Multicomponent synthesis of 4,4-dimethyl sterol analogues and their effect on eukaryotic cells

Article abstract of DOI:10.1016/j.steroids.2014.03.002

Most sterols, such as cholesterol and ergosterol, become functional only after the removal of the two methyl groups at C-4 from their biosynthetic precursors. Nevertheless, some findings suggest that 4,4-dimethyl sterols might be involved in specific physiological processes. In this paper we present the synthesis of a collection of analogues of 4,4-dimethyl sterols with a diamide side chain and a preliminary analysis of their in vitro activity on selected biological systems. The key step for the synthesis involves an Ugi condensation, a versatile multicomponent reaction. Some of the new compounds showed antifungal and cytotoxic activity.

Full text of DOI:10.1016/j.steroids.2014.03.002

Steroids 84 (2014) 1–6
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Multicomponent synthesis of 4,4-dimethyl sterol analogues and their
effect on eukaryotic cells
Fernando Alonso a, Adriana M. Cirigliano a, María Eugenia Dávola b, Gabriela M. Cabrera a,
Guadalupe E. García Liñares a, Carlos Labriola c, Andrea A. Barquero b, Javier A. Ramírez a,
⇑
a 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
b Departamento de Química Biológica and IQUIBICEN, (CONICET – Facultad de Ciencias Exactas y Naturales), Universidad de Buenos Aires,
Pabellón 2, Piso 4, Ciudad Universitaria, C1428EGA Buenos Aires, Argentina
c Laboratorio de Glicobilogía, Fundación Instituto Leloir e Instituto de Investigaciones Bioquímicas de Buenos Aires, Avenida Patricias Argentinas 435,
C1405BWE Buenos Aires, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
Most sterols, such as cholesterol and ergosterol, become functional only after the removal of the two
methyl groups at C-4 from their biosynthetic precursors. Nevertheless, some findings suggest that
4,4-dimethyl sterols might be involved in specific physiological processes. In this paper we present the
synthesis of a collection of analogues of 4,4-dimethyl sterols with a diamide side chain and a preliminary
analysis of their in vitro activity on selected biological systems. The key step for the synthesis involves an
Ugi condensation, a versatile multicomponent reaction. Some of the new compounds showed antifungal
and cytotoxic activity.
Received 27 October 2013
Received in revised form 31 January 2014
Accepted 3 March 2014
Available online 13 March 2014
Keywords:
Antifungal
Cytotoxic
Ó 2014 Elsevier Inc. All rights reserved.
4,4-Dimethyl sterols
Ugi reaction
1. Introduction
that a variety of C-4 methylated sterols have specific physiological
activity in different organisms. In animals, for example, 4,4-
Sterols have an extremely widespread distribution in living
organisms: protozoa, algae, fungi, higher plants and animals [1].
Although sterols have multiple roles, the most important is as
structural constituents of eukaryote membranes, where they
enhance the packing of the acyl chains of phospholipids in the
hydrophobic phase of the bilayer, increase its mechanical strength,
and reduce its permeability [2].
The outline for similarity and differences in sterol biosynthesis
across kingdoms has been established [3]. Although the most
abundant sterols are cholesterol, ergosterol and sitosterol [1],
chemical surveys of the sterol composition of eukaryotes show that
there are at least 250 different sterols, many of them being biosyn-
thetic intermediates, present in small amounts in tissues. Taking
into account that all sterols derive from the lanostane or
cycloartane skeletons, many of these intermediates possess methyl
groups at C-4 and/or C-14.
dimethyl-5a-cholesta-8,14,24-trien-3b-ol and related sterols can
activate the nuclear maturation of oocytes and markedly improve
fertilization rates in vitro [5]. On the other hand, recent studies
show that 4-methylsterols regulate Schizosaccharomyces pombe
fission under low oxygen and cell stress [6].
Synthetic, non natural 4,4-dimethylsterols may have biological
activities also: both 4,4-dimethylcholestrol (1) and 4,4-dimethyl-
cholest-5-en-3-one (2) (Fig. 1) are able to suppress the inflamma-
tory response induced by a phorbol derivative on mice skin [7].
In recent papers we have shown that some sterol analogues
with a diamide side chain are able to exert some interesting
biological activities. Particularly, compounds 3 and 4 (Fig. 2)
showed inhibitory effect on the growth of fungi associated with
plant pathologies, but have a low in vitro toxicity in mammalian
cells [8,9]. Furthermore, our previous findings suggest that an aro-
matic fragment bound to the N-22 is important for the activity.
Taking these previous results into account, in this paper we
describe the synthesis of a small library of 4,4-dimethyl sterol ana-
logues with a general structure 5, structurally related to 3 and 4,
and some preliminary in vitro studies about their effect on
eukaryotic cells.
Most sterols become functional only after the removal of the
two methyl groups at C-4 [4]. Nevertheless, some reports indicate
⇑
Corresponding author. Tel.: +54 01145763346; fax: +54 01145763385.
E-mail address: jar@qo.fcen.uba.ar (J.A. Ramírez).
http://dx.doi.org/10.1016/j.steroids.2014.03.002
0039-128X/Ó 2014 Elsevier Inc. All rights reserved.

2
F. Alonso et al. / Steroids 84 (2014) 1–6
added, and the resulting suspension was heated under reflux for
20 min until a clear solution was formed. Then H2O (50 ml) and
HCl 10% (50 ml) were added, and the precipitate extracted with
EtOAc (3 Â 20 ml). The organic fraction was dried over Na2SO4
and evaporated in vacuo. The crude product was purified by column
chromatography on silica (hexane/EtOAc gradient), to yield the
steroidal acid 8 (511 mg, 67% yield). M.p.: 217–218 °C. 1H NMR
(CDCl3): 0.73 (H18, 3H, s); 0.85 (H19, 3H, s); 1.21 (H4a, 3H, s);
1.23 (H4b, 3H, s); 1.51 (H8, 1H, dq, J = 10.6 and 5.4); 2.33 (H17,
Fig. 1. Known non natural 4,4-dimethylsterols with biological activity.
2. Experimental
1H, t, J = 9.3); 2.45 (H2b, 1H, m); 2.56 (H2a, 1H, m); 5.56 (H6, 1H,
2.1. Synthesis
dd, J = 5.2 and 2.5). 13C NMR (CDCl3): 12.6 (C18); 18.6 (C19); 20.4
(C11); 22.9 (C16); 23.7 (C15); 26.6 (C4a); 29.5 (C4b); 30.6 (C8);
30.9 (C7); 31.3 (C2); 33.0 (C1); 36.2 (C10); 37.2 (C12); 42.9 (C13);
47.7 (C4); 48.1 (C9); 54.4 (C17); 55.5 (C14); 119.0 (C6); 149.0
(C5); 175.2 (C20); 215.7 (C3). Anal. calculated for C22H32O3 C,
76.77; H, 9.36; found: C, 76.88; H, 9.45.
2.1.1. General
All the reagents were purchased from Sigma-Aldrich Chemical
Co. ESI-HRMS were measured on a Bruker micrOTOF-Q II. Melting
points were determined on a Fisher Johns apparatus and are uncor-
rected. All NMR spectra were recorded on a Bruker AM-500
(500 MHz for 1H and 125.1 MHz for 13C). Chemical shifts (d) 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 combus-
tion analysis data (purity P 98%) on an Exeter CE 440 Elemental
Analyzer.
2.1.4. (17b)-N-((t-butylcarbamoyl)methyl)-N-(4-methoxyphenyl)-
4,4-dimethyl-3-oxoandrost-5-ene-17-carboxamide (9a)
To a solution of the aniline (0.07 mmol, 1.1 eq.) in methanol
(1 ml), 5
was stirred for 30 min at room temperature. Then, 20 mg of the ste-
roidal acid 8 (0.06 mmol, 1 eq.) and t-butyl isocyanide (6.6 L,
lL of formaldehyde (37% aq) was added, and the mixture
l
0.07 mmol, 1.1 eq.) were added. The reaction was kept under the
same condition until total disappearance of the acid (72 h). The sol-
vent was evaporated under reduced pressure and the residue was
taken in EtOAc and washed with NaOH (5% aq.). The crude product
was purified by silica gel column chromatography (hexane/EtOAc
gradient) to give compound 9a with a 66% yield. M.p.: 174 °C. 1H
NMR (CDCl3): 0.83 (H18, 3H, s); 0.84 (H19, 3H, s); 1.19 (H4b, 3H,
s); 1.21 (H4a, 3H, s); 1.36 (–NH–C(CH3)3, 9H, s); 1.52 (H8, 1H, m);
2.1.2. Methyl (17b)-4,4-dimethyl-3-oxoandrost-5-ene-17-carboxylate
(7)
Methyl (17b)-3-oxoandrost-4-ene-17-carboxylate [10] (6, 1.6 g,
4.8 mmol, obtained from progesterone as previously described
[11]) was dissolved in anhydrous THF (100 ml) under argon, and
potassium t-butoxide (1.1 g, 9.6 mmol) was added in portions. After
stirring the yellow solution for 5 min at 0 °C, methyl iodide (13,7 g,
6.03 ml, 96 mmol) was added, after which the solution becomes
cloudy yellow. The reaction was monitored by TLC, and completed
within 60 min. Then, H2O (20 ml) and saturated solution of
ammonium chloride (20 ml) were added. The mixture was evapo-
rated in vacuo in order to get rid of the THF. The residue was taken
in ethyl acetate (200 ml), washed with brine, dried over Na2SO4 and
evaporated in vacuo to give 1.72 g of a yellow oil crude product,
which was subsequently purified by column chromatography on
silica (cyclohexane/EtOAc 95:5) to give the dimethylated product
7 (771 mg, 2.1 mmol, 63%). M.p.:123–125 °C. 1H NMR (CDCl3):
0.67 (H18, 3H, s); 0.85 (H19, 3H, s); 1.23 (H4a and H4b, 6H, s);
1.51 (H8, 1H, dq, J = 10.7 and 5.5); 2.36 (H17, 1H, t, J = 9.4); 2.47
2.47 (H2b, 1H, m); 2.50 (H17, 1H, t, J = 9.1); 2.56 (H2a, 1H, m);
3,83 (–N–Ar–OCH3, 3H, s); 3.86 (–N(Ar)–CHaHb–CO–, 1H, d,
J = 14.5); 4.38 (–N–CHaHb–CO–, 1H, d, J = 14.5); 5.50 (H6, 1H, dd,
J = 2.5 and 5.2); 6.42 (–NH–C(CH3)3, 1H, bs); 6.89 (2H, d, J = 9) and
7.12 (2H, m).
13C NMR (CDCl3): 13.8 (C18); 19.3 (C19); 21.0 (C11); 24.6 (C15);
26.1 (C16); 27.2 (C4a); 28.7 (–NH–C(CH3)3); 30.1 (C4b); 31.1 (C8);
31.6 (C7); 32.1 (C1); 33.6 (C2); 37.1 (C10); 38.3 (C12); 38.7 (C13);
45.3 (C4); 48.6 (C9); 51.0 (–NH–C(CH3)3); 51.5 (C17); 55.4 (–N–Ar–
OCH3); 56.1 (–N–CH2–CO–); 56.2 (C14); 114.6, 129.9, 136.3 and
158.9 (aromatic carbons); 119.6 (C6); 149.8 (C5); 168.7
(–N(Ar)CH2–CO–); 175.1 (C20); 216.5 (C3). HRMS (ESI): calculated
for [M + H+] C28H44NO4 458.3265, found 458.3289. Anal. calculated
for C33H48N2O3 C, 76.11; H, 9.29; N, 5.38; found: C, 76.21; H, 9.41;
N, 5.21.
(H2b, 1H, ddd, J = 18.9, 8.4 and 1.7); 2.56 (H2a, 1H, ddd, J = 18.9,
11.1 and 8.2); 3.03 (–CO–CH3, 3H, s); 5.55 (H6, 1H, dd, J = 5.3 and
2.5). 13C NMR (CDCl3): 13.6 (C18); 19.6 (C19); 21.4 (C11); 23.9
(C16); 24.7 (C15); 27.5 (C4b); 30.4 (C4a); 31.6 (C8); 31.9 (C7);
32.4 (C1); 33.9 (C2); 37.4 (C10); 38.4 (C12); 44.2 (C13); 48.9 (C4);
49.1 (C9); 51.5 (–CO–CH3); 55.4 (C17); 56.4 (C14); 119.9 (C6);
150.1 (C5); 174.7 (C20); 216.8 (C3). Anal. calculated for C23H34O3
C, 77.05; H, 9.56; found: C, 77.11; H, 9.67.
Further elution gave 12% of 2-(t-butylamino)-2-oxoethyl (17b)-
4,4-dimethyl-3-oxoandrost-5-ene-17-carboxylate (10), the Passe-
rini condensation product. M. p.: 131–133 °C. 1H NMR (CDCl3):
0.73 (H18, 3H, s); 0.73 (H18, 3H, s); 0.86 (H19, 3H, s); 1.24 (H4a
and H4b, 6H, s); 1.38 (–NH–C(CH3)3, 9H, s); 1.54 (H8, 1H, m);
2.45 (H17, 1H, t, J = 9.3); 2.48 (H2b, 1H, ddd, J = 18.9, 8.5 and
2.1.3. (17b)-4,4-Dimethyl-3-oxoandrost-5-ene-17-carboxylic acid. (8)
To a solution of ester 7 (771 mg, 2.1 mmol) in ethylene glycol
(10.5 ml) an aqueous solution of KOH (1.82 ml, 40% m/v) was
1.7); 2.57 (H2a, 1H, ddd, J = 18.9, 8.5 and 1.7); 4.42 –CO–CHaHb–
CO–, 1H, d, J = 15.1); 4.51 (–CO–CHaHb–CO–, 1H, d, J = 15.1); 5.56
(H6, 1H, dd, J = 5.24 and 2.5); 13C NMR (CDCl3): 13.7 (C18); 19.5
Fig. 2. Previously synthesised sterol analogs and general structure of new 4,4-dimethyl derivatives.

F. Alonso et al. / Steroids 84 (2014) 1–6
3
(C19); 21.3 (C11); 23.9 (C16); 24.5 (C15); 27.4 (C4a); 28.9 (C26);
2.1.14. (17b)-N-((t-butylcarbamoyl)methyl)-N-(4-chlorophenyl)-4,
4-dimethyl-3-hydroxyandrost-5-ene-17-carboxamide (5b)
30.3 (C4b); 31.5 (C8); 31.7 (C7); 32.3 (C1); 33.8 (C2); 37.3 (C10);
38.6 (C12); 44.4 (C13); 48.8 (C4); 49.0 (C9); 51.5 (–NH–C(CH3)3);
55.2 (C17); 56.4 (C14); 63.4 (–CO–CH2–CO–); 119.7 (C6); 150.1
(C5); 166.4 (–CO–CH2–CO–); 172.5 (C20); 216.6 (C3). HRMS
(ESI): calculated for [M + H+] C28H44NO4 458.3265, found
458.3265. Anal. calculated for C33H48N2O3 C, 76.11; H, 9.29; N,
5.38; found:C, 76.21; H, 9.41; N, 5.21.
The same procedure was repeated with different amines to give
compounds 9b–i, that in all cases were obtained with a small
amount (between 5% to 15%) of compound 10.
M.p.: 122–124 °C. HRMS (ESI+): calculated for [M+Na+]
C34H49ClN2NaO3 591.3324, found 591.3304.
2.1.15. (17b)-N-((t-butylcarbamoyl)methyl)-N-phenyl-4,4-dimethyl-3
-hydroxyandrost-5-ene-17-carboxamide (5c)
Colourless oil. HRMS (ESI+): calculated for [M+Na+]
C34H50N2NaO3 557.3714, found 557.3753.
2.1.16. (17b)-N-((t-butylcarbamoyl)methyl)-N-(4-methylphenyl)-4,
4-dimethyl-3-hydroxyandrost-5-ene-17-carboxamide (5d)
M.p.: 202–204 °C. HRMS (ESI+): calculated for [M+Na+]
2.1.5. (17b)-N-((t-butylcarbamoyl)methyl)-N-(4-chlorophenyl)-4,4-di
methyl-3-oxoandrost-5-ene-17-carboxamide (9b)
M.p.: 158–160 °C. HRMS (ESI+): calculated for [M+Na+]
C35H52N2NaO3 571.3870, found 571.3882.
C34H47ClN2NaO3 589.3167, found 589.3203.
2.1.17. (17b)-N-((t-butylcarbamoyl)methyl)-N-(2-chlorophenyl)-4,4-
dimethyl-3-hydroxyandrost-5-ene-17-carboxamide (5e)
Colourless oil. HRMS (ESI+): calculated for [M+Na+] C34H49
ClN2NaO3 591.3324, found 591.3270.
2.1.6. (17b)-N-((t-butylcarbamoyl)methyl)-N-phenyl-4,4-dimethyl-3-
oxoandrost-5-ene-17-carboxamide (9c)
Colourless oil. HRMS (ESI+): calculated for [M+Na+] C34H48
N2NaO3 555.3557, found 555.3562.
2.1.18. (17b)-N-((t-butylcarbamoyl)methyl)-N-(2-fluorophenyl)-4,4-d
imethyl-3-hydroxyandrost-5-ene-17-carboxamide (5f)
Colourless oil. HRMS (ESI+): calculated for [M+Na+] C34H49
FN2NaO3 575.3619, found 575.3618.
2.1.7. (17b)-N-((t-butylcarbamoyl)methyl)-N-(4-methylphenyl)-4,4-d
imethyl-3-oxoandrost-5-ene-17-carboxamide (9d)
M.p.: 184 °C. HRMS (ESI+): calculated for [M+Na+] C35H50
N2NaO3 569.3714, found 569.3734.
2.1.19. (17b)-N-((t-butylcarbamoyl)methyl)-N-(2-ethyl-6-methylphe
nyl-4,4-dimethyl-3-hydroxyandrost-5-ene-17-carboxamide (5g)
Colourless oil. HRMS (ESI+): calculated for [M+H+] C37H57N2O3
577.4364, found 577.4414.
2.1.8. (17b)-N-((t-butylcarbamoyl)methyl)-N-(2-chlorophenyl)-4,4-di
methyl-3-oxoandrost-5-ene-17-carboxamide (9e)
M.p.: 89–91 °C. HRMS (ESI+): calculated for [M+Na+]
C34H47ClN2NaO3 589.3167, found 589.3180.
2.1.9. (17b)-N-((t-butylcarbamoyl)methyl)-N-(2-fluorophenyl)-4,4-di
methyl-3-oxoandrost-5-ene-17-carboxamide (9f)
2.1.20. (17b)-N-((t-butylcarbamoyl)methyl)-N-(2-naphthyl)-4,4-dime
thyl-3-hydroxyandrost-5-ene-17-carboxamide (5h)
M.p.: 174 °C. HRMS (ESI+): calculated for [M+Na+] C38H52
N2NaO3 607.3870, found 607.3851.
Colourless oil. HRMS (ESI+): calculated for [M+Na+]
C34H47FN2NaO3 573.3463, found 573.3467.
2.1.10. (17b)-N-((t-butylcarbamoyl)methyl)-N-(2-ethyl-6-methylphe
nyl)-4,4-dimethyl-3-oxoandrost-5-ene-17-carboxamide (9g)
M.p.: 110 °C HRMS (ESI+): calculated for [M+Na+] C37H54N2NaO3
597.4027, found 597.4050.
2.1.21. (17b)-N-((t-butylcarbamoyl)methyl)-N-(4-fluorophenyl)-4,4-d
imethyl-3-hydroxyandrost-5-ene-17-carboxamide (5i)
Colourless oil. HRMS (ESI+): calculated for [M+Na+] C34H49
FN2NaO3 575.3619, found 575.3675.
2.1.11. (17b)-N-((t-butylcarbamoyl)methyl)-N-(2-naphthyl)-4,4-dime
thyl-3-oxoandrost-5-ene-17-carboxamide (9h)
2.1.22. 2-(t-butylamino)-2-oxoethyl-(17b)-4,4-dimethyl-3-hydroxyan
drost-5-ene-17-carboxylate (11)
Colourless oil. HRMS (ESI+): calculated for [M+Na+] C38H50
N2NaO3 605.3714, found 605.3679.
M.p.: 177 °C. HRMS (ESI+): calculated for [M+Na+] C28H45NNaO4
482.3241, found 482.3312.
2.1.12. (17b)-N-((t-butylcarbamoyl)methyl)-N-(4-fluorophenyl)-4,4-d
imethyl-3-oxoandrost-5-ene-17-carboxamide (9i)
3. Biological studies
Colourless oil. HRMS (ESI+): calculated for [M+Na+] C34H47
FN2NaO3 573.3463, found 573.3481.
3.1. Effect on Fusarium strains
2.1.13. (17b)-N-((t-butylcarbamoyl)methyl)-N-(4-methoxyphenyl)-4,
4-dimethyl-3-hydroxyandrost-5-ene-17-carboxamide (5a)
Compound 9a (13 mg, 0.02 mmol) was dissolved in 2 ml of
CH2Cl2/MeOH (1:1) and NaBH4 (2 eq.) were added. The resulting
slurry was stirred at room temperature for 15 min, until comple-
tion of the reaction. The solvent were removed in vacuo, and the
crude product was purified by column chromatography on silica
(hexane/EtOAc gradient) to give compound 5a with 95% yield.
M.p.: 248 °C. HRMS (ESI+): calculated for [M+Na+] C35H52N2NaO4
587.3819, found 587.3849.
3.1.1. Fungal inocula
Fusarium virguliforme (Centro de Referencia de Micología Facul-
tad de Ciencias Bioquímicas y Farmacéuticas. Universidad Nacional
de Rosario N° CCC220.05) and Fusarium solani (Instituto Spegazzini,
Facultad de Ciencias Naturales y Museo, Universidad Nacional de
La Plata N° LPSC868) were cultured in 3% (w/v) malt extract-agar
médium (Oxoid Ltd., Basingstoke Hants, England) in 9 cm Petri
dishes at 25 °C and in darkness. In order to obtain the spores, fungi
was cultured for 7–10 days. Harvesting was carried out by sus-
pending spores in sterilized water (with oligoelements). Colony
The same procedure was used for the reduction of the 3-oxo
moiety in compounds 9b-i and 10, to give the corresponding
3b-hydroxy analogs.
forming unit (CFU) values was determined by plating 10 ll of
10-times serially diluted suspensions and counting germination
spores/dilution concentration.

4
F. Alonso et al. / Steroids 84 (2014) 1–6
3.1.2. Minimum inhibitory concentration by broth microdilution
Test compound were dissolved in methanol to a concentration
of 1 lg/ll. Microdilution was performed in sterile disposable
Sigmoidal dose–response curves were fitted and the CC50 values
were calculated as the concentration of the compound that caused
a 50% reduction in absorbance.
microtitre plates (96 U-bottomed wells). Every well was filled with
0,2 ml of 3% (w/v) sterile malt extract broth. Aliquots of each com-
3.2.4. Antiproliferative assay
A549 cells were seeded at a density of 104 cells/well and solu-
tions containing different concentration of the tested compound
were added in 96-well plates. After a 48 h incubation period, via-
pound were dispensed in every well ranging from 0 to 100
Compounds were tested by duplicate. Stock inoculum suspension
were adjusted to 105 CFU/ml. Ten
l were dispensed into each well,
lM.
l
except the first row. The first row of the plate contains only sterile
malt extract broth as negative control of contamination. Microtitre
plates were incubated 72 hs at 25 °C in darkness and humidity con-
trol. Germ-tube inhibition was determined by direct observation.
The minimum inhibitory concentration (MIC) was determined as
the lowest test compound concentration that completely inhibits
spore germination [12]. The maximum concentration of ethanol
used did not elicit an inhibitory effect.
bility was determined by the addition of 20 lL of MTS solution
according to the manufacturer’s instructions (CellTiter 96 AQueous
One Solution Reagent; Promega, Leiden, The Netherlands). The
absorbance at 490 nm was measured on a BioTek microplate read-
er. Results were expressed as a percentage of absorbance of treated
cell cultures with respect to untreated ones. Sigmoidal dose–
response curves were fitted and the IC50 values were calculated
as the concentration of the compound that caused a 50% reduction
in absorbance.
3.1.3. Effect on Trypanosoma cruzi
Epimastigotes of Trypanosoma cruzi CL Brener strain were
grown in a culture medium containing 33 g/L brain-heart infusion;
3 g/L tryptose; 3 g/L Na2HPO4; 0.4 g/L KCl; 0.3 g/L glucose; pH was
about 7.5, without the need for adjustment. After sterilization
(10 min at 121 °C) penicillin (100 IU/mL), streptomycin (100
mg/mL) haemin (20 mg/mL, added as 1 ml of a 2 mg/mL solution
in 0.1 N NaOH per litre of medium) and 20% v/v heat-inactivated
(45 min at 57 °C) fetal calf serum were added. Cultures were per-
formed at 28 °C in 600 ml cylindrical flasks with screw caps, con-
taining 100 ml of medium. Shaking was performed manually,
twice a day. All cultures were started with inocula taken from
exponential growth parasite in the same medium. The susceptibil-
ities of the epimastigote form of T. cruzi to synthetic compounds
were tested by culturing them in cell-free medium at 28 °C with
4. Results and discussion
The synthesis of the novel compounds is shown in Scheme 1.
The key step implies the construction of the diamide side chain
applying an Ugi condensation, one of the most versatile multicom-
ponent reactions (MCR). The Ugi four-component reaction (U-4CR),
which is based on the exceptional reactivity of the isocyanides
functional group, occurs when a carbonyl compound, an amine, a
carboxylic acid and an isocyanides react together to give an
a-aminoacylamide. The structure of the reaction product can be
easily diversified by
a systematic variation of the starting
materials.
The carboxylic component for the U-4CR was obtained as de-
picted in Scheme 1. The known intermediate 6 [10], which was ob-
tained from progesterone in three steps, was treated with methyl
iodide and potassium t-butoxide to give the C-4 dimethylated ke-
tone 7. Further hydrolysis of the methyl ester gave the desired
17b-carboxylic acid 8 with a total yield of 12% from progesterone.
Treatment of compound 8 with formaldehyde, an aromatic
amine and t-butyl isocyanide gave the desired Ugi adducts. The
the inhibitors at a 100 lM final concentration. The inhibitory effect
on T. cruzi growth was observed by reading parasites in a Neubauer
chamber every two day for seven days [13].
3.2. Effect on mammalian cells
3.2.1. Cells cultures
Simian Vero cells were grown in Eagle’s minimal essential med-
ium (MEM) supplemented with 5% inactivated fetal bovine serum
(FBS) and 50 lg/ml gentamicin (MEM 5%) and maintained after
monolayer formation in MEM 1.5%. The human cancer cell line
A549 was grown in MEM 10% and maintained after monolayer for-
mation in MEM 5%.
3.2.2. Treatment solutions
Compounds were dissolved in dimethylsulfoxide and diluted
with MEM 1.5% or MEM 5% for testing. The maximum concentra-
tion of DMSO tested (1%) exhibited no toxicity under in vitro
conditions.
3.2.3. Cytotoxicity assay
The compounds were evaluated for cytotoxicity by
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrazolium (MTS) assay. Vero or A549 cells were
seeded in 96-well plates and grown at 37 °C with 5% CO2 until
monolayer was formed. The culture medium was replaced by fresh
medium containing the compounds at various concentrations and
cells were further incubated for 24 or 48 h. Then, monolayers were
subjected to MTS-based colorimetric assay for determining cell
viability, according to the manufacturer’s instructions (CellTiter
96 AQueous One Solution Reagent; Promega, Leiden, The Nether-
lands). The absorbance at 490 nm was measured on a BioTek
microplate reader. Results were expressed as a percentage of
absorbance of treated cell cultures with respect to untreated ones.
Scheme 1. Synthesis of new 4,4-dimethyl sterol analogues. Reactives and condi-
tions: (a) t-BuOK/CH3I/THF/0 °C, 1 h. (b) KOH/water/ethylene glycol/reflux, 20 min.
(c) R-NH2/HCHO/t-BuNC/MeOH/r.t., 3 days. (d) NaBH4/CH2Cl2/MeOH/r.t., 15 min.

F. Alonso et al. / Steroids 84 (2014) 1–6
5
U-4CR generally took place smoothly and in good yields, and
worked well with a set of structurally diverse anilines (Table 1).
The reaction was completely regioselective, providing that no reac-
tion of the 3-keto moiety was observed, probably due to the much
higher reactivity of formaldehyde as the carbonylic component.
Nevertheless all reactions gave a small amount of compound 10,
a by-product coming from the Passerini condensation [14].
Following this procedure a set of nine compounds was obtained
(9a–i), which were treated with sodium borohydryde to give the
corresponding 3b-hydroxy compounds 5a–i, the target sterol ana-
logues. Finally, reduction of compound 10 gave the analogue 11
with almost quantitative yield.
new compounds and allowed the determination of their structure
unambiguously.
Compounds both having 3-keto and 3b-hydroxy moieties were
tested in vitro for their inhibitory properties towards Fusarium
virguliforme, the causal agent of sudden death syndrome in soy
bean, and Fusarium solani, that produces root rots on various crops,
but is also involved in human mycoses [15]. Most of the new com-
pounds showed a measurable antifungal activity against either of
the two fungi. The results, expressed as their minimum inhibitory
concentration (MIC) are shown in Table 2. Values for compounds 3
and 4, non-methylated at C4 and previously reported, were
included for comparison.
The structures of intermediates 7, 8 and some representative
members of the library were fully characterized by NMR spectros-
copy. On the other hand, granted that the structures of the com-
pounds are closely related, and in order to obtain a complete and
unambiguous characterization of the library, we decided to per-
form a direct analysis of the purified steroids via HRESI MS. In all
cases, main peaks were observed for [M+H]+ and [M+Na]+. Fig. 3
shows a representative example spectrum for compound 9c.
The molecular formula was shown to be C34H48N2O3 on the ba-
sis of its HRMS ESI (m/z 533.3739 [M+H]+ and 555.3562 [M + Na]+).
In all cases a product ion resulting from the loss of t-butylamine
from the side chain was observed (structure A, in this example
m/z 460.2835). The structure of the side chain was revealed by
ESI MS/MS. For example, in the case of 9c, the ESI MS/MS of the
A
ion, used as
a precursor, yielded the products ions m/z
327.2297, 106.0639 and 299.2335, which can be assigned to struc-
´
tures B, B and C respectively. Fragments B and C are common to all
´
the compounds analyzed, whereas ions A and B are characteristic
for each particular structure because their contain the moiety com-
ing from each different amine. In addition, ions showing water loss
were observed for the 3b-hydroxy analogs 5a–i (Supplementary
material). The analysis described above was applied to all of the
Table 1
Yields of Ugi reactions.
R-NH2
Yield (%)a
63
9a
9b
MeO
NH2
71
Cl
NH2
9c
84
87
NH2
9d
Me
NH2
9e
9f
61
69
55
Cl
NH2
F
NH2
Me
9g
NH2
Et
F
9h
9i
89
83
NH2
NH2
a
Isolated yields.
Fig. 3. Representative example for the structural determination by HRMS.

6
F. Alonso et al. / Steroids 84 (2014) 1–6
Table 2
compounds against Trypanosoma cruzi, the ethiological agent of
Biological activity of the new 4,4-dimethyl sterol analogues.
´
Chagas disease [16]. Unfortunately, none of these analogs exerted
b
F. solani MICa
F. virguliforme MICa
Vero cells CC50
a significant inhibitory effect (at a 100
gote form of the parasite.
lM dose) on the epimasti-
9a
9b
9c
9d
9e
9f
9g
9h
9i
5a
5b
5c
5d
5e
5f
5g
5h
5i
27
35
96
37
88
46
52
26
18
18
35
>100
46
35
>100
>100
17
>100
44
48
92
88
>200
>200
>200
>200
59
In conclusion, in this paper we have confirmed that the applica-
tion of multicomponent reactions in the synthetic chemistry of ste-
roids is a powerful tool that allows a rapid and efficient generation
of collections of diverse compounds. On the other hand, we have
shown that the 4,4-dimethyl substitution, not fully explored in
previous research, may render novel synthetic analogs with a vari-
ety of biological effects, such as antifungal and cytotoxic activities.
Encouraged by these findings, we are currently performing further
studies in order to develop both structure-activity relationships
and to gain insight into the mechanism of action of the new com-
pounds in relevant biological systems.
93
65
61
>100
>100
>100
>100
88
96
92
44
>100
>100
>100
91
>200
85
>200
>200
92
>200
88
65
87
>200
91
Acknowledgements
36
This work was supported by grants from Universidad de Buenos
Aires (UBACyT X-084) and CONICET (PIP 0315). We are grateful to
UMYMFOR (UBA-CONICET) for the analytical and spectroscopic
determinations.
10
11
3
55
110
46
55
55
28
50
14
>200
>200
>200
4
50
a
Micromolar concentration. Mean results from duplicates. The MIC of the control
(Benomyl, a commercial antifungal) is 9 M for both species.
l
Appendix A. Supplementary data
b
Micromolar concentration. Mean results from duplicates.
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.steroids.
2014.03.002.
Firstly, methylation at C-4 seems to conserve, or even reduce,
the antifungal activity (compare compounds 5b and 5c vs. their
non-methylated counterparts 4 and 3 respectively). Nevertheless,
some compounds show a higher activity than that reported previ-
ously by us [9].
In sight of these findings, we decided to evaluate the effect of
the new analogs on the viability of mammalian cells, providing
that any compound with a potential use as an antifungal agent
should exert a selective toxicity. Thus, the in vitro cytotoxic con-
centration 50% (CC50) was determined on Vero cells; the results
are shown in Table 2. It can be seen that several of the new com-
pounds show a selective toxicity towards fungi, with no effect on
Vero cells at the maximum concentration tested.
Moreover, two of the compounds (9h and 5h) having a naphthyl
subtituent in the side chain, resulted toxic only against F. solani and
not against F. virguliforme or Vero cells, suggesting some selectivity
on their action.
On the other hand, compound 10, the by-product from the
Passerini reaction, showed a noteworthy toxic effect on Vero cells,
about five-fold higher than the rest of the analogs coming from the
Ugi condensations, suggesting that it might have cytotoxic proper-
ties. Thus, as a preliminary study, we tested its action on the A549
cells, an adenocarcinomic human epithelial cell line. Even though
compound 10 seems not to be quite toxic on this line (CC50 about
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