646; 1261; 1097. Analysis
calculated for C19H19FeNO3 (365.21): C, 62.49; H, 5.24; N, 3.84.
Found: C, 62.35; H, 5.11; N, 3.66 Rf (hexane/EtOAc = 3/1): 0.15.
Yield: 38%.
2.1.1. 16b-Azido-17a-hydroxy-5a-androstane (4)
1H NMR(d, CDCl3): 3.75 (ddd, J = 8.3 Hz, 7.5 Hz, 0.8 Hz, 1H,
16-H); 3.65 (d, J = 0.8 Hz, 1H, 17-H); 2.15–2.25 (m, 1H, 15-Ha);
0.65–1.70 (m, 22H, ring protons, OH), 0.82 (s, 3H, 18-H3), 0.80 (s,
3H, 19-H3). 13C NMR(d, CDCl3): 84.7; 69.1; 54.2; 48.9; 46.9; 44.3;
38.7; 36.3; 35.3; 32.5; 32.3; 31.6; 29.0; 28.9, 26.7; 22.1; 19.9;
2.3. Synthesis of 16b-(4-ferrocenyl-1,2,3-triazol-1-yl)-17-hydroxy-5-
androstane (8a)
Method A: A mixture of the steroidal azide 4 (0.2 mmol,
63.4 mg), the alkyne 7a (0.2 mmol, 42.0 mg), CuI (0.03 mmol,
5.7 mg) and DIPEA (4 ml) was stirred under argon at room temper-
ature for 8 h. The solvent was removed in vacuo, the residue was
dissolved in CH2Cl2 (8 ml) and was washed neutral with 5% HCl.
The organic phase was washed with water (3 ꢂ 4 ml), dried over
Na2SO4 and concentrated. The product was purified by column
chromatography (silica, eluent: toluene/methanol = 6/1). Yield:
55%.
17.1; 12.2. IR (KBr, m
(cmꢀ1)): 3371, 2093, 1256. Analysis calculated
for C19H31N3O (317.47): C, 71.88; H, 9.84; N, 13.24. Found: C,
71.67; H, 9.95; N, 13.01. Rf (toluene/MeOH = 6/1): 0.71. Yield: 98%.
2.1.2. 2b-Azido-3a-hydroxy-5a-androstan-17-one (5)
1H NMR(d, CDCl3): 3.83–3.86 (m, 1H, 3-H); 3.75–3.78 (m, 1H,
2-H); 2.40 (ddd, J = 0.9 Hz, 9.0 Hz, 19.0 Hz, 1H, 15-Ha); 0.71–2.09
(m, 20 H, ring protons, OH); 0.99 (s, 3H, 18-H3); 0.84 (s, 3H, 19-
H3). 13C NMR(d, CDCl3): 221.1; 68.3; 61.6; 55.1; 51.4; 47.8; 38.7;
36.1; 35.8; 35.7; 34.5; 31.7; 31.5; 30.7; 27.7; 21.7; 20.2; 13.8;
Method B: A mixture of the steroidal azide 4 (0.2 mmol,
63.4 mg), the alkyne 7a (0.2 mmol, 42.0 mg), CuI (0.03 mmol,
5.7 mg), CH3CN (4 ml) and DIPEA (0.5 mmol, 87 ll) was stirred un-
13.0. IR (KBr,
19H29N3O2 (331.46): C, 68.85; H, 8.82; N, 12.68. Found: C, 68.59;
H, 8.95; N, 12.42 Rf (toluene/MeOH = 6/1): 0.48. Yield: 62%.
m
(cmꢀ1)): 3427, 2092, 1255. Analysis calculated for
C
der argon at 60 °C for 8 h. The solvent was removed in vacuo, the
residue was dissolved in CH2Cl2 (8 ml) and was washed with 5%
HCl (4 ml) and water (3 ꢂ 4 ml), dried over Na2SO4 and concen-
trated. The product was purified by column chromatography (sil-
ica, eluent: toluene/methanol = 6/1). Yield: 60%.
Method C: A mixture of the steroidal azide 4 (0.2 mmol,
63.4 mg), the alkyne 7a (0.2 mmol, 42.0 mg), CuSO4.5H2O (0.03
mmol, 7.5 mg), sodium ascorbate (0.076 mmol, 15.0 mg), iPrOH
(2 ml) and water (2 ml) was stirred under argon at 60 °C for 8 h.
The product was extracted with CH2Cl2 (3 ꢂ 4 ml). The combined
organic phase was washed with water (3 ꢂ 4 ml) dried over
Na2SO4 and concentrated. The product was purified by column
chromatography (silica, eluent: toluene/methanol = 6/1). Yield:
70%.
2.1.3. 6b-Azido-5a,3b-dihydroxyandrostan-17-one (6)
1H NMR(d, CDCl3): 3.99–4.11 (m, 1H, 3-H); 3.41 (brs, 1H, 6-H);
2.45 (dd, J = 9.1 Hz, 19.4 Hz, 1H, 15-Ha); 1.20–2.12 (m, 20 H, ring
protons, OH); 1.14 (s, 3H, 18-H3); 0.87 (s, 3H, 19-H3). 13C NMR(d,
CDCl3): 220.5; 76.1; 67.3; 66.5; 50.7; 47.8; 45.6; 41.4; 38.8; 35.7;
32.3; 31.4; 30.7; 30.5; 30.0; 21.6; 20.3; 16.7; 13.9. IR (KBr,
(cmꢀ1)): 3443, 3345, 2096, 1258. Analysis calculated for
19H29N3O3 (347.46): C, 65.68; H, 8.41; N, 12.09. Found: C, 65.81;
m
C
H, 8.18; N, 11.89. Rf (toluene/MeOH = 5/1): 0.37. Yield: 86%.
2.2. Synthesis of (E)-N-(prop-2-ynyl)-2-ethoxycarbonyl-3-ferrocenyl-
2-propenamide (7b)
Method D: A mixture of the steroidal azide 4 (0.2 mmol,
63.4 mg), the alkyne 7a (0.2 mmol, 42.0 mg), CuSO4.5H2O
(0.03 mmol, 7.5 mg), sodium ascorbate (0.076 mmol, 15.0 mg),
CH2Cl2 (2 ml) and water (2 ml) was stirred under argon at room
temperature for 8 h. Then CH2Cl2 (6 ml) was added to the reaction
mixture. The combined organic phase was washed with water
(3 ꢂ 4 ml), dried over Na2SO4 and concentrated. The product was
purified by column chromatography (silica, eluent: toluene/meth-
anol = 6/1). Yield: 76%.
Ferrocenecarboxaldehyde (5 mmol, 1.07 g), propargyl amine
(5 mmol, 343 ll) and methanol (25 ml) was stirred under argon
in the presence of 4 Å molecular sieves (8–10 pieces) at room tem-
perature for 4 h. The reaction mixture was filtered, the solvent was
removed in vacuo. The product was crystallized from diethyl ether
to yield N-(prop-2-ynyl)-ferrocenylideneamine (12). 1H NMR(d,
CDCl3): 8.41 (s, 1H,=C–H); 4.67 (brs, 2H, 2,5-Cp) 4.39 (brs, 2H, 3,
4-Cp); 4.30 (brs, 2H, C–CH2); 4.18 (s, 5H, unsubstituted Cp); 2.48
(t, J = 2.3 Hz, 1H, CꢁCH). MS (m/z/rel. int.): 251 (M+)/100; 211/
1H NMR(d, CDCl3): 7.35 (s, 1H,=CH); 5.10 (brs, 2H, 2,3-Cp); 4.67
(brs, 2H, 3,4-Cp); 4.50 (ddd, J = 8.2 Hz, 7.5 Hz, 0.8 Hz, 1H, 16-H);

740
K. Fehér et al. / Steroids 77 (2012) 738–744
4.36 (s, 5H, unsubstituted Cp); 3.95 (d, J = 0.8 Hz, 1H 17-H);
0.69–2.33 (m, 23H, ring protons, OH); 0.89 (s, 3H, 18-H3); 0.79 (s,
3H, 19-H3). 13C NMR(d, CDCl3): 146.7; 120.8; 85.3; 75.5; 73.2;
71.7; 71.2; 69.7; 54.2; 49.3; 46.9; 44.4; 38.6; 36.3; 35.0; 32.8;
32.4; 31.9; 28.9; 28.8; 26.7; 22.1; 19.9; 18.0; 12.2. MS (m/z/rel.
int.): 550 [(M+Na)+]/6; 528/[(M+H)+]/100; 527 (M+)/98. Analysis
calculated for C31H41FeN3O (527.53) C, 70.58; H, 7.83; N, 7.97.
Found: C, 70.42; H, 8.07; N, 8.10. Rf (toluene/MeOH = 6/1): 0.33.
17-H); 0.75–2.58 (m, 23H, ring protons, OH); 2.05 (s, 3H,
C(O)CH3); 0.92 (s, 3H, 18-H3); 0.79 (s, 3H, 19-H3). 13C NMR(d,
CDCl3): 170.9; 142.2; 124.5; 85.2; 70.2; 57.6; 54.2; 49.3; 46.9;
44.5; 38.6; 36.3; 35.0; 32.8; 32.3; 31.8; 28.9; 28.8; 26.7; 22.1;
20.9; 19.9; 17.9; 12.2. MS (m/z/rel. int.): 438 [(M+Na)+]/95; 416/
[(M+H)+]/100. IR (KBr,
m
(cmꢀ1)): 3302, 1738. Analysis calculated
for C24H37N3O3 (415.57): C, 69.37; H, 8.97; N, 10.11. Found: C,
69.51; H, 8.69; N, 10.23. Rf (toluene/MeOH = 6/1): 0.26. Yield: 75%.
2.4. General method for the synthesis of triazolyl steroids 8 b–f, 9 a–f
and 10 b, f (method D)
2.4.5. 17-Hydroxy-16b-(4-(methoxycarbonyl)-1,2,3-triazol-1-yl)-5-
androstane (8f)
1H NMR(d, CDCl3): 8.14 (s, 1H,=CH); 4.64 (ddd, J = 8.5 Hz, 7.6 Hz,
0.8 Hz, 1H, 16-H); 4.03 (d, J = 0.8 Hz, 1H, 17-H); 3.93 (s, 3H, OCH3);
0.69–2.47 (m, 23H, ring protons, OH); 0.89 (s, 3H, 18-H3); 0.79 (s,
3H, 19-H3). 13C NMR(d, CDCl3): 161.3; 142.2; 127.2; 85.4; 70.3;
54.1; 52.2; 49.3; 46.9; 44.6; 38.6; 36.3; 35.0; 32.8; 32.3; 31.7;
28.9; 28.8; 26.7; 22.1; 19.9; 17.9; 12.2. MS (m/z/rel. int.): 424
A mixture of the steroidal azide (4–6) (0.2 mmol), the alkyne
(7a–f) (0.2 mmol), CuSO4 5H2O (0.03 mmol, 7.5 mg), sodium ascor-
bate (0.076 mmol, 15.0 mg), CH2Cl2 (2 ml) and water (2 ml) was
stirred under argon at room temperature for 8 h. Then CH2Cl2
(6 ml) was added to the reaction mixture. The combined organic
phase was washed with water (3 ꢂ 4 ml), dried over Na2SO4 and
concentrated. The product was purified by column chromatogra-
phy (silica, eluent: toluene/methanol = 6/1).
[(M+Na)+]/82; 402/[(M+H)+]/100. IR (KBr,
m
(cmꢀ1)): 3387, 1727.
Analysis calculated for C23H35N3O3 (401.55): C, 68.80; H, 8.79; N,
10.46. Found: C, 68.95; H, 8.65; N, 10.51. Rf (toluene/MeOH = 6/
1): 0.23. Yield: 72%.
2.4.1. 16b-(4-((E)-Ethyl 3-ferrocenylacrylate-2-methylcarbamoyl)-
1,2,3-triazol-1-yl)-17-hydroxy-5-androstane (8b)
1H NMR(d, CDCl3): 7.78 (s, 1H,=CH); 7.54 (s, 1H,=CH); 6.89 (brs,
1H, NH); 4.55–4.66 (m, 3H, 16-H, NCH2); 4.43 (brs, 2H, 2,5-Cp);
4.39 (brs, 2H, 3,4-Cp); 4.09–4.26 (m, 7H, OCH2, unsubstituted
Cp); 3.95 (s, 1H, 17-H); 0.69–2.41 (m, 23H, ring protons, OH);
1.24 (t, J = 7.1 Hz, 3H, CH3); 0.87 (s, 3H, 18-H3); 0.79 (s, 3H,
19-H3). 13C NMR(d, CDCl3): 166.9; 165.1; 144.7; 144.1; 123.2;
122.2; 85.2; 75.5; 71.8; 70.7; 70.6; 70.2; 69.9; 61.1; 54.1; 49.3;
46.8; 44.4; 44.2; 38.6; 36.3; 35.0; 32.6; 32.3; 31.8; 28.9; 28.8;
26.7; 22.1; 19.9; 18.0; 14.2; 12.2. MS (m/z/rel. int.): 705
[(M+Na)+]/49; 683/[(M+H)+]/4; 682 (M+)/3; 311/100; 137/41. IR
2.4.6. 2b-(4-Ferrocenyl-1,2,3-triazol-1-yl)-3-hydroxy-5-androstane-
17-one (9a)
1H NMR(d, CDCl3): 7.35 (s, 1H,=CH); 5.01 (brs, 2H, 2,5-Cp); 4.81
(m, 1H, 2-H); 4.58 (m, 2H, 3,4-Cp); 4.46 (m, 1H, 3-H); 4.28 (s, 5H,
unsubstituted Cp); 2.38–2.45 (m, 1H, 16-Ha); 0.75–2.23 (m, 20H,
ring protons, OH); 0.81 (s, 3H, 18-H3); 0.51 (s, 3H, 19-H3). 13C
NMR(d, CDCl3): 220.9; 144.6; 116.9; 68.9; 67.9; 66.3; 65.5; 64.4;
59.2; 53.1; 49.2; 45.9; 38.7; 37.2; 34.1; 33.9; 32.7; 31.7; 29.6;
28.7; 25.8; 19.8; 18.5; 12.0; 11.2. MS (m/z/rel. int.): 564
[(M+Na)+]/19; 542 [(M+H)+]/96; 541(M+)/100. IR (KBr,
m
(cmꢀ1)):
(KBr,
m
(cmꢀ1)): 3436, 3231, 1704, 1636, 1619. Analysis calculated
3440, 1718. Analysis calculated for C31H39FeN3O2 (541.52): C,
68.76; H, 7.26; N, 7.76. Found: C, 68.61; H, 7.11; N, 7.89. Rf (tolu-
ene/MeOH = 6/1): 0.26. Yield: 64%.
for C38H50FeN4O4 (682.68) C, 66.86; H, 7.38; N, 8.21. Found: C,
67.05; H, 7.56; N, 8.02. Rf (toluene/MeOH = 6/1): 0.23. Yield: 67%.
2.4.2. 17-Hydroxy-16b-(4-phenyl-1,2,3-triazol-1-yl)-5-androstane
(8c)
2.4.7. 2b-(4-((E)-Ethyl 3-ferrocenylacrylate-2-methylcarbamoyl))-
1,2,3-triazol-1-yl)-3-hydroxy-5-androstan-17-one (9b)
1H NMR(d, CDCl3): 7.85 (s, 1H,=CH); 7.84 (d, J = 7.3 Hz, 2H, Ph);
7.43 (t, J = 7.3 Hz, 2H, Ph); 7.34 (t, J = 7.3 Hz, 1H, Ph); 4.65 (ddd,
J = 8.3 Hz, 7.6 Hz, 0.8 Hz, 1H, 16-H); 4.10 (d, J = 0.8 Hz, 1H, 17-H);
0.78–2.48 (m, 23H, ring proton, OH); 0.97 (s, 3H, 18-H3); 0.83 (s,
3H, 19-H3). 13C NMR(d, CDCl3): 147.4; 130.6; 128.7; 127.9; 125.5;
119.3; 85.3; 69.8; 54.1; 49.3; 46.8; 44.4; 38.5; 36.2; 34.9; 32.6;
32.2; 31.7; 28.8; 28.7; 26.6; 22.0; 19.8; 17.8; 12.1. MS (m/z/rel.
int.): 442 [(M+Na)+]/22; 420/[(M+H)+]/100. Analysis calculated
for C27H37N3O (419.61) C, 77.29; H, 8.89; N, 10.01. Found: C,
77.45; H, 8.99; N, 9.79.Rf (toluene/MeOH = 6/1): 0.44. Yield: 72%.
1H NMR(d, CDCl3): 7.70 (s, 1H,=CH); 7.54 (s, 1H,=CH); 6.55 (s,
1H, NH); 4.72 (m, 1H, 2-H); 4.62 (m, 2H, NCH2); 4.45–4.51 (m,
3H, 3-H, 2,5-Cp); 4.40 (brs, 2H, 3,4-Cp); 4.17–4.20 (m, 7H, OCH2,
unsubstituted Cp) 0.82–2.56 (m, 21H, ring protons, OH); 1.26 (t,
J = 7.0 Hz, 3H, CH3); 0.75 (s, 3H, 18-H3); 0.52 (s, 3H, 19-H3). 13C
NMR(d, CDCl3): 220.8; 166.9; 165.2; 144.3; 144.2; 123.2; 122.2;
75.6; 71.9; 70.8; 69.9; 66.6; 61.5; 61.1; 55.1; 51.3; 47.7; 40.9;
39.1; 36.0; 35.7; 35.4; 34.6; 33.8; 31.5; 30.4; 27.7; 21.7; 20.4;
14.2; 13.8; 13.7. MS (m/z/rel. int.):719 [(M+Na)+]/18; 697
[(M+H)+]/6; 696 (M+)/5; 311/100; 137/73. IR (KBr,
m
(cmꢀ1)):
3440, 1705, 1645. Analysis calculated for C38H48FeN4O5 (696.67):
C, 65.51; H, 6.94; N, 8.04. Found: C, 65.71; H, 7.12; N, 8.21. Rf (tol-
uene/MeOH = 5/1): 0.33. Yield: 63%.
2.4.3. 16b-(4-Hexyl-1,2,3-triazol-1-yl)-17-hydroxy-5-androstane (8d)
1H NMR(d, CDCl3): 7.40 (s, 1H,=CH); 4.60 (ddd, J = 8.5 Hz, 7.5 Hz,
0.8 Hz, 1H, 16-H); 4.00 (d, J = 0.8 Hz, 1H, 17-H); 0.75–2.75 (m, 33H,
ring protons, (CH2)5, OH); 0.91 (s, 3H, 18-H3); 0.87 (t, J = 7.8 Hz, 3H,
CH3); 0.79 (s, 3H, 19-H3). 13C NMR(d, CDCl3): 148.1; 120.6; 85.2;
69.9; 54.2; 49.3; 46.9; 44.4; 38.6; 36.3; 35.0; 32.6; 32.3; 31.9;
31.5; 29.3; 28.9 (2C); 28.8; 26.7; 25.6; 22.5; 22.1; 19.9; 17.9;
14.0; 12.2. MS (m/z/rel. int.): 450 [(M+Na)+]/5; 428/[(M+H)+]/100.
Analysis calculated for C27H45N3O (427.67) C, 75.83; H, 10.61; N,
9.83. Found: C, 75.59; H, 10.87; N, 9.72. Rf (toluene/MeOH = 6/1):
0.50. Yield: 59%.
2.4.8. 3-Hydroxy-2b-(4-phenyl-1,2,3-triazol-1-yl)-5-androstan-17-
one (9c)
1H NMR(d, CDCl3): 7.83 (s, 1H,=CH); 7.81 (d, J = 7.2 Hz, 2H, Ph);
7.40 (t, J = 7.2 Hz, 2H, Ph); 7.32 (t, J = 7,2 Hz, 1H, Ph); 4.85 (m, 1H,
2-H); 4.59 (m, 1H, 3-H); 0.78–2.52 (m, 21H, ring protons, OH); 0.95
(s, 3H, 18-H3); 0.85 (s, 3H, 19-H3). 13C NMR(d, CDCl3): 220.8; 146.9;
132.1; 128.9; 128.3; 125.8; 119.9; 66.5; 62.0; 55.1; 51.3; 47.8;
39.1; 35.8; 34.6; 34.3; 33.8; 31.6; 30.9; 30.5; 27.7; 21.7; 20.4;
13.8; 13.0. MS (m/z/rel. int.): 456 [(M+Na)+]/23; 434 [(M+H)+]/
2.4.4. 16b-(4-(Acetoxy-methyl)-1,2,3-triazol-1-yl)-17-hydroxy-5-
androstane (8e)
100. IR (KBr,
27H35N3O2 (433.59): C, 74.79; H, 8.14; N, 9.69. Found: C, 74.71;
H, 8.30; N, 9.52. Rf (toluene/MeOH = 6/1): 0.27. Yield: 63%.
m
(cmꢀ1)): 3420, 1722. Analysis calculated for
1H NMR(d, CDCl3): 7.79 (s, 1H,=CH); 5.20 (s, 2H, OCH2); 4.62
(ddd, J = 8.4 Hz, 7.2 Hz, 0.8 Hz, 1H, 16-H); 4.04 (d, J = 0.8 Hz, 1H,
C

K. Fehér et al. / Steroids 77 (2012) 738–744
741
2.4.9. 2b-(4-Hexyl-1,2,3-triazol-1-yl)-3-hydroxy-5-androstan-17-one
(9d)
1H NMR(d, CDCl3): 7.31 (s, 1H,=CH); 4.77–4.82 (m, 1H, 2-H);
2.5. Computational details
All the geometries were calculated without any symmetry con-
straints by using the gradient-corrected exchange functional
developed by Perdew, Burke and Ernzerhof in combination with
a correlation functional also developed by the same authors and
denoted as PBEPBE [20]. The 6–31G(d,p) basis set [21] was em-
ployed throughout this study. For all stationary points the Hessian
was evaluated to characterize the genuine minima (no imaginary
frequency). Thermal correction for the Gibbs free energy (at
298 K) has been estimated on the basis of the frequency calcula-
tions at the optimization level. For the calculations the Firefly (for-
merly known as PC GAMESS) [22] software was used.
4.46–4.50 (m, 1H, 3-H); 2.68 (t, J = 7.6 Hz, 2H, CH2); 2.38–2.45
(m, 1H, 16-Ha); 0.81–2.21 (m, 28H, ring protons, (CH2)4, OH);
0.86 (t, J = 7.6 Hz, 3H, CH3); 0.81 (s, 3H, 18-H3); 0.50 (s, 3H,
19-H3). 13C NMR(d, CDCl3): 220.9; 146.5; 121.2; 66.3; 62.1; 55.1;
51.3; 47.8; 40.8; 39.1; 36.0; 35.8; 34.6; 33.8; 31.5 (2C); 30.4;
29.2; 28.8; 27.7; 25.1; 22.5; 21.7; 20.4; 14.0; 13.8; 13.7. MS
(m/z/rel. int.): 464 [(M+Na)+]/65; 442/[(M+H)+]/100. IR (KBr,
m
(cmꢀ1)): 3450, 1728. Analysis calculated for C27H43N3O2 (441.66)
C, 73.43; H, 9.81; N, 9.51. Found: C, 73.22; H, 10.01; N, 9.43. Rf
(toluene/MeOH = 6/1): 0.30. Yield: 47%.
2.4.10. 2b-(4-(Acetoxy-methyl)-1,2,3-triazol-1-yl)-3-hydroxy-5-
androstan-17-one (9e)
3. Results and discussion
1H NMR(d, CDCl3): 7.76 (s, 1H,=CH); 5.21 (s, 2H, OCH2); 4.72–
4.81 (m, 1H, 2-H); 4.51–4.60 (m, 1H, 3-H); 2.38–2.44 (m, 1H, 16-
Ha); 0.80–2.21 (m, 20H, ring protons, OH); 2.08 (s, 3H, C(O)CH3);
0.82 (s, 3H, 18-H3); 0.54 (s, 3H, 19-H3). 13C NMR(d, CDCl3): 221.0;
170.8; 142.3; 124.7; 66.4; 62.2; 57.5; 55.0; 51.2; 47.7; 40.6;
39.0; 35.9; 35.7; 34.5; 33.7; 31.4; 30.4; 27.6; 21.6; 20.8; 20.3;
13.8; 13.6. MS (m/z/rel. int.): 452 [(M+Na)+]/63; 430/[(M+H)+]/
Three steroids (4–6), bearing the azido group in different posi-
tions of the steroidal skeleton, were synthesised as model com-
pounds by a known procedure [23], starting from epoxides 1–3
(Scheme 2). The ring-opening was completely selective in each
case, leading to the 16b- (4), 2b- (5) and 6b-azido-androstanes
(6) in moderate to excellent yields. No formation of other epimers
could be detected.
First, two alkynyl-ferrocene derivatives, ethynylferrocene (7a)
and a ferrocenylmethylidene–malonic acid derivative (7b) were
used as reaction partners (Scheme 3).
100. IR (KBr,
24H35N3O4 (429.56): C, 67.11; H, 8.21; N, 9.78. Found: C, 67.35;
m
(cmꢀ1)): 3430, 1735, 1720. Analysis calculated for
C
H, 8.35; N, 9.91. Rf (toluene/MeOH = 5/1): 0.37. Yield: 67%.
Compound 7b was obtained by the cobalt-catalyzed domino
reaction developed in our group (Scheme 4) [24]. The cobalt-cata-
lyzed carbonylation of ethyl diazoacetate (11) leads to ethoxycar-
bonyl ketene that reacts rapidly with ferrocenylimine 12 to yield
2.4.11. 3-Hydroxy-2b-(4-methoxycarbonyl-1,2,3-triazol-1-yl)-5-
androstan-17-one (9f)
1H–NMR(d, CDCl3): 8.22 (s, 1H,=CH); 4.82 (m, 1H, 2-H); 4.60 (m,
1H, 3-H); 3.95 (s, 3H, OCH3); 0.75–2.50 (m, 21H, ring protons, OH);
0.80 (s, 3H, 18-H3); 0.74 (s, 3H, 19-H3). 13C NMR(d, CDCl3): 220.9;
162.1; 143.9; 127.9; 66.5; 62.6; 55.0; 52.3; 51.2; 47.8; 40.5;
39.0; 36.0; 35.7; 34.5; 33.7; 31.4; 30.4; 27.6; 21.6; 20.4; 13.8;
13.6. MS (m/z/rel. int.): 438 [(M+Na)+]/30; 416 [(M+H)+]/100. IR
(KBr,
23H33N3O4 (415.53): C, 66.48; H, 8.00; N, 10.11. Found: C, 66.31;
m
(cmꢀ1)): 3430, 1724, 1713. Analysis calculated for
C
H, 8.25; N, 10.27. Rf (toluene/MeOH = 5/1): 0.35. Yield: 68%.
2.4.12. 6b-(4-((E)-Ethyl 3-ferrocenylacrylate-2-methylcarbamoyl)-
1,2,3-triazol-1-yl)-3,5-dihydroxy-androstan-17-one (10b)
1H NMR(d, CDCl3): 7.59 (s, 1H,=CH); 7.36 (s, 1H,=CH); 6.68 (brs,
1H, NH); 4.64–4.71 (m, 2H, NCH2); 4.02–4.58 (m, 12H, Fc, OCH2,
3-H); 3.40 (brs, 1H, 6H); 0.75–2.60 (m, 21H, ring protons, OH);
1.28 (t, J = 7.2 Hz, 3H, CH3); 0.95 (s, 3H, 18-H3); 0.66 (s, 3H,
19-H3). 13C NMR(d, CDCl3): 221.0; 167.3; 165.2; 144.3; 143.3;
124.9; 122.8; 75.7; 75.4; 72.1; 70.8; 70.5; 70.0; 67.1; 65.0; 61.3;
53.4; 51.3; 48.0; 45.1; 41.7; 38.6; 35.8; 32.7; 31.8; 31.5; 30.4;
29.3; 21.5; 20.4; 15.5; 14.1, 13.8. MS (m/z/rel. int.):735
[(M+Na)+]/24; 713 [(M+H)+]/5; 712 (M+)/2; 311/100; 137/39. IR
Scheme 2. Synthesis of azido-androstanes 4–6.
(KBr,
m
(cmꢀ1)): 3430, 1715, 1704, 1681, 1632. Analysis calculated
for C38H48FeN4O6 (712.67): C, 64.04; H, 6.79; N, 7.86. Found: C,
64.19; H, 6.87; N, 8.00. Rf (toluene/MeOH = 5/1): 0.25. Yield: 57%.
2.4.13. 3b,5-Dihydroxy-6b-(4-methoxycarbonyl-1,2,3-triazol-1-yl)-
androstan-17-one (10f)
1H NMR(d, CDCl3): 8.25 (s, 1H,=CH); 4.35 (m, 1H, 3-H); 3.85 (s,
3H, OCH3); 3.45 (brs, 1H, 6-H); 1.25–2.60 (m, 21H, ring protons,
OH); 0.92 (s, 3H, 18-H3); 0.66 (s, 3H, 19-H3). 13C NMR(d, CDCl3):
220.7; 207.0; 141.5; 127.8; 76.1; 67.3; 66.5; 51.3; 50.7; 47.8;
45.6; 41.4; 38.8; 35.7; 32.3; 31.4; 30.9; 30.4; 30.0; 21.7; 20.3;
16.8; 13.9. MS (m/z/rel. int.): 454 [(M+Na)+]/100; 432 [(M+H)+]/
70. IR (KBr,
23H33N3O5 (431.53): C, 64.02; H, 7.71; N, 9.74. Found: C, 64.23;
H, 7.99; N, 9.95. Rf (toluene/MeOH = 5/1): 0.33. Yield: 47%.
m
(cmꢀ1)): 3414, 1734, 1718. Analysis calculated for
C
Scheme 3. CuAAC reaction of steroids 4–6 and alkynes 7a–f.

742
K. Fehér et al. / Steroids 77 (2012) 738–744
Scheme 4. Synthesis of 7b by the domino reaction of ethyl diazoacetate (11), ferrocenylimine 12 and carbon-monoxide.
Table 1
Table 2
a
CuAAC reaction of alkyne 7a and steroid 4 under different conditionsa.
CuAAC reaction of steroids 4–6 with alkynes 7a–f
.
Entry Catalyst Additive
Solvent
r. temperature
Yield of 8a
Entry
Steroid
Alkyne
R
Product
Yield (%)
(°C)
(%)
1
2
4
4
7a
7b
Fc
8a
8b
76
67
1
2
3
CuI
CuI
CuSO4
DIPEAb
DIPEAb
Na-
ascorbate
Na-
DIPEA
CH3CN
iPrOH/
H2O
CH2Cl2/
H2O
rt
60
60
55
60
70
EtO2C
H
O
Fc
NH
H2C
4
CuSO4
rt
76
ascorbate
3
4
5
6
7
8
4
4
4
4
5
5
7c
7d
7e
7f
7a
7b
Ph
C6H13
CH2OC(O)CH3
C(O)OCH3
Fc
8c
8d
8e
8f
9a
9b
72
59
75
72
64
63
a
Reaction conditions: 4/7a/Cu = 0.2 mmol/0.2 mmol/0.03 mmol in 4 ml solvent,
DIPEA: N,N-diisopropylethylamine.
8 h.
b
EtO2C
H
O
Fc
b-lactam 13. An immediate N(1)-C(4) cleavage of the latter com-
pound results in the formation of 7b. Compound 7b might have
preferential applications in click reactions, since its use leads to
steroidal triazoles connected to the bulky ferrocene moiety
through a linker, which may be desirable in order not to hamper
NH
H2C
9
5
5
5
5
6
6
7c
7d
7e
7f
7a
7b
Ph
C6H13
CH2OC(O)CH3
C(O)OCH3
Fc
9c
9d
9e
9f
63
47
67
68
10
11
12
13
14
the attachment of the steroid to a receptor (vide infra). 17
ocenyl-17b-estradiol and 17 -ferrocenylethynyl-17b-estradiol
were found to have a relative binding affinity of 8% and 28% to
the estrogen -receptor, respectively [1]. This shows a favorable ef-
a-ferr-
traces
57
a
10b
EtO2C
H
a
O
Fc
fect of the introduction of a linker between the steroidal skeleton
and the ferrocenyl moiety.
NH
H2C
In the CuAAC reaction the key intermediate is a Cu(I) acetylide
that is obtained in the reaction of the precursor Cu(I) salt and the
terminal acetylene [6]. The Cu(I) catalyst can also be obtained
in situ, starting from a Cu(II) salt and sodium ascorbate.
Cycloaddition of 7a and 4 (Scheme 4) was used as a model reac-
tion to determine the optimal conditions (Table 1). The activities of
CuI + base (entries 1,2) and CuSO4 + Na–ascorbate catalyst systems
(entries 3,4), commonly used for similar reactions, were compared.
The application of the Cu(II) precursor was found to be more effi-
cient, so the subsequent reactions were carried out under the con-
ditions used in entry 4 (method D).
15
16
17
6
6
6
7c
7e
7f
Ph
no reaction
no reaction
47
CH2OC(O)CH3
C(O)OCH3
10f
a
Reaction conditions catalyst: 15% CuSO4, 38% Na-ascorbate in CH2Cl2/H2O, 24 h,
rt.
unchanged in both cases. Triazole 10f was obtained in moderate
yield using the activated alkyne 7f (Table 2, entry 17).
According to these experiments, the reactivity of steroidal
azides decreased in the order 4 > 5 >> 6. Although steric crowding
caused by the proximity of the angular methyl group seems to
be similar in the 2b and 6b positions, the steric hindrance hamper-
ing the addition of alkyne to the azide moiety might be reduced by
the flipping of the corresponding ring of the steroid scaffold from
conformers 5-ax and 6-ax to conformers 5-eq, and 6-eq, respec-
tively (Fig. 1.). As a consequence, the carbon atom in position 2
(compound 5) and in position 6 (compound 6) is puckered out of
plane arranging the A and B rings in twisted boat conformation,
respectively. Consequently, the azido groups are allocated in equa-
torial positions with significantly less steric hindrance. The free en-
ergy difference between 5-ax and 5-eq is 3.4 kcal/mol in favor of
the former structure, with all the cyclohexane rings in chair confor-
mation. For species 6-ax, however, the ring flipping is a far less fa-
vored process. The resulting structure 6-eq, with the B ring in
twisted boat conformation, is higher in free energy by 11.6 kcal/
mol. Thus, it can be stated that in spite of the presumably higher
reactivity of 6-eq, its population is almost negligible resulting in
slower reaction of 6 towards the addition of alkynes.
Similar yields were obtained in the CuAAC reaction of 4 and 7b
(Table 2, entry 2), as well as in the cycloaddition of 5 and alkynes
7a,b (Table 2, entries 7,8).
At the same time, a different behavior of the 6b-azido derivative
6 was observed. The target compound was detected only in traces
by TLC in the CuAAC reaction of 6 with ethynylferrocene 7a. The
application of other conditions, corresponding to those used for
the cycloaddition of 4 and 7a (Table 1, entries 1–3), did not im-
prove the course of the cycloaddition, either. On the other hand,
cycloaddition of 6 with 7b, where the alkyne and the bulky ferro-
cene moieties are separated by a spacer, led to product 10b with
acceptable yield (Table 2, entry 14).
In order to explore if this difference in the reactivity of the ste-
roids 4–6 is restricted to the click reactions of the bulky ferrocenyl
alkynes, cycloaddition of the azides 4–6 with simple alkynes (7c–f)
were also investigated.
The reactions of azides 4 and 5 gave the corresponding triazoles
8c–f and 9c–f in good yields (Table 2, entries 3–6 and 9–12, respec-
tively). The results obtained with 4 were slightly better. Azide 6
was proved to be considerably less reactive again. No reaction
was observed with 7c and 7e and the substrate was recovered
For species 4, however, only one conformer was found to be a
genuine minimum (depicted also in Fig. 1) with the D ring in

K. Fehér et al. / Steroids 77 (2012) 738–744
743
Fig. 1. Conformations of steroidal azides 4, 5 and 6.
[3] Osella D, Nervi C, Galeotti F, Cavigiolio G, Vessieres A, Jaouen G. The
half-chair conformation. No local minimum was found for the
envelope conformation possessing the azido moiety in a clearly ax-
ial position. At the same time, the azido group is sterically the least
hindered compared to all conformers of 5 and 6 which may explain
its higher reactivity.
ferrocenylethynyl unit:
3289–98.
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Copper-catalyzed azide–alkyne cycloaddition can efficiently be
used for the synthesis of ferrocene-labeled steroids with triazole
groups attached directly to the 2b and 16b positions. At the same
time, considerably lower reactivity of azide 6 towards both ferroce-
nyl and simple alkynes was observed. According to the computa-
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the 16b azido-steroid 4. Flipping of ring A of steroid 5 into a
twisted boat conformer with the 2b azido group in equatorial posi-
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the free energies of the twisted boat and the chair conformations of
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The authors thank the Hungarian National Science Foundation
(OTKA NK71906) and project ‘Developing Competitiveness of Uni-
versities in the South Transdanubian Region’ (SROP-4.2.1.B-10/2/
KONV-2010-0002) for financial support. Z.C. thanks the Bolyai
Grant of the Hungarian Academy of Sciences. The support of the
project TÁMOP-4.2.2/B-10/1-2010-0025, realized with the support
of the Hungarian Government and the European Union, with the
co-funding of the European Social Fund, is also acknowledged.
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Synthesis and antimicrobial activity of b-lactam-bile acid conjugates linked via
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