Synthesis and preliminary biological screening of sterol analogues as new antifungal agents against plant pathogens

Article abstract of DOI:10.1016/j.steroids.2010.04.002

In this paperwereport the synthesis of anewfamily of sterol analogues that have two amidic bonds on the side chain. These azasterols were obtained by a straightforward procedure including an Ugi condensation that allows the facile attachment of a polyfunctionalized side chain into the steroidal framework. Some of the new compounds showed an interesting inhibitory effect on the growth of two pathogenic fungi involved in plant diseases.

Full text of DOI:10.1016/j.steroids.2010.04.002

Steroids 75 (2010) 659–664
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis and preliminary biological screening of sterol analogues as new
antifungal agents against plant pathogens
Fernando Alonso, Adriana M. Cirigliano, Gabriela M. Cabrera, Javier A. Ramírez∗
Departamento de Química Orgánica and UMYMFOR (CONICET - Facultad de Ciencias Exactas y Naturales), Universidad de Buenos Aires, Pabellón 2, Piso 3, Ciudad Universitaria,
C1428EGA Buenos Aires, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
In this paper we report the synthesis of a new family of sterol analogues that have two amidic bonds on the
side chain. These azasterols were obtained by a straightforward procedure including an Ugi condensation
that allows the facile attachment of a polyfunctionalized side chain into the steroidal framework.
Some of the new compounds showed an interesting inhibitory effect on the growth of two pathogenic
fungi involved in plant diseases.
Received 27 February 2010
Received in revised form 5 April 2010
Accepted 8 April 2010
Available online 5 May 2010
© 2010 Elsevier Inc. All rights reserved.
Keywords:
Antifungal
Azasterols
Plant pathogens
Ugi reaction
1. Introduction
reaction product can be easily diversified by a systematic variation
of the starting materials [6].
Plants are constantly exposed to a variety of pathogenic
microorganisms present in their environments. Diseases caused
by pathogens, including bacteria, fungi and viruses, significantly
contribute to the overall loss in crop yield worldwide and consti-
tute an emerging threat to the global food security [1]. Many of the
currently available antimicrobial agents for agriculture are highly
toxic and non-biodegradable and cause extended environmental
pollution [2].
One of the most versatile MCRs is the Ugi four-component reac-
tion (U-4CR), which is based on the exceptional reactivity of the
isocyanide functional group [7]. The U-4CR constitutes a homoge-
neous group of reactions in which an amino component, an acid, a
carbonyl compound and an isocyanide react together to give an ␣-
aminoacylamide [6,7]. We have recently shown that this approach
can be successfully applied to the synthesis of heterocyclic steroids
[8,9].
Several sterol analogues are known to behave as antiprolifera-
tive agents against fungi, yeast and protozoa [3,4]. An important
class of antifungal steroids are azasteroids in which one or more
nitrogen atoms are present in the side chain [5]. The natural alka-
loid verazine 1 and the synthetic piperinidyl derivative 2 (Fig. 1)
are two compounds belonging to this class.
2. Experimental
2.1. Synthesis
Taking these previous results into account, our group started a
research programme devoted to the synthesis of new sterol ana-
logues with activity against plant pathogens.
We aimed at choosing a synthetic strategy based on a simple
and fast generation of new compounds having a high structural
diversity. Multicomponent reactions (MCRs), which are generally
defined as reactions where more than two starting materials react
to form a product by incorporating essentially all the atoms of the
adducts, are best suited to achieve this goal. The structure of the
2.1.1. General
All the reagents were purchased from Sigma–Aldrich Chemical
Co. ESI-HRMS were measured on a Bruker micrOTOF-Q II. Melt-
ing points were determined on a Fisher Johns apparatus and are
uncorrected. All NMR spectra were recorded on a Bruker AM-500
(500 MHz for 1H and 125.1 MHz for 13C). Chemical shifts (ı) are
given in ppm downfield from TMS as the internal standard. Cou-
pling constant (J) values are in Hz. All solvents and reagents were
of analytical grade. All new compounds gave satisfactory NMR and
mass spectral/combustion analysis data. Assignments of the NMR
signals of the side chain correspond to the numbering shown in
Figs. 2 and 3.
∗
Corresponding author. Tel.: +54 01145763346; fax: +54 01145763385.
E-mail address: jar@qo.fcen.uba.ar (J.A. Ramírez).
0039-128X/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2010.04.002

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F. Alonso et al. / Steroids 75 (2010) 659–664
2.1.2. General synthetic procedure
The steroidal acid 3 (3␤-hydroxyandrost-5-en-17␤-carboxylic
acid, 50 mg, 0.16 mmol [10]) was suspended in 1 mL of methanol
and 1.1 equivalent of the corresponding amine and 15 ␮L of
formaldehyde (37% aq.) were added. The mixture was stirred for
15 min at room temperature and then 1.1 equivalent of the isoni-
trile was added. The reaction was kept under the same condition
until total disappearance of the acid (usually 48 h). The solvent
Fig. 1. Antifungal azasteroids.
Fig. 2. Structures of the synthesized 21-azasteroids.
Fig. 3. Structures of the synthesized 23-azasteroids.

F. Alonso et al. / Steroids 75 (2010) 659–664
661
was evaporated under reduced pressure and the residue was taken
in EtOAc and washed with NaOH (5% aq.). The compounds were
purified by silica gel column chromatography (hexane/EtOAc gra-
dient).
When starting from acetate 4 (3␤-acetoxy-23,24-dinorchol-5-
en-20-carboxylic acid [11]) the same procedure was followed, but
after 48 h of reaction 0.2 mL of K2CO3 (satd.) was added to the reac-
tion. The mixture was stirred for an additional period of 24 h and
worked up as usual.
4.37 (H1ꢀ, 1H each signal, d, 14.4); 5.28 (H6, 1H, d, 5.0); 6.65 (H3ꢀ,
1H, bs); 7.28 (H9ꢀ, 1H, t, 7.9); 7.36 (H7ꢀ, 2H, bs); 7.52 (H8ꢀ, 2H, t, 7.5).
13C NMR (CDCl3): 13.5 (C18); 19.1 (C19); 20.6 (C11); 24.5 (C16);
25.8 (C15); 28.4 (C5ꢀ); 31.5 (C7); 31.6 (C2); 31.6 (C8); 36.3 (C10);
37.0 (C1); 38.2 (C12); 41.6 (C4); 45.3 (C13); 49.6 (C9); 51.1 (C4ꢀ);
51.7 (C17); 55.3 (C1ꢀ); 56.0 (C14); 71.1 (C3); 121.0 (C6); 127.7 (C7ꢀ);
127.9 (C9ꢀ); 129.4 (C8ꢀ); 140.7 (C5); 143.2 (C6ꢀ); 168.6 (C2ꢀ); 175.2
(C20). HRMS (ESI): calculated for [M+H+] C32H47N2O3 507.3587,
found 507.3580. Anal. calculated for C32H46N2O3 C, 75.85; H, 9.15;
N, 5.53; found C, 75.73; H, 9.21; N, 5.42.
2.1.3. N-((tert-butylcarbamoyl)methyl)-N-benzyl-3ˇ-
hydroxyandrost-5-en-17ˇ-carboxamide
(5a)
2.1.7.
N-((cyclohexylcarbamoyl)methyl)-N-(3,4-dimethoxyphenethyl)-
3ˇ-hydroxyandrost-5-en-17ˇ-carboxamide
(5e)
Colourless oil. 1H NMR (CDCl3): 0.82 (H18, 3H, s); 0.97 (H19, 3H,
s); 1.36 (H5ꢀ, 9H, s); 2.51 (H17, 1H, t, 9.5); 3.45 (H3, 1H, m); 3.95 and
4.21 (H1ꢀ, 1H each signal, d, 14.0); 4.65 and 4.72 (H6ꢀ, 2H, d, 15.9);
5.28 (H6, 1H, d, 5.0); 6.55 (H3ꢀ, 1H, bs); 7.21 (H8ꢀ, 2H, dd, 7.1 and
1.0); 7.35 (H9ꢀ, 2H, m); 7.38 (H10ꢀ, 1H, m). 13C NMR (CDCl3): 13.5
(C18); 19.1 (C19); 20.6 (C11); 24.5 (C16); 25.8 (C15); 28.5 (C5ꢀ); 31.5
(C7); 31.6 (C2); 31.6 (C8); 36.3 (C10); 37.1 (C1); 38.2 (C12); 41.6
(C4); 45.3 (C13); 49.6 (C9); 51.2 (C4ꢀ); 51.7 (C17); 52.8 (C6ꢀ); 55.3
(C1ꢀ); 56.0 (C14); 71.2 (C3); 121.1(C6); 127.1 (C10ꢀ); 128.0 (C8ꢀ);
128.6 (C9ꢀ); 136.5 (C7ꢀ); 140.7 (C5); 168.8 (C2ꢀ); 175.2 (C20). HRMS
(ESI): calculated for [M+H+] C33H49N2O3 521.3743, found 521.3754.
Anal. calculated for C33H48N2O3 C, 76.11; H, 9.29; N, 5.38; found:
C, 76.21; H, 9.41; N, 5.21.
Mp: 107 ◦C. 1H NMR (CDCl3): 0.73 (H18, 3H, s); 1.01 (H19, 3H, s);
2.48 (H17, 1H, t, 9.1); 2.79 (H9ꢀ, 2H, m); 3.52 (H3, 1H, m); 3.45 and
3.91 (H8ꢀ, 1H each signal, m); 3.61 and 4.42 (H1ꢀ, 1H each signal,
d, 15.0); 3.86 and 3.87 (–OMe, 3H each signal, s); 5.35 (H6, 1H, d,
5.0); 6.59 (H3ꢀ, 1H, d, 8.0); 6.65 (H11ꢀ, 1H, d, 2.1); 6.68 (H15ꢀ, 1H, dd,
8.0 and 2.1); 6.81 (H14ꢀ, 1H, d, 8.0). 13C NMR (CDCl3): 13.9 (C18);
19.4 (C19); 21.0 (C11); 24.6 (C16); 24.7 (C6ꢀ); 25.4 (C7ꢀ); 25.9 (C15);
31.5 (C2); 31.8 (C7); 31.9 (C8); 32.9 (C5ꢀ); 34.7 (C9ꢀ); 36.5 (C10);
37.2 (C1); 38.8 (C12); 42.2 (C4); 45.4 (C13); 48.1 (C4ꢀ); 50.0 (C9);
51.1 (C17); 51.1 (C8ꢀ); 51.6 (C1ꢀ); 55.9 (C14); 55.9 (–OMe); 71.6
(C3); 111.5 (C14ꢀ); 112.0 (C11ꢀ); 120.8 (C15ꢀ); 121.3 (C6); 130.4
(C10ꢀ); 140.8 (C5); 147.9 (C13ꢀ); 149.1 (C12ꢀ); 168.9 (C2ꢀ); 174.8
(C20). HRMS (ESI): calculated for [M+H+] C38H57N2O5 621.4267,
found 621.4240. Anal. calculated for C38H56N2O5 C, 73.51; H, 9.09;
N, 4.51; found C, 73.47; H, 8.99; N, 4.67.
2.1.4. N-((tert-butylcarbamoyl)methyl)-N-(3-hydroxyphenyl)-
3ˇ-hydroxyandrost-5-en-17ˇ-carboxamide
(5b)
Mp: 149 ◦C. 1H NMR (CDCl3): 0.82 (H18, 3H, s); 0.96 (H19, 3H,
s); 1.37 (H5ꢀ, 9H, s); 2.56 (H17, 1H, t, 9.0); 3.49 (H3, 1H, m); 4.03 and
4.36 (H1ꢀ, 1H each signal, d, 14.1); 5.28 (H6, 1H, d, 5.0); 6.06 (H3ꢀ, 1H,
bs); 6.54 (H11ꢀ, 1H, bs); 6.68 (H9ꢀ, 1H, d, 7.7); 6.96 (H7ꢀ, 1H, bs); 7.09
(H8ꢀ, 1H, t, 8.7); 8.6 (H12ꢀ, 1H, bs). 13C NMR (CDCl3): 13.9 (C18); 19.4
(C19); 20.9 (C11); 24.7 (C16); 26.1 (C15); 28.7 (C5ꢀ); 31.5 (C2); 31.8
(C7); 31.8 (C8); 36.5 (C10); 37.2 (C1); 38.3 (C12); 42.1 (C4); 45.5
(C13); 49.7 (C9); 51.4 (C4ꢀ); 51.8 (C17); 55.8 (C1ꢀ); 56.1 (C14); 71.7
(C3); 115.3 (C9ꢀ); 116.3 (C11ꢀ); 118.5 (C7ꢀ); 121.4 (C6); 130.1(C8ꢀ);
140.7 (C5); 144.0 (C6ꢀ); 158.0 (C10ꢀ); 169.9 (C2ꢀ); 175.5 (C20). HRMS
(ESI): calculated for [M+H+] C32H47N2O4 523.3536, found 523.3518.
Anal. calculated for C32H46N2O4 C, 73.53; H, 8.87; N, 5.36; found C,
73.30; H, 8.75; N, 5.31.
2.1.8. N-((cyclohexylcarbamoyl)methyl)-N-benzyl-3ˇ-
hydroxypregn-5-ene-20-carboxamide
(6a)
Mp: 114 ◦C. 1H NMR (CDCl3): 0.62 (H18, 3H, s); 0.99 (H19, 3H,
s); 1.11 (H21, 3H, d, 6.4); 2.77 (H20, 1H, m); 3.50 (H3, 1H, m); 3.70
(H4ꢀ, 1H, m); 3.88 and 4.03 (H1ꢀ, 1H each signal, d, 14.5); 4.67 and
4.75 (H8ꢀ, 1H each signal, d, 16.0); 5.34 (H6, 1H, m); 6.60 (H3ꢀ, 1H, d,
7.5); 7.20 (H10ꢀ, 2H, dd, 7.4 and 1.2); 7.31 (H11ꢀ, 2H, m); 7.38 (H12ꢀ,
1H, t, 7.7). 13C NMR (CDCl3): 12.3 (C18); 17.5 (C21); 19.3 (C19);
21.0 (C11); 24.6 (C16); 24.7 (C6ꢀ); 25.4 (C15); 25.4 (C7ꢀ); 31.3 (C7);
31.7 (C2); 31.8 (C8); 32.9 (C5ꢀ); 36.4 (C10); 37.2 (C1); 37.7 (C12);
38.0 (C20); 40.9 (C13); 42.0 (C4); 48.0 (C4ꢀ); 49.4 (C9); 51.2 (C1ꢀ);
52.8 (C8ꢀ); 53.0 (C17); 56.1 (C14); 71.4(C3); 121.4 (C6); 126.7 (C10ꢀ);
127.9 (C11ꢀ); 128.9 (C12ꢀ); 136.2 (C9ꢀ); 140.8 (C5); 168.3 (C2ꢀ); 178.6
(C22). HRMS (ESI): calculated for [M+H+] C37H55N2O3 575.4213,
found 575.4224. Anal. calculated for C37H54N2O3 C, 77.31; H, 9.47;
N, 4.87; found C, 77.25; H, 9.62; N, 4.99.
2.1.5. N-((tert-butylcarbamoyl)methyl)-N-(4-chlorophenyl)-3ˇ-
hydroxyandrost-5-en-17ˇ-carboxamide
(5c)
Mp: 116 ◦C. 1H NMR (CDCl3): 0.81 (H18, 3H, s); 0.98 (H19, 3H,
s); 1.36 (H5ꢀ, 9H, s); 2.47 (H17, 1H, t, 9.0); 3.48 (H3, 1H, m); 3.84 and
4.34 (H1ꢀ, 1H each signal, d, 14.4); 5.29 (H6, 1H, d, 5.0); 6.27 (H3ꢀ,
1H, bs); 7.18 (H7ꢀ, 2H, bs); 7.36 (H8ꢀ, 2H, d, 8.7). 13C NMR (CDCl3):
13.7 (C18); 19.4 (C19); 20.8 (C11); 24.7(C16); 26.0 (C15); 28.7 (C5ꢀ);
31.6 (C2); 31.8 (C7); 31.8 (C8); 36.5 (C10); 37.2 (C1); 38.4 (C12); 42.2
(C4); 45.6 (C13); 49.8 (C9); 51.2 (C4ꢀ); 51.9 (C17); 55.8 (C1ꢀ); 56.2
(C14); 71.7 (C3); 121.3 (C6); 129.4 (C7ꢀ); 129.8 (C8ꢀ); 133.7 (C9ꢀ);
140.8 (C5); 142.2 (C6ꢀ); 168.3 (C2ꢀ); 174.7(C20). HRMS (ESI): cal-
culated for [M+H+] C32H46ClN2O3 541.3197, found 541.3181. Anal.
calculated for C32H45ClN2O3 C, 71.02; H, 8.38; N, 5.18; found C,
71.21; H, 8.41; N, 5.21.
2.1.9. N-((cyclohexylcarbamoyl)methyl)-N-(2-(1H-indol-3-
yl)ethyl)-3ˇ-hydroxypregn-5-ene-20-carboxamide
(6b)
Mp: 195 ◦C. 1H NMR (CDCl3): 0.44 (H18, 3H, s); 0.91 (H21, 3H, d,
6.3); 0.94 (H19, 3H, s); 2.53 (H20, 1H, m); 3.07 (H9ꢀ, 2H, t, 7.0); 3.42
(H3, 1H, m); 3.62 (H4ꢀ, 1H, m); 3.71 and 3.81 (H8ꢀ, 2H, m); 3.86 and
4.07 (H1ꢀ, 1H each signal, d, 15.3); 5.27 (H6, 1H, m); 6.78 (H3ꢀ, 1H, d,
7.4); 7.01 (H11ꢀ, 1H, s); 7.11 (H15ꢀ, 1H, dt, 7.2 and 1.1); 7.18 (H16ꢀ,
1H, dt, 7.2 and 1.1); 7.39 (H14ꢀ, 1H, bd, 8.2); 7.57 (H17ꢀ, 1H, bd, 7.8).
13C NMR (CDCl3): 11.8 (C18); 17.0 (C21); 19.0 (C19); 20.8 (C11);
24.5 (C9ꢀ); 24.6 (C16); 24.7 (C6ꢀ); 25.4 (C15); 25.4 (C7ꢀ); 31.6 (C2);
31.6 (C7); 31.8 (C8); 32.9 (C5ꢀ); 36.3 (C10); 37.0 (C1); 37.5 (C12);
37.5 (C20); 40.9 (C13); 42.0 (C4); 48.0 (C4ꢀ); 49.4 (C9); 50.2 (C8ꢀ);
52.8 (C17); 52.8 (C1ꢀ); 56.0 (C14); 71.2 (C3); 110.6 (C10ꢀ); 111.3
(C14ꢀ); 117.7 (C17ꢀ); 119.2 (C15ꢀ); 121.3 (C6); 121.9 (C16ꢀ); 122.7
(C11ꢀ); 126.9 (C18ꢀ); 136.5 (C13ꢀ); 140.7 (C5); 169.1 (C2ꢀ); 178.6
(C22). HRMS (ESI): calculated for [M+H+] C40H58N3O3 628.4478,
2.1.6. N-((tert-butylcarbamoyl)methyl)-N-phenyl-3ˇ-
hydroxyandrost-5-en-17ˇ-carboxamide
(5d)
Mp: 110 ◦C. 1H NMR (CDCl3): 0.82 (H18, 3H, s); 0.97 (H19, 3H,
s); 1.36 (H5ꢀ, 9H, s); 2.51 (H17, 1H, t, 9.0); 3.45 (H3, 1H, m); 3.92 and

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F. Alonso et al. / Steroids 75 (2010) 659–664
(H11ꢀ, 1H, t, 7.7); 7.39 (H9ꢀ, 2H, m); 7.42 (H10ꢀ, 2H, m). 13C NMR
(CDCl3): 12.4 (C18); 17.5 (C21); 19.3 (C19); 21.0 (C11); 24.6 (C16);
25.4 (C15); 28.6 (C5ꢀ); 31.6 (C7); 31.7 (C2); 31.8 (C8); 36.3 (C10);
37.2 (C1); 37.6 (C20); 37.7 (C12); 39.5 (C13); 42.2 (C4); 42.2 (C7ꢀ);
49.9 (C9); 51.3 (C4ꢀ); 52.7 (C6ꢀ); 52.9 (C1ꢀ); 53.0 (C17); 56.1 (C14);
71.7 (C3); 121.5 (C6); 126.8 (C11ꢀ); 128.6 (C10ꢀ); 128.8 (C9ꢀ); 137.7
(C8ꢀ); 140.7 (C5); 168.9 (C2ꢀ); 177.7 (C22). HRMS (ESI): calculated
for [M+H+] C36H55N2O3 563.4213, found 563.4233. Anal. calculated
for C36H54N2O3 C, 76.82; H, 9.67; N, 4.98; found C, 76.70; H, 9.51;
N, 4.90.
found 628.4473. Anal. calculated for C40H57N3O3 C, 76.51; H, 9.15;
N, 6.69; found C, 76.44; H, 9.27; N, 6.42.
2.1.10. N-((cyclohexylcarbamoyl)methyl)-N-(3-(4-
nitrophenylamino)propyl)-3ˇ-hydroxypregn-5-ene-20-
carboxamide
(6c)
Mp: 124 ◦C. 1H NMR (CDCl3): 0.65 (H18, 3H, s); 1.00 (H19, 3H, s);
1.12 (H21, 3H, d, 6.4); 1.80 and 2.02 (H9ꢀ, 2H, m); 2.67 (H20, 1H, m);
3.27 (H10ꢀ, 2H, m); 3.47 and 3.55 (H8ꢀ, 2H, m); 3.53 (H3, 1H, m); 3.71
(H4ꢀ, 1H, m); 3.86 and 4.02 (H1ꢀ, 1H each signal, d, 14.1); 5.34 (H6,
1H, m); 5.47 (H11ꢀ, 1H, bt); 6.55 (H13ꢀ, 2H, dd, 8.9 and 1.9); 8.05 and
8.08 (H14ꢀ, 2H, m). 13C NMR (CDCl3): 11.8 (C18); 17.0 (C21); 19.0
(C19); 20.8 (C11); 24.6 (C16); 24.7 (C6ꢀ); 25.3 (C7ꢀ); 25.4 (C15);
28.4 (C9ꢀ); 31.6 (C2); 31.6 (C7); 31.8 (C8); 32.8 (C5ꢀ); 36.3 (C10);
37.0 (C1); 37.5 (C12); 37.9 (C20); 40.6 (C10ꢀ); 40.9 (C13); 42.0 (C4);
44.3 (C8ꢀ); 48.1 (C4ꢀ); 49.4 (C9); 52.6 (C1ꢀ); 52.8 (C17); 56.0 (C14);
71.7 (C3); 111.0 (C13ꢀ); 121.4 (C6); 126.5 (C14ꢀ); 137.5 (C15ꢀ); 140.7
(C5); 153.4 (C12ꢀ); 169.2 (C2ꢀ); 178.1 (C22). HRMS (ESI): calculated
for [M+H+] C39H59N4O5 663.4485, found 663.4489. Anal. calculated
for C39H58N4O5 C, 70.66; H, 8.82; N, 8.45; found C, 70.52; H, 8.95;
N, 8.33.
2.1.14. N-((tertbutylcarbamoyl)methyl)-N-benzyl-3ˇ-
hydroxypregn-5-ene-20-carboxamide
(6g)
Mp: 97 ◦C. 1H NMR (CDCl3): 0.63 (H18, 3H, s); 1.00 (H19, 3H, s);
1.13 (H21, 3H, d, 6.4); 1.31 (H5ꢀ, 9H, s); 2.82 (H20, 1H, m); 3.52 (H3,
1H, m); 3.78 and 4.02 (H1ꢀ, 1H each signal, d, 14.1); 4.64 and 4.74
(H6ꢀ, 1H each signal, d, 16.5); 5.34 (H6, 1H, m); 6.36 (H3ꢀ, 1H, bs);
7.25 (H10ꢀ, 1H, t, 7.7); 7.32 (H8ꢀ, 2H, m); 7.42 (H9ꢀ, 2H, m). 13C NMR
(CDCl3): 12.3 (C18); 17.6 (C21); 19.4 (C19); 21.0 (C11); 24.3 (C16);
28.3 (C15); 28.4 (C5ꢀ); 31.6 (C7); 31.8 (C2); 31.9 (C8); 36.5 (C10);
37.2 (C1); 37.2 (C11ꢀ); 37.7 (C12); 38.5 (C20); 41.8 (C13); 42.3 (C4);
50.0 (C9); 51.4 (C4ꢀ); 52.4 (C1ꢀ); 52.8 (C6ꢀ); 53.0 (C17); 56.1 (C14);
71.7 (C3); 121.5 (C6); 126.9 (C8ꢀ); 127.9 (C10ꢀ); 128.9 (C9ꢀ); 136.4
(C7ꢀ); 140.7 (C5); 168.4 (C2ꢀ); 178.2 (C22). HRMS (ESI): calculated
for [M+H+] C35H53N2O3 549.4056, found 549.4070. Anal. calculated
for C35H52N2O3 C, 76.60; H, 9.55; N, 5.10; found C, 76.51; H, 9.40;
N, 5.32.
2.1.11. N-((tertbutylcarbamoyl)methyl)-N-phenyl-3ˇ-
hydroxypregn-5-ene-20-carboxamide
(6d)
Colourless oil. 1H NMR (CDCl3): 0.34 (H18, 3H, s); 0.96 (H19,
3H, s); 1.09 (H21, 3H, d, 6.3); 2.44 (H20, 3H, m); 3.51 H3, 1H, m);
3.99 and 4.29 (H1ꢀ, 1H each signal, d, 14.5); 5.32 (H6, 1H, m); 6.49
(H3ꢀ, 1H, bs); 7.21 (H9ꢀ, 1H, t, 7.7); 7.39 (H7ꢀ, 2H, m); 7.42 (H8ꢀ,
2H, m). 13C NMR (CDCl3): 11.8 (C18); 17.4 (C21); 19.3 (C19); 21.0
(C11); 24.6 (C16); 25.4 (C15); 28.6 (C5ꢀ); 31.3 (C7); 31.7 (C2); 31.8
(C8); 36.3 (C10); 37.1 (C1); 37.7 (C12); 38.3 (C20); 39.4 (C13); 42.1
(C4); 49.9 (C9); 51.0 (C4ꢀ); 53.0 (C17); 53.0 (C1ꢀ), 55.9 (C14); 71.6
(C3); 121.4 (C6); 127.6 (C9ꢀ), 128.2 (C7ꢀ); 129.8 (C8ꢀ); 140.6 (C5);
142.9 (C6ꢀ); 168.2 (C2ꢀ); 177.6 (C22). HRMS (ESI): calculated for
[M+H+] C34H51N2O3 535.3900, found 535.3911. Anal. calculated for
2.1.15. Diethyl {[N-benzyl-N-[(3ˇ-hydroxypregn-5-ene-20-yl)-
carbonyl]-glycyl]-amino}-methylphosphonate
(6h)
Colourless oil. 1H NMR (CDCl3): 0.64 (H18, 3H, s); 1.01 (H19,
3H, s); 1.18 (H21, 3H, d, 6.5); 1.35 (H6ꢀ, 6H, m); 2.79 (H20, 1H, m);
3.54 (H3, 1H, m); 3.71 (H4ꢀ, 2H, m); 4.04 (H1ꢀ, 2H, m); 4.15 (H5ꢀ, 4H,
m); 4.67 and 4.74 (H7ꢀ, 1H each signal, d, 16.6); 5.36 (H6, 1H, m);
6.72 (H3ꢀ, 1H, bs); 7.21 (H9ꢀ, 2H, dd, 7.1 and 1.0); 7.35 (H10ꢀ, 2H,
m); 7.38 (H11ꢀ, 1H, m). 13C NMR (CDCl3): 12.2 (C18); 16.4 (C6ꢀ, d,
JC–P = 6.1); 17.5 (C21); 19.5 (C19); 21.0 (C11); 24.4 (C16); 25.4 (C15);
31.3 (C7); 31.4 (C2); 31.8 (C8); 34.7, (C4ꢀ, d, JC–P = 158.9); 36.8 (C10);
37.6 (C1); 37.6 (C12); 37.6 (C20); 41.3 (C13); 41.8 (C4); 49.4 (C9);
50.4 (C1ꢀ); 52.5 (C7ꢀ); 52.6 (C17); 56.1 (C14); 62.1 (C5ꢀ, d, JC–P = 18.3);
71.7 (C3); 121.5 (C6); 126.8 (C9ꢀ); 128.0 (C10ꢀ); 129.0 (C11ꢀ); 136.2
(C8ꢀ); 140.7 (C5), 169.3(C2ꢀ); 178.5(C22). HRMS(ESI): calculated for
[M+H+] C36H56N2O6P 643.3876, found 643.3876. Anal. calculated
for C36H55N2O6P C, 67.27; H, 8.62; N, 4.36; found C, 67.06; H, 8.79;
N, 4.15.
C34H50N2O3 C, 76.36; H, 9.42; N, 5.24; found C, 76.25; H, 9.59; N,
5.12.
2.1.12. N-((tertbutylcarbamoyl)methyl)-N-((1H-indol-6-
yl)methyl)-3ˇ-hydroxypregn-5-ene-20-carboxamide
(6e)
Mp: 126 ◦C. 1H NMR (CDCl3): 0.66 (H18, 3H, s); 0.95 (H19, 3H,
s); 1.17 (H21, 3H, d, 6.4); 1.29 (H5ꢀ, 9H, s); 2.94 (H20, 1H, m); 3.53
(H3, 1H, m); 3.94 and 4.17 (H1ꢀ, 1H each signal, d, 14.1); 4.47 and
4.93 (H6ꢀ, 1H each signal, d, 16.8); 5.34 (H6, 1H, m); 6.34 (H3ꢀ, 1H,
bs); 6.51 (H11ꢀ, 1H, m); 6.51 (H12ꢀ, 1H, m); 7.01 (H8ꢀ, 1H, dd, 8.5 and
1.5); 7.37 (H9ꢀ, 1H, d, 8.0); 7.58 (H15ꢀ, 1H, bs). 13C NMR (CDCl3): 12.3
(C18); 17.7 (C21); 19.3 (C19); 21.0 (C11); 24.3 (C16); 28.3 (C15);
28.7 (C5ꢀ); 31.6 (C7); 31.7 (C2); 31.8 (C8); 36.5 (C10); 37.2 (C1);
37.7 (C12); 38.5 (C20); 41.8 (C13); 42.2 (C4); 50.0 (C9); 51.1 (C4ꢀ);
52.0 (C1ꢀ); 53.0 (C17); 53.1 (C6ꢀ); 56.1 (C14); 71.7 (C3); 101.4 (C11ꢀ);
111.5 (C15ꢀ); 119.3 (C8ꢀ); 121.5 (C6); 121.5 (C9ꢀ); 122.9 (C12ꢀ); 128.9
(C10ꢀ); 129.0 (C7ꢀ); 135.5 (C14ꢀ); 140.7 (C5); 168.6 (C2ꢀ); 178.1
(C22). HRMS (ESI): calculated for [M+H+] C37H54N3O3 588.4165,
found 588.4203. Anal. calculated for C37H53N3O3 C, 75.60; H, 9.09;
N, 7.15; found C, 75.48; H, 9.13; N, 7.25.
2.1.16. N-benzyl-N-[(3ˇ-hydroxypregn-5-ene-20-yl)-carbonyl]-
glycylglycine
(6i)
Mp: 156 ◦C. 1H NMR (CDCl3): 0.62 (H18, 3H, s); 0.99 (H19, 3H,
s); 1.11 (H21, 3H, d, 6.4); 1.35 (H6ꢀ, 6H, m); 2.82 (H20, 1H, m); 3.47
(H3, 1H, m); 3.92 and 4.09 (H4ꢀ, 1H each signal, d, 17.6); 4.00 and
4.11 (H1ꢀ, 1H each signal, d, 15.4); 4.51 and 4.82 (H6ꢀ, 1H each sig-
nal, d, 14.5); 5.34 (H6, 1H, m); 6.72 (H3ꢀ, 1H, bs); 7.23 (H8ꢀ, 2H,
dd, 7.1 and 1.0); 7.31 (H9ꢀ, 2H, m); 7.38 (H11ꢀ, 1H, t, 7.7). 13C NMR
(CDCl3–CD3OD 9:1): 11.7 (C18); 16.8 (C21); 18.8 (C19); 20.5 (C11);
23.9 (C16); 25.4 (C15); 31.3 (C7); 31.4 (C2); 31.8 (C8); 36.8 (C10);
37.6 (C1); 37.6 (C12); 37.6 (C20); 41.3 (C13); 41.8 (C4); 49.1 (C6ꢀ);
49.2 (C4ꢀ); 49.3 (C1ꢀ); 49.4 (C9); 52.6 (C17); 55.8 (C14); 70.8 (C3);
120.9 (C6); 126.4 (C8ꢀ); 127.5 (C9ꢀ); 128.6 (C10ꢀ); 136.2 (C7ꢀ); 140.4
(C5); 169.0 (C2ꢀ); 174.1 (C5ꢀ), 178.6 (C22). HRMS (ESI): calculated
for [M+H+] C33H47N2O5 551.3485, found 551.3497. Anal. calculated
for C33H46N2O5 C, 71.97; H, 8.42; N, 5.09; found C, 71.79; H, 8.31;
N, 5.17.
2.1.13. N-((tertbutylcarbamoyl)methyl)-N-phenethyl-3ˇ-
hydroxypregn-5-ene-20-carboxamide
(6f)
Colourless oil. 1H NMR (CDCl3): 0.70 (H18, 3H, s); 1.02 (H19, 3H,
s); 1.06 (H21, 3H, d, 6.4); 1.31 (H5ꢀ, 9H, s); 2.64 (H20, 1H, m); 2.90
(H7ꢀ, 2H, m); 3.52 (H3, 1H, m); 3.67 (H6ꢀ, 2H, m); 3.72 and 4.03 (H1ꢀ,
1H each signal, d, 15.2); 5.34 (H6, 1H, m); 6.51 (H3ꢀ, 1H, bs); 7.21

F. Alonso et al. / Steroids 75 (2010) 659–664
663
Scheme 1. Synthesis of compounds 5a–5e and 6a–6h.
Scheme 2. Synthesis of compound 6i.
2.1.17. Antifungal activity
Fusarium virguliforme (causal agent of sudden death syndrome in
soy bean) and Fusarium lateritium (causal agent of sweetpotato
chlorotic leaf distortion). The results are shown in Table 1.
Most of the new compounds showed a measurable antifun-
gal activity against either of the two fungi tested. Although this
is a preliminary screening, the activity might be species-specific
(F. virguliforme seems to be more susceptible towards this family
of compounds). On the other hand, it is interesting to note that
whereas the homologous pair 5d and 6d elicited a significant activ-
ity, compound 6g was inactive, in contrast to its homologue 5a,
which induced a small but measurable halo on the plate. This may
indicate that the antifungal properties depend not only on the moi-
eties attached to the side chain but also on the steroidal skeleton.
Finally, we determined the minimum inhibitory concentration
(MIC) of two of the most active compounds. Compounds 5d and 6i
had MIC values against F. lateritium of 8.6 and 10 nmol/spot respec-
tively whereas 5d showed a MIC value against F. virguliforme of
7.7 nmol/spot.
Direct bioautography on TLC was used to assess the inhibitory
activity. A concentration level 50 ␮g/spot of each compound
assayed was used. Benomyl was used as a positive control. When
appropriate, as large inhibitory halos were observed, minimum
inhibitory concentration (MIC) was measured by the same method.
3. Results
The synthesis of the novel compounds is shown in Scheme 1.
The known steroidal acids 3 and 4 were obtained as described pre-
viously [10,11]. Treatment of 3 with formaldehyde, an amine and
an isonitrile led to the desired compounds of general structures
5a–5e.
When the 3␤-acetate 4 was used as the carboxylic component in
the U-4CR, the resulting adducts were hydrolysed in situ to achieve
the compounds 6a–6h. As expected, the Ugi product obtained from
ethyl isocyanoacetate gave the free acid 6i after the hydrolysis step
(Scheme 2).
Table 1
The U-4CR generally took place smoothly and in good yields,
and worked well with a set of structurally diverse amines, which
can also include additional functional groups (Table 1).
The structure of all new compounds (Figs. 2 and 3) was unequiv-
ocally assigned by the usual methods (1 and 2D NMR experiments
and mass spectrometry). In some cases, the NMR spectra showed
that the compounds were present in the solution as a mixture of
two conformers. These conformers are expected to originate from
the cis–trans rotation around the N-substituted side chain amide
bond [12]. Because the interchange between the cis and trans iso-
mers of these bonds is generally slow in the NMR time scale, the
NMR spectrum is the composite of the NMR spectra of the two con-
figurational isomers (Fig. 4). For clarity, only the chemical shifts and
coupling constants for the most populated conformer in each case
was described in Section 2.
Yields of the new compounds and their antifungal activity.
Yield (%)a
F. lateritiumb
F. virguliformeb
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
6f
76
69
75
82
79
63
62
69
71
69
78
77
73
63
0.3 0.1
–
–
1.2 0.2
–
–
–
–
0.5 0.1
0.4 0.1
1.1 0.1
1.0 0.2
–
0.8 0.1
–
0.5 0.1
0.4 0.1
0.3 0.1
–
–
0.7 0.2
–
–
–
1.1 0.2
1.3 0.1
6g
6h
6i
–
–
a
Isolated yields.
Bioautography offers a rapid and convenient approach to iden-
tify novel antifungal compounds and requires only microgram
quantities [13]. The new compounds were tested in vitro using
a bioautography method for their inhibitory properties towards
b
Diameters of inhibition zone in cm. Mean values standard error from four inde-
pendent experiments testing 50 ␮g of compound/spot. The inhibition halo of the
control (Benomyl, a commercial antifungal) is 2.7 0.1 cm at the same concentra-
tion.

664
F. Alonso et al. / Steroids 75 (2010) 659–664
Fig. 4. Side chain methylene signals (1H NMR, 500 MHz) for compound 6g showing a 2:1 mixture of two conformers. Assignments of the signals correspond to the numbering
depicted in Fig. 3.
Although examples of sterol analogues bearing amide bonds
in the side chain are known [3], they were obtained using stan-
dard peptide chemistry, which imposes some limitations to the
type of accessible structures. In this preliminary study, we demon-
strated that the use of the U-4CR allows the facile attachment of
a polyfunctionalized side chain into the steroidal framework ren-
dering complex azasteroids by a simple procedure. In addition, our
approach also leads to novel structures with remarkable antifungal
activity against two pathogenic species of agricultural relevance.
Some natural and synthetic steroids with a nitrogen atom in
their side chains were found to inhibit fungal growth through the
inhibition of the enzyme sterol 24-methyl transferase, responsible
for alkylation at C-24 during ergosterol biosynthesis [14]. Due to
the structural features of our novel compounds, this mechanism
of action cannot be discarded. Further studies are under way to
expand this procedure to obtain a larger library of analogues in
order to establish a structure–activity relationship and their possi-
ble mechanism of action on fungal growth.
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Acknowledgements
This work was supported by grants from the Universidad de
Buenos Aires (UBACyT X-190 and X-029), and the Agencia Nacional
de Promoción Científica y Técnica (ANPCyT PICT 00194/07). We
are grateful to UMYMFOR (UBA-CONICET) for the analytical and
spectroscopic determinations.
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