Synthesis of conjugated bile acids/azastilbenes as potential antioxidant and photoprotective agents

Article abstract of DOI:10.1016/j.steroids.2015.03.009

A series of 14 bile acids/azastilbenes conjugates (1a-g and 2a-g) was prepared through the condensation of bile amides (1 and 2) and aromatic aldehydes. The newly synthesized conjugates were evaluated in vitro for their antioxidant and photoprotective activities. Six compounds (1, 1a, 1b, 2, 2a and 2b) showed promising antioxidant activity with IC₅₀ values of 19.60-31.83 μg mL^-1. The synthesized compounds presented a varied photoprotection profile, with the SPF ranging from 2 to 9. Among the 16 compounds tested for the protection against UVB sunrays, 3 compounds (2c, 2e and 2g) presented more significant protection than resveratrol and the free azastilbene 3; while the UVAPF increased from 2 in resveratrol and 5 in 3 to 5-11 in the majority of the conjugates.

Full text of DOI:10.1016/j.steroids.2015.03.009

Steroids 98 (2015) 114–121
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis of conjugated bile acids/azastilbenes as potential antioxidant
and photoprotective agents
Juliana Alves dos Santos , Hudson Caetano Polonini b,c, Érika Yoko Suzuki , Nádia R.B. Raposo ,
Adilson David da Silva
a
a,
⇑
b
b
a
Departamento de Química, Universidade Federal de Juiz de Fora, Campus Universitário, Juiz de Fora 36036-900, Brazil
NUPICS Núcleo de Pesquisa e Inovação em Ciências da Saúde, Universidade Federal de Juiz de Fora, Juiz de Fora, MG 36036-900, Brazil
Chemical and Food Engineering Department, Federal University of Santa Catarina, Florianópolis, SC 88040-090, Brazil
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 14 bile acids/azastilbenes conjugates (1a–g and 2a–g) was prepared through the condensation
of bile amides (1 and 2) and aromatic aldehydes. The newly synthesized conjugates were evaluated
Received 24 October 2014
Received in revised form 9 February 2015
Accepted 12 March 2015
in vitro for their antioxidant and photoprotective activities. Six compounds (1, 1a, 1b, 2, 2a and 2b)
ꢀ1
showed promising antioxidant activity with IC50 values of 19.60–31.83
l
g mL . The synthesized
Available online 23 March 2015
compounds presented a varied photoprotection profile, with the SPF ranging from 2 to 9. Among the
6 compounds tested for the protection against UVB sunrays, 3 compounds (2c, 2e and 2g) presented
more significant protection than resveratrol and the free azastilbene 3; while the UVAPF increased from
in resveratrol and 5 in 3 to 5–11 in the majority of the conjugates.
1
Keywords:
Bile acids
Azastilbenes
Schiff bases
2
Ó 2015 Published by Elsevier Inc.
Antioxidant activity
Photoprotective activity
1
. Introduction
Steroids are natural compounds with several important
the liver and also improving the intestinal absorption of poorly
or non absorbed drugs [2,12].
Within this context, a number of works [13–21] described the
conjugation of active substances with bile derivatives through
chemical modifications at the 3- and 24-positions of the sterol
nucleus. For instance, Pore et al. [17] designed novel fluconazole/bile
acid conjugates and the new molecules showed good antifungal
activity against Candida species. Tolle-Sander et al. [19], in turn,
described increased acyclovir oral bioavailability (from 20% to
54%) through a bile acid conjugate: the antiviral drug was
conjugated with chenodeoxycholane acid via a valine linker.
Our research group has previously described the conjugation of
biliar acid with other bioactive molecules, such as thiopurine [20]
and aminoquinoline [21], and the results highlight the importance
of steroids as carriers.
In this work, we focused on the conjugation of these steroids
with azastilbenes. Stilbenes are a class of plant polyphenols pro-
duced in a number of unrelated plant species. They are generally
known as phytoalexins, which can be biosynthesized in response
to pathogen or herbivore attacks [22]. These compounds are of
noticeable interest for medicinal chemistry because of their diverse
biological activities [23]; resveratrol, a potent antioxidant agent, is
the major stilbene present in grapes and wines. Particularly,
azastilbenes comprise a special group of stilbenes derivatives
that possess biological properties such as antioxidant [24,25],
applications in many fields of research, such as pharmacology,
biomimetic and supramolecular chemistry, and nanotechnology
[
(
1]. In particular, bile acids such as cholic and deoxycholic acids
Fig. 1) have been considered quite useful in the preparation of
new pharmaceutic drugs due to their chemical and biological
properties [2,3]. They are end products of cholesterol metabolism
in the liver and exhibit a facially amphiphilic nature, due to the
polar hydroxyl groups and non-polar hydrocarbon body.
The literature describes a vast amount of pharmacological
applications for bile acids derivatives, notably antimicrobial
[
4–6], antifungal [4,7,8], carbonic anhydrase inhibition [9,10],
and antitumor [11]. Yet, one of the most promising applications
of bile acid derivatives is as drug carriers, a direct consequence
of their amphiphilic character. They have already been reported
to improve the permeability of cell membranes, including the
bacterial wall [2,4]. The high specificity and capacity of bile acid
transport systems during their entero-hepatic circulation allows
the use of drug-bile acid conjugates for specific drug targeting to
⇑
Corresponding author. Tel.: +55 21023310; fax.: +55 21023314.
E-mail address: julianalves07@yahoo.com.br (J.A.d. Santos).
http://dx.doi.org/10.1016/j.steroids.2015.03.009
039-128X/Ó 2015 Published by Elsevier Inc.
0

J.A.d. Santos et al. / Steroids 98 (2015) 114–121
115
OH
H
21
o
2
4
m
m
OH
O
OH
12
1
8
24
24
O
o' m'
NH
N
O
OH
23
p'
1
9
OH
o
3
H
H
R
3
o'
R
m'
HO
3
7
H
OH
R= OH (cholic) R= H (deoxycholic)
HO
R
H
Fig. 3. Structure and numbering of bile acids/azastilbenes analogues conjugates
and bile amides.
Fig. 1. Chemical structures of cholic and deoxycholic acids.
antibacterial [26,27], antimicrobial [28,29], antifungal [30], and
antiproliferative [31]. In a recent effort, our research group has
been reporting the antileishmanial [32], depigmenting [33] and
photoprotective [34] activities of azastilbenes. In these reports,
the azastilbenes are presented as bioisosters of stilbene since
may be seen as resulting from the substitution of the central
C@C linkage with a C@N double bond (Fig. 2).
In this work we synthesized and characterized new bile acids/
azastilbenes conjugates, in which the azastilbenes have been built
at carboxylic acid C-24 at cholic and deoxycholic acids through the
intermediates amides 1 and 2.
0
(
1
.80 (s, 3H, 19-CH
m, 22H, CH and CH skeleton); 2.16 (m, 2H, 23-CH
H, H-3b); 3.61 (br s, 1H, H-7b); 3.79 (br s, 1H, H-12b); 4.01
3
); 0.95 (d, 3H, 21-CH
3
, J = 5.4); 1.29–1.77
2
2
); 3.17 (br s,
(
br s, 1H, 7-OH); 4.10 (br s, 1H, 12-OH); 4.34 (br s, 1H, 3-OH);
4
2
.82 (br s, 2H, ANH
H-o, J = 6.0); 9.42 (s, 1H, ANH). C-NMR (75 MHz, DMSO-d
2
); 6.47 (d, 2H, 2H-m, J = 6.0); 7.19 (d, 2H,
13
6
), d
(
ppm): 12.4 (C-18); 17.2 (C-21); 22.6 (C-19); 22.8–46.2 (CH and
CH skeleton); 66.2 (C-7); 70.4 (C-3); 71.0 (C-12); 113.8 (2C-m);
20.8 (2C-o); 128.7 (CarANH); 144.4 (C-p); 170.8 (C-24). HRMS
2
1
+
(
47 2 4
LC-MS): calcd. for [M + H] (C30H N O ) requires m/z 499.3536,
found m/z 499.3550.
(
R)-N-(4-aminophenyl)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-
,12-dihydroxy-10,13-dimethylhexadecahydro-1H-
cyclopenta[ ]phenanthren-17-yl)pentanamide (2): The bile amide 2
was obtained as a lilac solid. Yield: 44%. M.p.: 134.4–136.0 °C. I.R.
3
2
. Experimental
a
2.1. General
ꢀ1 1
(
(
(
KBr)
300 MHz, DMSO-d
s, 3H, 19-C ); 0.96 (d, 3H, 21-CH
v
: 3433, 3398, 2933, 2864, 1650, 1515, 1512 cm . H-NMR
), d (ppm), J (Hz): 0.59 (s, 3H, 18-CH ); 0.84
, J = 5.4); 1.19–1.79 (m, 23H,
6
3
Melting points (M.p.) were determined using a Microquímica
3
3
MQAPF-301 digital apparatus and are uncorrected. Infrared spectra
ꢀ1
CH2 and CH skeleton); 2.18 (m, 2H, 23-CH ); 3.80 (s, 1H, H-12b);
2
(
wave numbers in cm ) were recorded on a Schimadzu 8400
1
4.22 (s, 1H, 12-OH); 4.50 (s,1H, 3-OH); 4.84 (br s, 2H, ANH );
2
series FTIR instrument. H NMR spectra were recorded on a
1
3
6.47 (d, 2H, J = 6.0, 2H-m); 7.19 (d, 2H, J = 6.0, 2H-o); 9,42 (s, 1H,
Bruker AC-300 and C NMR spectra were recorded on a Bruker
AC-300 at 75 MHz. The chemical shifts (d) are given in parts per
million relative to tetramethylsilane (TMS). Spectra were acquired
1
3
ANH). C-NMR (75 MHz, DMSO-d
6
), d (ppm): 12.0 (C-18); 17.0
(
C-21); 23.0 (C-19); 26.9–46.2 (CH and CH skeleton); 47.4 (C-7);
2
6
9.9 (C-3); 71.0 (C-12); 113.8 (2C-m); 120.8 (2C-o); 128.7
in DMSO-d
6
for all compounds. Column chromatography was
(
[
C
arANH); 144.4 (C-p); 170.7 (C-24). HRMS (LC-MS): calcd. for
performed on Merck silica gel (70–230 mesh). All reagents used
were of analytical reagent grade. High resolution Mass spectra
+
M + H] (C30
H
47
N
2
O
3
) requires m/z 483.3587, found m/z 483.3618.
(
HRMS) were recorded in Bruker Daltonics – micrOTOF.
2
.2.2. General procedure for the synthesis of bile acids/azastilbenes
2
2
.2. Chemistry
conjugates (1a–g and 2a–g)
Bile amide (0.100 g; 0.20 mmol of amide 1 or 0.21 mmol of
amide 2) was dissolved in 3 mL of methanol and then, under con-
stant stirring, 1 M equivalent of an aromatic aldehyde was added.
The mixture was kept at room temperature and after a short time
interval (5 min–10 h) the formation of a precipitate was noticed.
The reaction medium was then filtered and the precipitate was
washed with methanol and dried in an oven. All compounds of this
class were obtained as colored solid.
.2.1. General procedure for the synthesis of amides 1 and 2
Bile acid (4.89 mmol of cholic or deoxycholic acid) was dis-
solved in 70 mL of tetrahydrofuran (THF). To this solution 1,4-phe-
nylenediamine (4.89 mmol) and dicyclohexylcarbodiimide (DCC)
(
5.07 mmol) were added. The mixture was stirred for 24 h, when
the formations of the desired bile acid amides were found by thin
layer chromatography (TLC). Ethyl acetate was then added in order
to promote the precipitation of the byproduct N,N -dicyclohexy-
lurea, which was filtered off. The solvents were evaporated and
the solid crude obtained was chromatographed on silica gel using
a mixture of dichloromethane/methanol as eluent.
The NMR peaks were assigned according to the numbered gen-
eral structure (Fig. 3).
0
2-((E)-(4-((R)-4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-tri-
hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[ ]phenanth-
ren-17-yl)pentanamido)phenylimino)methyl)benzoic acid (1a): The
compound 1a was obtained as a beige solid. Yield: 100%. M.p.:
1
a
75.5–177.0 °C. I.R. (KBr)
v
: 3348, 2931, 2860, 1747, 1660, 1523,
H-NMR (300 MHz, DMSO-d ), d (ppm), J (Hz): 0.59
); 0.80 (s, 3H, 19-CH ); 0.97 (d, 3H, 21-CH , J = 5.4);
.33–2.00 (m, 22H, CH and CH skeleton); 2.17 (m, 2H, 23-CH );
.17 (br s, 2H, H-3b); 3.61 (br s, 1H, H-7b); 3.81(br s, 1H, H-12b);
ꢀ1 1
1
465 cm
.
6
(
R)-N-(4-aminophenyl)-4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-
,7,12-trihydroxy-10,13-dimethylhexadecahydro-1H-
cyclopenta[ ]phenanthren-17-yl)pentanamide (1): The bile amide 1
was obtained as a gray solid. Yield: 41%. M.p.: 157.8–158.3 °C. I.R.
(
s, 3H, 18-CH
3
3
3
3
1
3
2
2
a
ꢀ1
4.04 (br s, 1H, 7-OH); 4.14 (br s, 1H, 12-OH); 4.37 (br s, 1H, 3-
OH); 6.88 (br s, 2H, 2H-m); 7.14 (br s, 2H, 2H-o); 7.44 (br s, 2H,
H-o and H-m ); 7.70 (m, 2H, H-m and H-p ); 7.88 (br s, 2H,
(
KBr) v: 3406, 2935, 2866, 1654, 1541, 1515, 1431, 1251 cm .
1
H-NMR (300 MHz, DMSO-d
6
3
), d (ppm), J (Hz): 0.59 (s, 3H, 18-CH );
0
0
0
0
1
3
Bioisosterism
N
HC@N and ACOOH); 9.68 (s, 1H, ANH).
DMSO-d ), d (ppm):13.0 (C-18); 17.9 (C-21); 23.3 (C-19); 23.9–
6.5 (CH and CH skeleton); 66.8 (C-7); 70.9 (C-3); 71.5 (C-12);
15.0 (2C-o); 120.9 (2C-m); 124.5 (C-m ); 125.1 (C-o ); 130.9 (C-
C-NMR (75 MHz,
6
5
1
2
0
0
Natural stilbene
Azastilbene
0
0
0
p ); 132.3 (C-o ACOOH); 134.7 (C-m ); 141.2 (CarACH@N); 145.1
(CarANH); 146.3 (CarAN@CH); 169.5 (HC@N); 171.7 (C-24).
Fig. 2. Comparison of the basic structures of stilbene and azastilbene skeletons.

1
16
J.A.d. Santos et al. / Steroids 98 (2015) 114–121
HRMS (LC-MS): calcd. for [M + H]+ (C38
31.3747, found m/z 631.3750.
-((E)-(4-((R)-4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-tri-
hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[ ]phenan-
H
51
N
2
O
6
) requires m/z
M.p.: 204.2–205.5 °C. I.R. (KBr)
v
: 3442, 2921, 2866, 1666, 1616,
H-NMR (300 MHz, DMSO-d ), d (ppm), J (Hz): 0.59
(s, 3H, 18-CH ); 0.80 (s, 3H, 19-CH ); 0.98 (d, 3H, 21-CH , J = 5.4);
1.20–1.98 (m, 22H, CH and CH skeleton); 2.31 (m, 2H, 23-CH );
ꢀ
1 1
6
1535 cm
.
6
4
3
3
3
a
2
2
thren-17-yl)pentanamido)phenylimino)methyl)benzoicacid (1b): The
compound 1b was obtained as a yellow solid. Yield: 71%. M.p.:
3.16 (br s, 2H, H-3b and AOH); 3.61 (br s, 1H, H-7b); 3.80 (br s,
1H, H-12b); 4.04 (br s, 1H, 7-OH, 4.14 (s, 2H, 12-OH and AOH);
0
0
3
1
0
38.0–339.5 °C. I.R. (KBr)
v
: 3350, 2921, 2867, 1699, 1672, 1598,
H-NMR (300 MHz, DMSO-d ), d (ppm), J (Hz):
); 0.79 (s, 3H, 19-CH ); 0.97 (d, 3H, 21-CH
and CH skeleton); 2.19 (m, 2H,
); 3.17 (br s, 1H, H-3b); 3.60 (br s, 1H, H-7b); 3.79 (br s,
H, H-12b); 4.02 (br s, 1H, 7-OH); 4.13 (br s, 1H, 12-OH); 4.36
4.36 (s, 1H, 3-OH); 6.28 (s, 1H, H-m ); 6.38 (d, 1H, H-m , J = 6.6);
ꢀ1
1
0
537, 1510 cm
.
6
7.30 (d, 2H, 2H-m, J = 8.8); 7.39 (d, 1H, H-o , J = 8.8); 7.64 (d, 2H,
.58 (s, 3H, 18-CH
3
3
3
,
2H-o, J = 8.5); 8.77 (s, 1H, HC@N); 9.97 (s, 1H, ANH); 10.23 (s,
1
3
J = 5.4); 1.33–2.00 (m, 22H, CH
2
6
1H, AOH). C-NMR (75 MHz, DMSO-d ), d (ppm): 12.5 (C-18);
2
3-CH
2
17.1 (C-21); 22.6 (C-19); 22.8–46.1 (CH and CH
2
skeleton); 66.2
0
1
(C-7); 70.4 (C-3); 71.0 (C-12); 102.4 (C-m between AOH groups);
0
(
8
br s, 1H, 3-OH); 7.32 (br s, 2H, 2H-m); 7.67 (br s, 2H, 2H-o);
107.7 (C-m ); 112.1 (CarACH@N); 119.8 (2C-o); 121.3 (2C-m);
0
0
0
.03 (s, 4H, 2H-o and 2H-m ); 8.73 (s, 1H, HC@N); 9.99 (s, 1H,
134.2 (C-o ); 137.9 (CarANH); 142.7 (C-p); 161.1 (HC@N), 162.2
1
3
0
0
ANH). C-NMR (75 MHz, DMSO-d
6
), d (ppm): 12.3 (C-18); 17.2
C-21); 22.6 (C-19); 26.2–46.1 (CH and CH skeleton); 66.2 (C-7);
0.4 (C-3); 71.0 (C-12); 119.6 (2C-o); 121.8 (2C-m); 128.4
(C-p AOH); 162.9 (C-o AOH); 171.7 (C-24). HRMS (LC-MS): calcd.
+
(
7
2
for [M + H] (C37
619.3765.
51 2 6
H N O ) requires m/z 619.3747, found m/z
0
0
0
(
(
(
2C-o ); 129.7 (2C-m ); 132.7 (C-p ); 138.3 (Car-NH); 139.9
C
C-24). HRMS (LC-MS): calcd. for [M + H] (C38
(R)-N-(4-((E)-4-nitrobenzylideneamino)phenyl)-4-((3R,5S,7R,8R,
9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13-dimethylhexadec-
ar-CH@H); 145.4 (C-p); 157.9 (HC@N); 166.9 (COOH); 171.8
+
51
H N
2
O
6
) requires
ahydro-1H-cyclopenta[
compound 1f was obtained as a yellow solid. Yield: 81%. M.p.:
223.5–224.3 °C. I.R. (KBr) : 3463, 2970, 2923, 2866, 1633, 1598,
H-NMR (300 MHz, DMSO-d ), d (ppm), J (Hz):
); 0.79 (s, 3H, 19-CH ); 0.98 (d, 3H, 21-CH
J = 5.4); 1.20–2.36 (m, 24H, 23-CH , CH and CH skeleton); 3.17
a]phenanthren-17-yl)pentanamide (1f): The
m/z 631.3747, found m/z 631.3747.
R)-N-(4-((E)-5-bromo-2-hydroxybenzylideneamino)phenyl)-4-
(3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13-
dimethylhexadecahydro-1H-cyclopenta[ ]phenanthren-17-yl)pen-
tanamide (1c): The compound 1c was obtained as a yellow solid.
Yield: 66%. M.p.: 301.5–303.0 °C. I.R. (KBr) : 3456, 3317, 2925,
H-NMR (300 MHz, DMSO-
), d (ppm), J (Hz): 0.59 (s, 3H, 18-CH ); 0.80 (s, 3H, 19-CH );
.97 (d, 3H, 21-CH , J = 5.4); 1.33–2.00 (m, 22H, CH and CH skele-
); 3.17 (m, 1H, H-3b); 3.60 (br s, 1H, H-
b); 3.79 (br s, 1H, H-12b); 4.01 (d, 1H, 7-OH, J = 3,0); 4.12 (d,
(
v
ꢀ
1 1
(
1515, 1344 cm
.
6
a
0.59 (s, 3H, 18-CH
3
3
3
,
2
2
v
(br s, 1H, H-3b); 3.61 (br s, 1H, H-7b); 3.79 (s, 1H, H-12b); 4.04
(br s, 1H, 7-OH); 4.14 (br s, 1H, 12-OH); 4.34 (d, 1H, 3-OH,
J = 3.8); 7.36 (d, 2H, 2H-m, J = 8.2); 7.68 (d, 2H, 2H-o, J = 7.7);
ꢀ1 1
2
866, 1644, 1600, 1539, 1508 cm .
d
6
3
3
0
0
0
3
2
8.15 (d, 2H, 2H-o , J = 8.8); 8.34 (d, 2H, 2H-m , J = 8.5); 8.82 (s,
1
3
ton); 2.27 (m, 2H, 23-CH
2
1H, HC@N); 10.02 (s, 1H, ANH). C-NMR (75 MHz, DMSO-d
(ppm): 12.3 (C-18); 17.1 (C-21); 22.6 (C-19); 22.8–46.1 (CH and
CH skeleton); 66.2 (C-7); 70.4 (C-3); 71.0 (C-12); 119.6 (2C-o);
122.0 (2C-m); 124.0 (2C-m ); 129.4 (2C-o ); 138.8 (CarANH);
141.8 (CarACH@N); 144.9 (C-p); 148.6 (C-p ); 156.8 (HC@N);
6
), d
7
1
0
H, 12-OH, J = 3.0); 4.33 (d, 1H, 3-OH, J = 3.0); 6.92 (d, 1H, H-m ,
2
0
0
0
J = 8.8); 7.38 (d, 2H, 2H-m, J = 8.5); 7.52 (dd, 1H, H-p , J = 8.6,
0
0
J = 2.5); 7.68 (d, 2H, 2H-o, J = 8.8); 7.82 (d, 1H, H-o , J = 2.2); 8.92
s, 1H, HC@N); 10.0 (s, 1H, ANH). 13C-NMR (75 MHz, DMSO-d
), d
ppm): 12.3 (C-18); 17.2 (C-21); 22.6 (C-19); 22.8–46.1 (CH and
171.8 (C-24). HRMS (LC-MS): calcd. for [M + H] (C37
+
(
(
6
50
H N
3
O
6
)
requires m/z 632.3700, found m/z 632.3720.
CH
2
skeleton); 66.3 (C-7); 70.5 (C-3); 71.0 (C-12); 109.8
(R)-N-(4-((E)-2-nitrobenzylideneamino)phenyl)-4-((3R,5S,7R,8R,
9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13-dimethylhexadec-
0
0
(
C-m -Br); 119.0 (C-m ); 119.8 (CarACH@N); 121.3 (2C-o); 121.9
0
0
(
1
2C-m); 133.8 (C-o ); 135.1 (C-p ); 138.9 (CarANH); 142.1 (C-p);
59.3 (HC@N), 160.1 (CarAOH); 171.9 (C-24). HRMS (LC-MS): calcd.
ahydro-1H-cyclopenta[
compound 1g was obtained as a yellow solid. Yield: 55%. M.p.:
285.9–287.0 °C. I.R. (KBr) : 3404, 2925, 2864, 1668, 1593, 1409,
H-NMR (300 MHz, DMSO-d ), d (ppm), J (Hz): 0.59
(s, 3H, 18-CH ); 0.80 (s, 3H, 19-CH ); 0.98 (d, 3H, 21-CH , J = 5.4);
1.25–2.33 (m, 22H, CH and CH skeleton); 3.20 (br s, 1H, H-3b);
a]phenanthren-17-yl)pentanamide (1g): The
+
for [M + H] (C37
81.2941.
R)-N-(4-((E)-4-(dimethylamino)benzylideneamino)phenyl)-4-
(3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13-
dimethylhexadecahydro-1H-cyclopenta[ ]phenanthren-17-
yl)pentanamide (1d): The compound 1d was obtained as a yellow
solid. Yield: 79%. M.p.: 268.4–269.3 °C. I.R. (KBr) : 3433, 2933,
H-NMR (300 MHz,
), d (ppm), J (Hz): 0.59 (s, 3H, 18-CH ); 0.80 (s, 3H,
, J = 5.4); 1.33–2.22 (m, 22H, CH
and CH skeleton); 2.33 (m, 2H, 23-CH ); 2.99 (s, 6H, N(CH );
.20 (br s, 1H, H-3b); 3.60 (br s, 1H, H-7b); 3.80 (br s, 1H,
H-12b); 4.04 (br s, 1H, 7-OH); 4.13 (br s, 1H, 12-OH); 4.37 (d,1H,
2
H50BrN O
5
) requires m/z 681.2903, found m/z
v
ꢀ
1 1
6
1340 cm
.
6
(
3
3
3
(
2
a
3.62 (br s, 1H, H-7b); 3.81 (br s, 1H, H-12b); 4.02 (br s, 1H, 7-
OH); 4.12 (br s, 1H, 12-OH); 4.33 (br s, 1H, 3-OH); 7.29 (d, 2H,
0
v
2H-m, J = 8.5); 7.67 (d, 2H, 2H-o, J = 8.5); 7.80 (m, 2H, H-m and
ꢀ1
1
0
0
0
2
866, 1662, 1608, 1598, 1533, 1504 cm
.
H-p ); 8.09 (d, 1H, H-o , J = 7.9); 8.17 (d, 1H, H-m -NO
2
, J = 7.1);
C-NMR (75 MHz,
), d (ppm): 12.4 (C-18); 17.2 (C-21); 22.6 (C-19); 22.8–
46.1 (CH and CH skeleton); 66.3 (C-7); 70.5 (C-3); 71.0 (C-12);
119.7 (2C-o); 121.9 (2C-m); 124.5 (C-m ); 129.4 (C-o ); 130.2 (C-
1
3
DMSO-d
6
3
8.87 (s, 1H, HC@N); 10.00 (s, 1H, ANH).
DMSO-d
1
9-CH
3
); 0.98 (d, 3H, 21-CH
3
2
6
2
3
)
2
2
0
0
3
0
0
0
p ); 131.7 (C-o ANO
2
); 133.7 (C-m ); 138.6 (CarACH@N); 145.3
0
0
3
-OH, J = 3.8); 6.76 (d, 2H, H-m , J = 8.2); 7.15 (d, 2H, 2H-o ,
(CarANH); 149.2 (CarAN@CH); 154.8 (HC@N); 171.8 (C-24).
+
J = 8.5); 7.59 (d, 2H, 2H-m, J = 8.5); 7.71 (d, 2H, 2H-o, J = 8.5);
50 3 6
HRMS (LC-MS): calcd. for [M + H] (C37H N O ) requires m/z
13
8
.40 (s, 1H, HC@N); 9.89 (s, 1H, ANH).
C-NMR (75 MHz,
632.3700, found m/z 632.3723.
DMSO-d ), d (ppm): 12.8 (C-18); 17.6 (C-21); 23.1 (C-19); 23.3–
6
2-((E)-(4-((R)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-dihy-
droxy-10,13-dimethylhexadecahydro-1H-cyclopenta[ ]phenan-
thren-17-yl)pentanamido)phenylimino)methyl)benzoicacid (2a):
4
6.6 (CH and CH
2
skeleton); 66.7 (C-7); 70.9 (C-3); 71.5 (C-12);
a
0
111.9 (2C-m ); 120.2 (2C-o); 121.6 (2C-m); 124.5 (Car-CH@N); 130.5
0
(2C-o ); 137.3 (CarANH); 147.5 (CarAN@CH); 152.7 (CarAN(CH
3
)
2
);
The compound 2a was obtained as a beige solid. Yield: 38%. M.p.:
+
1
58.9 (HC@N); 172.0 (C-24). HRMS (LC-MS): calcd. for [M + H]
) requires m/z 630.4271, found m/z 630.4296.
R)-N-(4-((E)-2,4-dihydroxybenzylideneamino)phenyl)-4-((3R,5S,
R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13-dimethylhex-
]phenanthren-17-yl)pentanamide (1e):
The compound 1e was obtained as a yellow solid. Yield: 80%.
163.5–165.0 °C. I.R. (KBr)
v
: 3406, 2931, 2858, 1747, 1664, 1612,
H-NMR (300 MHz, DMSO-d ), d (ppm), J (Hz): 0.61
(s, 3H, 18-CH ); 0.85 (s, 3H, 19-CH ); 0.98 (d, 3H, 21-CH , J = 5.4);
1.20–1.79 (m, 23H, CH and CH skeleton); 2.22 (m, 2H, 23-CH );
3.81 (s, 1H, H-12b); 4.22 (s, 1H, 12-OH); 4.48 (s, 1H, 3-OH); 6.90
ꢀ
1 1
(C
39
H
56
N
3
O
4
1517 cm
.
6
(
3
3
3
7
2
2
adecahydro-1H-cyclopenta[
a
0
0
(br s, 2H, 2H-m); 7.14 (br s, 2H, 2H-o); 7.44 (d, 2H, 1H-o , 1H-m ,

J.A.d. Santos et al. / Steroids 98 (2015) 114–121
117
0
0
J = 6.0); 7.70 (m, 2H, 1H-m and 1H-p ); 7.86 (m, 2H, HC@N and
(R)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-dihydroxy-10,13-
dimethylhexadecahydro-1H-cyclopenta[ ]phenanthren-17-yl)-N-(4-
((E)-2,4-dihydroxybenzylideneamino)phenyl)pentanamide (2e): The
13
COOH); 9.66 (s, 1H, ANH).
ppm):12.4 (C-18); 17.1 (C-21); 23.1 (C-19); 23.5–47.5 (CH and
CH skeleton); 69.9 (C-3); 71.0 (C-12); 114.6 (2C-o); 120.8 (2C-
m); 124.1 (C-m ); 124.7 (C-o ); 130.8 (C-p ); 131.9 (C-o ACOOH);
34.1 (C-m ); 140.8 (CarACH@N); 144.0 (CarANH); 145.8
C-NMR (75 MHz, DMSO-d
6
), d
a
(
2
compound 2e was obtained as a yellow solid. Yield: 86%. M.p.:
0
0
0
0
185.5–187.7 °C. I.R. (KBr)
v
: 3438, 2929, 2864, 1622, 1515,
H-NMR (300 MHz, DMSO-d ), d (ppm), J (Hz): 0.60
(s, 3H, 18-CH ); 0.84 (s, 3H, 19-CH ); 0.96 (d, 3H, 21-CH , J = 5.4);
1.18–1.78 (m, 23H, CH and CH skeleton); 2.23 (m, 2H, 23-CH );
0
ꢀ1 1
1
1458 cm
.
6
(
C
arAN@CH); 169.0 (HC = N); 171.1 (C-24). HRMS (LC-MS): calcd.
3
3
3
+
for [M + H] (C38
H
51
N
O
2 5
) requires m/z 615.3798, found m/z
2
2
6
15.3802.
-((E)-(4-((R)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-dihy-
droxy-10,13-dimethylhexadecahydro-1H-cyclopenta[ ]phenanth-
3.17 (br s, 1H, H-3b); 3.80 (br s, 1H, H-12b); 4.23 (br s, 1H, 12-
0
4
OH); 4.49 (br s, 1H, 3-OH); 6.28 (br s, 1H, H-m between AOH
0
a
groups); 6.38 (br s, 1H, H-m ); 7.30 (br s, 2H, 2H-m); 7.39 (br s,
0
ren-17-yl)pentanamido)phenylimino)methyl)benzoicacid (2b): The
compound 2b was obtained as a yellow solid. Yield: 76%. M.p.:
1H, H-o ); 7.63 (d, 2H, 2H-o); 8.78 (s, 1H, HC@N); 9.97 (br s, 1H,
1
3
6
ANH); 10.23 (br s, 1H, AOH). C-NMR (75 MHz, DMSO-d ), d
3
1
20.1–321.0 °C. I.R. (KBr)
v
: 3353, 2925, 2864, 1708, 1676, 1623,
(ppm): 17.1 (C-18); 23.1 (C-21); 23.5 (C-19); 26.1–46.2 (CH and
ꢀ1
1
0
598, 1535, 1508 cm
.
H-NMR (300 MHz, DMSO-d
); 0.84 (s, 3H, 19-CH ); 0.96 (d, 3H,
, J = 6.3); 1.14–1.79 (m, 23H, CH and CH skeleton); 2.24
m, 2H, 23-CH ); 3.16 (br s, 1H, H-3b); 3.80 (s, 1H, H-12b); 4.22
s, 1H, 12-OH); 4.49 (s, 1H, 3-OH); 7.32 (d, 2H, 2H-m, J = 8.8);
6
), d (ppm),
CH
2
skeleton); 47.5 (C-7); 69.9 (C-3); 71.0 (C-12); 102.4 (C-m );
0
J (Hz): 0.60 (s, 3H, 18-CH
1-CH
3
3
107.7 (C-m ); 112.1 (Car-CH@N); 119.8 (2C-o); 121.3 (2C-m);
134.2 (C-o ); 137.9 (Car-NH); 142.7 (C-p); 161.1 (HC@N), 162.2
(C-p AOH); 162.9 (C-o AOH); 171.7 (C-24). HRMS (LC-MS): calcd.
for [M + H]
0
2
(
(
3
2
0
0
2
+
(C37H
51
N
2
O
5
)
requires m/z 603.3798, found
0
0
7
1
.66 (d, 2H, 2H-o, J = 8.8); 8.03 (m, 4H, 2H-o and 2H-m ); 8,73 (s,
m/z 603.3799.
H, HC@N); 9.98 (s, 1H, ANH). 13C-NMR (75 MHz, DMSO-d
), d
(R)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-dihydroxy-10,13-
dimethylhexadecahydro-1H-cyclopenta[ ]phenanthren-17-yl)-N-(4-
6
(
ppm): 12.5 (C-18); 17.1 (C-21); 23.1 (C-19); 23.5–46.2 (CH and
a
CH skeleton); 47.4 (C-7); 69.9 (C-3); 71.0 (C-12); 119.6 (2C-o);
2
((E)-4-nitrobenzylideneamino)phenyl)pentanamide (2f): The compound
0
0
0
1
21.8 (2C-m); 128.4 (2C-o ); 129.7 (2C-m ); 132.7 (C-p ); 138.3
arANH); 139.9 (CarACH@N); 145.4 (C-p); 157.9 (HC@N); 166.9
2f was obtained as a yellow solid. Yield: 82%. M.p.: 141.9–142.5 °C.
ꢀ
1
(
(
(
C
I.R. (KBr)
v
: 3417, 2935, 2867, 1660, 1600, 1539, 1517, 1344 cm
H-NMR (300 MHz, DMSO-d ), d (ppm), J (Hz): 0.60 (s, 3H, 18-CH
0.84 (s, 3H, 19-CH ); 0.96 (d, 3H, 21-CH , J = 5.4); 1.18–1.78 (m,
23H, CH and CH skeleton); 2.23–2.36 (m, 2H, 23-CH ); 3.16 (br
.
+
1
COOH); 171.7 (C-24). HRMS (LC-MS): calcd. for [M + H]
C
6
3
);
38
H
51
N
2
O
5
) requires m/z 615.3798, found m/z 615.3804.
3
3
(
R)-N-(4-((E)-5-bromo-2-hydroxybenzylideneamino)phenyl)-4-
2
2
(
(3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-dihydroxy-10,13-dimethyl-
]phenanthren-17-yl)pentanamide
2c): The compound 2c was obtained as a beige solid. Yield: 64%.
M.p.: 262.7–264.2 °C. I.R. (KBr) : 3421, 3282, 3066, 2923, 2860,
H-NMR (300 MHz, DMSO-d ), d (ppm),
J (Hz): 0.59 (s, 3H, 18-CH ); 0.83 (s, 3H, 19-CH ); 0,96 (d, 3H,
1-CH , J = 5.4); 1.14–1.79 (m, 23H, CH and CH skeleton); 2.22–
.49 (m, 2H, 23-CH ); 3.55 (br s, 1H, H-3b); 3.79 (s, 1H, H-12b);
.22 (d, 1H, 12-OH, J = 3.6); 4.49 (d, 1H, 3-OH, J = 3.9); 6.93 (d,
s, 1H, H-3b); 3.80 (br s, 1H, H-12b); 4.22 (br s, 1H, 12-OH); 4.49
(br s, 1H, 3-OH); 7.35 (br s, 2H, 2H-m); 7.67 (br s, 2H, 2H-o);
8.15 (d, 2H, 2H-o , J = 5.8); 8.34 (br s, 2H, 2H-m ); 8.83 (s, 1H,
hexadecahydro-1H-cyclopenta[
(
a
0
0
1
3
v
HC@N); 10.01 (s, 1H, ANH).
(ppm): 12.4 (C-18); 17.1 (C-21); 23.0 (C-19); 23.5–46.2 (CH and
CH skeleton); 47.4 (C-7); 69.9 (C-3); 71.0 (C-12); 119.6 (2C-o);
6
C-NMR (75 MHz, DMSO-d ), d
ꢀ
1 1
1
664, 1618, 1542 cm
.
6
3
3
2
0
0
2
2
4
1
3
2
122.0 (2C-m); 124.0 (2C-m ); 129.3 (2C-o ); 138.7 (CarANH);
141.8 (CarACH@N); 144.9 (C-p); 148.6 (C-p ); 156.7 (HC@N);
171.8 (C-24). HRMS (LC-MS): calcd. for [M + H] (C37
0
2
+
50
H N
3
O
5
)
0
0
H, H-m , J = 9.0); 7.38 (d, 2H, 2H-m, J = 8.7); 7.51 (dd, 1H, H-p ,
requires m/z 616.3750, found m/z 616.3747.
0
J = 8.7, J = 2.4); 7.69 (d, 2H, 2H-o, J = 8.4); 7.83 (d, 1H, H-o ,
J = 2.4); 8.92 (s, 1H, HC@N); 10.03 (s, 1H, ANH); 13.2 (s, 1H,
AOH). C-NMR (75 MHz, DMSO-d
(R)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-dihydroxy-10,13-
dimethylhexadecahydro-1H-cyclopenta[ ]phenanthren-17-yl)-N-(4-
a
1
3
6
), d (ppm): 12.9 (C-18); 17.6
((E)-2-nitrobenzylideneamino)phenyl)pentanamide (2g): The com-
(
7
(
1
C-21); 23.5 (C-19); 23.9–46.7 (CH and CH
0.4 (C-3); 71.5 (C-12); 110.3 (C-m -Br); 119.4 (C-m ); 120.1
CACH@N); 121.7 (2C-o); 122.3 (2C-m); 134.3 (C-o ); 135.6 (C-p );
39.4 (CarANH); 142.7 (C-p); 159.8 (HC@N); 160,5(C-o AOH);
2
skeleton); 47.9 (C-7);
pound 2g was obtained as a brown solid. Yield: 75%. M.p.:
0
0
257.7–259.8 °C. I.R. (KBr)
v
: 3463, 2927, 2864, 1666, 1598, 1525,
H-NMR (300 MHz, DMSO-d ), d (ppm), J (Hz): 0.60
(s, 3H, 18-CH ); 0.84 (s, 3H, 19-CH ); 0.97(d, 3H, 21-CH , J = 5.4);
1.22–1.77 (m, 23H, CH and CH skeleton); 2.23–2.31 (m, 2H, 23-
CH ); 3.16 (br s, 1H, H-3b); 3.79 (br s, 1H, H-12b); 4.22 (br s, 1H,
12-OH); 4.49 (br s, 1H, 3-OH); 7.29 (d, 2H, 2H-m, J = 7.2); 7.67 (d,
0
0
ꢀ1 1
1508 cm
.
6
0
3
3
3
+
1
72.3 (C-24). HRMS (LC-MS): calcd. for [M + H] (C37
H50BrN
2
O
4
)
2
requires m/z 665.2954, found m/z 665.2959.
R)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-dihydroxy-10,13-
dimethylhexadecahydro-1H-cyclopenta[ ]phenanthren-17-yl)-N-(4-
(E)-4-(dimethylamino)benzylideneamino)phenyl)pentanamide (2d):
The compound 2d was obtained as a yellow solid. Yield: 86%.
M.p.: 253.3–255.0 °C. I.R. (KBr) : 3438, 2937, 2864, 1664, 1610,
H-NMR (300 MHz, DMSO-d ), d (ppm), J (Hz):
); 0.84 (s, 3H, 19-CH ); 0.97 (d, 3H, 21-CH
2
(
0
0
0
a
2H, 2H-o, J = 5.8); 7.69–8.16 (m, 4H, 2H-m , H-o and H-p ); 8.88
1
3
(
(s, 1H, HC@N); 10.01 (s, 1H, ANH). C-NMR (75 MHz, DMSO-d
d (ppm): 12.5 (C-18); 17.2 (C-21); 23.1 (C-19); 23.5–46.2 (CH
and CH skeleton); 47.5 (C-7); 70.0 (C-3); 71.1 (C-12); 119.8
(2C-o); 121.9 (2C-m); 124.5 (C-m ); 129.4 (C-o ); 130.2 (C-p );
6
),
v
2
ꢀ1
1
0
0
0
1
0
537, 1502 cm
.
6
0
0
.60 (s, 3H, 18-CH
3
3
3
,
2
131.7 (C-o ANO ); 133.7 (C-m ); 138.6 (CarACH@N); 145.3
J = 5.4); 1.16–1.78 (m, 23H, CH
3-CH ); 3.00 (br s, 6H, N(CH
2
and CH skeleton); 2.26 (m, 2H,
); 3.18 (br s, 1H, H-3b); 3.80 (br
(CarANH); 149.1 (CarAN@CH); 154.8 (HC@N); 171.9 (C-24).
HRMS (LC-MS): calcd. for [M + H] (C37H N O ) requires m/z
50 3 5
+
2
2
3
)
2
s, 1H, H-12b); 4.23 (br s, 1H, 12-OH); 4.50 (br s, 1H, 3-OH); 6.76
616.3750, found m/z 616.3745.
0
0
(
m, 2H, 2H-m ); 7.16 (d, 2H, 2H-o , J = 5.8); 7.59 (d, 2H, 2H-m,
J = 8.5); 7.71 (d, 2H, 2H-o); 8.40 (s, 1H, HC@N); 9.89 (s, 1H,
2.2.3. Procedure for the synthesis of 5-bromosalicylideneaniline (3)
Azastilbene 3 was prepared by condensation of aniline with the
equimolar amount of 5-bromosalicylaldehyde in ethanol at room
temperature. After a short time interval the formation of a precipi-
tate was noticed. The solid was washed with ethanol and dried in
an oven. Azastilbene 3 was obtained in 86% yield, as a orange solid,
1
3
ANH). C-NMR (75 MHz, DMSO-d
6
), d (ppm): 12.4 (C-18); 17.1
(
6
C-21); 23.0 (C-19); 23.5–46.2 (CH and CH skeleton); 47.4 (C-7);
2
0
9.9 (C-3); 71.0 (C-12); 111.4 (2C-m ); 119.7 (2C-o); 121.1 (2C-
0
m); 124.0 (CarACH@N); 130.1 (2C-o ); 136.8 (CarANH); 147.0
(
C
arAN@CH); 152.2 (CarAN(CH
HRMS (LC-MS): calcd. for [M + H] (C39
14.4322, found m/z 614.4320.
3 2
) ); 158.5 (HC@N); 171.5 (C-24).
+
1
H
56
N
O
3 3
) requires m/z
and characterized by H NMR, I.R. and melting point (M.p.) and was
in accordance with data in the literature [35].
6

1
18
J.A.d. Santos et al. / Steroids 98 (2015) 114–121
2.3. Antioxidant activity
For each product, three plates were prepared, which were kept
protected from light exposure in a dark chamber at room tempera-
ture (ꢂ20 °C) for 15 min, in order to facilitate the formation of a
standard stabilized sunscreen film.
The antioxidant activity was evaluated using the 2,2-diphenyl-
-picrylhydrazyl (DPPH; Sigma–Aldrich, USA) assay, described by
1
Sreejayan and Rao [36] with minor modifications. This method is
After this period, the plates containing the product were placed
in the light-path of the transmittance analyzer. The transmission of
UV radiation through the sample was measured from 290 to
450 nm at 1 nm intervals on 9 different sites of each plate (total
measurement area = 2.0 cm ). The blank was prepared using the
HD6 plates covered with 15-lL of glycerin, because of its non-
based on the DPPH reduction in the presence of an antioxidant
+
(
AH), a proton donor (H ) for a non-radical (DPPH-H) [37]. An
aliquot of 150
added to 50 L of ethanol solution of bile acids/azastilbenes conju-
gates at concentrations from 0.97 to 250
lL of a DPPH ethanolic solution of 0.05 mM were
2
l
ꢀ
1
l
g mL in 96-well plates.
Resveratrol (trans-resveratrol 99.0%, Attivos Magistrais, Brazil) was
fluorescence and UV transparency.
used as a standard at the same concentrations.
Using the generated data, SPFin vitro was calculated using Eq. (2):
Reactions elapsed at room temperature for 30 min in the dark
and then the absorbance was read in a spectrophotometer
R
k¼400nm
EðkÞ ꢁ IðkÞ ꢁ dk
k¼290nm
SPFin vitro ¼ R
ð2Þ
k¼400nm
k¼290nm
ꢀA ðkÞ
0
(
=510 nm).
EðkÞ ꢁ IðkÞ ꢁ 10
ꢁ dk
Inhibition of DPPH radical was calculated using the equation:
where E(k) is the erythema action spectrum,10 I(k) is the spectral
irradiance of the UV source, A (k) is the mean monochromatic
0
%
of inhibition ¼ 100 ꢁ ðA
0
—A
s
Þ=A
0
ð1Þ
Being: A
of test samples.
0
absorbance as the negative control and A
s
the absorbance
absorbance measurements per plate of the test product layer before
UV exposure, and dk is the wavelength step (1 nm).
The IC50 value was calculated from the line in the equation of
the linear dispersion graph. All tests were performed in triplicate.
We performed a descriptive statistical analysis and ANOVA fol-
lowed by the Tukey test, with the aid of Statistical Package for
Social Sciences (SPSS) v.14.0 for Windows software, to compare
the average values of IC50 calculated between the prepared com-
pounds versus the standard. The level of significance was 5%.
In order to generate the UVAPF value, the coefficient of
adjustment ‘‘C’’ was calculated as shown in Eq. (3) and using the
SPF label as the value generated by the UV-2000’s software.
SPFin vitro;adj ¼ SPF label
R
k¼400nm
EðkÞ ꢁ IðkÞ ꢁ dk
k¼290nm
¼
R
ð3Þ
k¼400nm
k¼290nm
ꢀA ðkÞꢁC
0
EðkÞ ꢁ IðkÞ ꢁ 10
ꢁ dk
2.4. Photoprotection assay
Using the ‘‘C’’ value, initial UVAPF was calculated using Eq. (4), and
the dose ‘‘D’’ of UV irradiation was determined by Eq. (5).
In vitro photoprotection experiments were conducted in an
R
k¼400nm
UV-2000S Ultraviolet Transmittance Analyzer (Labsphere, USA),
composed of two photodiode array spectrographs and equipped
with an integrating sphere and a xenon flash lamp, which emits
a continuous spectrum of radiation with no peaks and supplies
energy for a 290–450 nm spectral range, with a wavelength
increment step of 1 nm, having low irradiance such that the
photostability of the product is not unduly challenged.
PðkÞ ꢁ IðkÞ ꢁ dk
k¼320nm
UVAPF
0
¼ R
ð4Þ
ð5Þ
k¼400nm
k¼290nm
ꢀA ðkÞꢁC
0
PðkÞ ꢁ IðkÞ ꢁ 10
ꢁ dk
D ¼ UVAPF
0
ꢁ D
0
where P(k) is the PPD action spectrum10 and D0 = 1.2 J cm 2
ꢀ
.
The plates were inserted into the UV irradiation source (tem-
perature maintained below 40 °C) and then exposed to the calcu-
lated UV dose D. After that, new transmission measurements of
the sunscreen samples were conducted, for acquisition of the sec-
ond UV spectrum. The final UVAPF was calculated according to Eq.
(6). If the coefficient of variation (CV) between the UVAPF’s of the
individual plates exceeded 20%, then further plates were measured
until the CV threshold was achieved.
A
long-arc xenon Suntest™ insolator, CPS + type (Atlas,
Germany), filtered with its original UV short cut-off filter combined
with a Special UV Glass filter (limiting radiation at approximately
90 nm), providing a VIS + UVA + UVB spectrum, was used as the
artificial UV source for irradiation of the sunscreen samples.
PMMA plates were supported firmly throughout the irradiation
by a SunTray holder, which also provided a dark background
behind each plate to reduce the risk of any back exposure. An
electronic analytical balance AY-220 (Shimadzu, Japan) and a posi-
tive-displacement manual pipette (Mettler Toledo, USA) were used
for the preparation of the samples PMMA plates.
2
R
k¼400nm
PðkÞ ꢁ IðkÞ ꢁ dk
PðkÞ ꢁ IðkÞ ꢁ 10
k¼320nm
UVAPF ¼ R
ð6Þ
k¼400nm
k¼290nm
ꢀAðkÞꢁC
ꢁ dk
The sunscreens were compounded by adding 15% of the
each substance to a lanette inert lotion, in isolation. Each sample
was accurately and quickly weighed (to reduce product
evaporation and dryness) to satisfy the application rate of
where A(k) is the mean monochromatic absorbance of the test pro-
duct layer after UV exposure.
c
For calculation of the Critical Wavelength Value (k ), a series of
absorbance values were calculated for each of the three separate
plates to which the samples were applied. Absorbance at each
wavelength increment A(k) was calculated using Eq. (7), and the
ꢀ2
1
.3 mg cm
HD6 plates (HelioScreen, France) with roughened surface on one
side (Sa ꢂ 6 m) (actual quantity applied: 32.5 mg, determined
in polymethylmethacrylate (PMMA) Helioplate™
l
k
c
using Eq. (8).
by weighing the plates before and immediately after applying
the products). The samples were directly weighed on the plate sur-
face, applied as a large number of small droplets of approximately
equal mass, and distributed in an even manner on the roughened
surface of the plate. Then, the products were spread over the whole
surface with a fingertip covered with a vinyl glove and pre-satu-
rated with the product, to prevent possible losses of the amount
weighed. The spreading was achieved in two steps: (i) quick
distribution of the product, without pressure (20–30 s); and (ii)
rubbing it into the rough surface using pressure (20–30 s too).
A
k
¼ logðCk=Pk
Þ
ð7Þ
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
n
where
C
k
¼
ðC
k
½1ꢃ ꢁ C
k
k
½2ꢃ ꢁ . . . ꢁ C
k
½nꢃÞ
and
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
n
P
k
¼
kc
ðP
k
½1ꢃ ꢁ P
k
½2ꢃ ꢁ . . . ꢁ P ½nꢃÞ.
Z
Z
400nm
Ak ꢁ dk ¼ 0:9
Ak ꢁ dk
ð8Þ
2
90nm
290nm
Finally, the UVA/UVB Ratio was calculated as the ratio between the
final UVAPF and the SPF label.

J.A.d. Santos et al. / Steroids 98 (2015) 114–121
119
The verification of the validity of the results was obtained using
the Cosmetics Europe Reference Sunscreen S2 (determined
SPF = 18 ± 1.5, UVAPF = 12 ± 1.1, = 381 nm, and UVA/UVB
Ratio = 0.88). All results were expressed as mean of 27
determinations (3 plates, 9 readings each, at different sites) for
lotions containing 15% of each bile acid/azastilbene.
HO
HO
(
a)
NH2
O
N
k
c
a
3
Br
Br
Scheme 2. Synthetic route to preparation of non conjugated azastilbene 3.
Reagents and conditions: (a) ethanol, r.t.
3
. Results and discussions
Table 1
3.1. Chemistry
Antioxidant activity of bile acids/azastilbenes conjugates and the bile amides.
0
00
ꢀ1
Compound
R
–
R
–
IC50
(
l
g mL
)
Initially, the bile acids (cholic and deoxycholic) were subjected
1
19.60a
to an amide forming reaction using DCC and aromatic amine
,4-phenylenediamine in THF. Thus, once bile amides 1 and 2
moderate yields) were formed, the targeted conjugates 1a–g and
a–g were obtained through the condensation of aromatic
1
1
a
b
o-CO
p-CO
o-OH
p-N(CH
o-OH
p-NO
o-NO
–
o-CO
p-CO
o-OH
p-N(CH
o-OH
p-NO2
o-NO
2
H
H
H
H
m-Br
H
p-OH
H
23.35a
1
(
2
31.83a
2
a
1c
1d
232.10
3
)
2
247.18a
a
1
1
1
2
e
f
g
246.93
aldehydes in ethanol in good yields. (Scheme 1).
449.10a
2
Additionally, the non conjugated azastilbene 3 was prepared
using classical methods of synthesis for this class of molecules,
by the condensation of 5-bromosalicylaldehyde and aniline in very
good yield (Scheme 2). The compound 3 was prepared so that we
have a representative azastilbene as reference drug, making
possible to compare the photoprotective potential of steroids
conjugated species to the free specie, non conjugated.
a
2
H
124.87
19.12
20.45
a
–
a
2a
2
H
H
H
m-Br
H
p-OH
H
a
2
2
2
2
b
c
d
e
2
H
30.60
307.61a
a
3
)
2
238.35
a
200.27
2f
325.00a
123.06a
9.18
2g
2
H
Resveratrol
–
–
3.2. Antioxidant activity
a
Statistically different from resveratrol (p < 0.05).
The results of the activity can be seen in Table 1. From that, one
0
can see that the intermediate amides 1 and 2 were those which
required the lowest concentrations in order to inhibit the DPPH
radical, among all the compounds. However, this requirement
was still higher when compared to that shown by the reference
standard.
The antioxidant activity of the azastilbene conjugates versus the
reference standard showed that these are statistically different,
being that the conjugates presented a lower antioxidant activity
when compared the resveratrol (p < 0.001).
The choice for adding azastilbenes was based on the fact that
polyphenolic compounds such as stilbenes and, particularly,
resveratrol present chemical structures which play roles in neu-
tralizing free radicals, as well as in the chelation of transition
metals, both in the initiation step and in the propagation of oxida-
tive stress [38]. Nevertheless, there are some factors in addition to
the chemical structure, for instance: a reactive agent as a proton
and electron donor agent, the ability to chelate transition metals
and the solubility and interaction with biological membranes [39].
Compounds 1a, 1b, 2a and 2b possess a carboxyl group in R or
0
0
R , which may be the responsible for the uptake of free radical
DPPH, although this occurred in a milder way than it happened
in 1 and 2. The carboxyl groups can interact with metal ions,
responsible for initiating the Fenton reaction, which is responsible
for the production of hydroxyl radical. Thus, molecules containing
carbonyl group would be useful as metal chelators [40].
Compounds 1f, 1g, 2f and 2g present a nitro group in their
structure. Most of the nitro compounds show a bio enzymatic
reduction mechanism and this results in the formation of free radi-
cals, but with preferential toxicity to bacteria and parasites [41].
3.3. Photoprotective activity
In this work, the photoprotective activity of the conjugates was
determined in terms of SPF, UVAPF and Critical Wavelength, with
the objective of determining the full UV spectrum of the
OH
OH
24
2
4
OH
NH
NH2
H
a
H
O
O
H
H
R
H
H
R
HO
HO
1
2
R= OH
R= H
H
H
R= OH (cholic)
R= H (deoxycholic)
b
OH
H
2
4
1
a/2a R' = o-CO H; R'' = H
NH
N
R'
2
1
b/2b R' = p-CO H; R'' = H
2
1
c/2c R' = o-OH; R'' = m-Br
O
1
d/2d R' = p-N(CH ) ; R'' = H
R"
3
2
H
H
R
1
e/2e R' = o-OH; R'' = p-OH
1
a-g
1f /2f R' = p-NO ; R'' = H
HO
2
H
2 a-g
1g/2g R' = o-NO ; R'' = H
2
Scheme 1. Synthetic route to preparation of bile acids/azastilbenes conjugates and bile amides. Reagents and conditions: (a) 1,4-phenylenediamine, DCC, THF, r.t.; (b)
aromatic aldehydes, MeOH, r.t.

1
20
J.A.d. Santos et al. / Steroids 98 (2015) 114–121
Table 2
Photoprotection results of bile acids/azastilbenes conjugates and bile amides
0
00
Compound
R
–
R
–
SPF
UVAPF
k
c
(nm)
Initial UVA/UVB ratio
Post exposure UVA/UVB ratio
1
3 ± 0.1
2 ± 0.1
5 ± 0.4
7 ± 0.9
5 ± 0.5
2 ± 0.1
5 ± 0.6
3 ± 0.2
2 ± 0.4
3 ± 0.2
5 ± 0.4
9 ± 1.6
2 ± 0.1
8 ± 0.8
3 ± 0.2
8 ± 0.3
3 ± 0.5
7 ± 1.7
6 ± 0.6
2 ± 0.1
5 ± 0.2
11 ± 0.6
5 ± 0.3
2 ± 0.1
5 ± 0.1
4 ± 0.3
5 ± 0.6
4 ± 0.5
5 ± 0.6
11 ± 1.1
2 ± 0.1
8 ± 0.7
4 ± 0.3
7 ± 0.2
5 ± 0.5
2 ± 0.2
377
383
389
388
389
387
389
388
383
385
389
388
389
388
389
389
387
362
0.31
0.48
1.01
1.17
1.09
1.07
0.89
0.87
0.43
0.65
1.04
1.11
1.02
1.00
0.89
0.94
1.10
0.44
0.30
0.48
1.01
1.16
1.08
1.06
0.90
0.87
0.41
0.62
1.02
1.09
1.02
1.00
0.86
0.93
1.11
0.46
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
3
a
b
c
d
e
f
o-CO
p-CO
o-OH
p-N(CH
o-OH
p-NO
o-NO
–
o-CO
p-CO
o-OH
p-N(CH
o-OH
p-NO
o-NO
–
2
H
H
H
H
m-Br
H
p-OH
H
2
3
)
2
2
g
2
H
–
a
b
c
d
e
f
2
H
H
H
m-Br
H
p-OH
H
2
H
3
)
2
2
g
2
H
–
–
Resveratrol
–
same. Finally, it is important to mention that all the synthesized
conjugates were better photoprotectors than their bile amides
starters, which suggests that azastilbene moiety positively
influences the photoprotective activity.
Table 3
Boots Star Rating criteria for classification of UVA protection in sunscreens
Initial Mean UVA/UVB ratio
All conjugates were also subjected to the determination of their
UVAPF. Conjugates from 1b to 1d, 1f, 2b-2c, 2e and 2g presented
very similar protection against UVA rays among themselves, and
it is important to highlight that the structural modifications led
to a very significant improvement in the activity in this solar
spectrum range, as the UVAPF increased from 2 in resveratrol
and 5 in 3 to 5–11 in the cited conjugates. Moreover, all other
conjugates were equally or at least two times more active than
resveratrol, although some of them remained with the same
UVAPF as 3. This is of sheer relevance, since UVA rays are known
to play an important role on skin cancers.
0
0
.0–
.59
0.6–
0.79
0.8–
0.89
0.89 and
over
Post exposure mean
UVA/UVB ratio
0.0–0.56
No
rating
No
rating
No
rating
No
No
rating
www
No
rating
www
No rating
0
0
0
0
0
.57–
.75
.76–
.85
www
www
www
wwww wwww
.86 and
wwww wwwww
over
rating
protection. SPF is the ratio between the time of exposure to UVB
radiation needed to develop erythema on a skin site protected by
the sunscreen and the time demanded to produce the same effect
on a bare skin site, without the application of any products [42].
UVAPF, in turn, refers to the protection against UVA radiation,
which is important since UVA sunrays penetrate to the deeper lay-
ers of the skin and damage the DNA and tissues via the production
of reactive oxygen species (ROS) [43]. In this context, the Critical
Wavelength is a parameter which determines if a filter has a broad
spectrum of protection or not. It is another measure of the UVA
Critical Wavelength was also determined. The US Food and
Drug Administration (FDA) [44] adopts the same classification cre-
ated by Springsteen et al. [45], which considers the broad spectrum
test as a pass/fail test, based on the critical wavelength value of
3
70 nm. Under such classification, all conjugates and the bile pre-
cursor amides are considered broad spectrum filters. The impor-
tance of a broad-spectrum sunscreens has already been shown to
be more effective when compared to those which are not broad-
spectrum, with regards to protection against DNA damage and in
the prevention of photo carcinogenesis [46].
protection property in
a product, defined as the shortest
We have also determined the UVA/UVB ratio. This is another
measure of the protection spectrum and provides a good idea of
which UV region is better blocked by the substances. The
UVA/UVB ratio is associated with the completeness of the
protection, when the ratio exceeds 0.92 [42]. The UVA/UVB ratio
can also be used to provide the so-called Boots Star Rating, which
classifies the products in categories from 0 to 5 stars [47]. Thus, the
results can be displayed with stars, and the greatest number
matches greatest protection [42]. This classification should be done
according to Table 3. Conjugates 1b–f, 2b–f and 2g are, therefore, 5
stars; 1g and 2f are 4 stars; 2a is 3 stars and, finally, intermediate
amides 1 and 2, as well as conjugate 1a, receive no stars.
In summary, in the present study we found that the inclusions
of nitro, methoxy and amine groups were not modifications of
interest in the improvement of the antioxidant activity. However,
bile acids/azastilbenes conjugates may have an important source
for a newer modification to improve the antioxidant activity of
the final compounds. Regarding the photoprotective activity, we
verified that the compounds presented a varied photoprotection
profile. While protection against UVB sunrays of conjugates 2c,
wavelength in which the product absorption corresponds to 90%
of total absorption. In order to be indicated as appropriate in
protecting against UVA radiation, a sunscreen should present a k
c
value greater than 370 nm [42].
The conjugates and bile amides presented a varied photoprotec-
tion profile, with the SPF ranging from 2 to 9 and the UVAPF from 2
to 11, as shown in Table 2. All the molecules presented some
degree of photoprotection for a single UV-filter substance. Their
SPF, i.e., their protection against UVB sunrays, was similar, but
the activity in conjugates 2c, 2e and 2g were more significant than
in resveratrol and 3 themselves. This demonstrates that the pres-
ence of hydroxyl, bromide and nitro groups in the terminal ring
can be seen as positive influences to their satisfactory SPF values.
However, regarding their respective cholic conjugated derivatives,
compounds 1c, 1e and 1g, only the 1c conjugate showed a satisfac-
tory SPF. 1a and 1e showed the lowest SPF. Yet, since conjugates 1c
and 1f showed photoprotection similar to that of resveratrol and
superior to 3, the presence of the mentioned substituent groups
is relevant to significant protection against UVA rays. However,
when one compares the results of the conjugates with the free aza-
stilbene 3, only the SPF value increased, as the UVAPF remained the
2
e and 2g was more significant than that of resveratrol or 3, the

J.A.d. Santos et al. / Steroids 98 (2015) 114–121
121
UVAPF increased from 2 in resveratrol and 5 in 3 to 5–11 in the
majority of the conjugates.
[17] Pore VS, Aher NG, Kumar M, Shukla PK. Design and synthesis of
fluconazole/bile acid conjugate using click reaction. Tetrahedron 2006;62:
11178–86.
These results suggest that the conjugation with bile acids influ-
ences positively the photoprotection, once their sunscreen activity
was similar or greater than that of the free azastilbene. Although
the profiles for UVA and UVB radiations varied among the
conjugates, it seems that the conjugation was important for
increasing protection within the both ultraviolet ranges, as 1c
and 2c have twice the UVAPF of the free specie and 2c, 2e and
[
[
[
18] Balakrishnan A, Polli JE. Apical sodium dependent bile acid transporter (ASBT,
SLC10A2): a potential prodrug target. Mol Pharm 2006;3:223–30.
19] Tolle-Sander S, Lentz KA, Maeda DY, Coop A, Polli JE. Increased acyclovir oral
bioavailability via a bile acid conjugate. Mol Pharm 2003;1:40–8.
20] Corrales RCNR, de Souza NB, Pinheiro LS, Abramo C, Coimbra ES, Da Silva AD.
Thiopurine derivatives containing triazole and steroid: synthesis, antimalarial
and antileishmanial activities. Biomed Pharmacother 2011;65:198–203.
21] Antinarelli L, Carmo A, Pavan F, Leite CQ, Da Silva A, Coimbra E, et al. Increase
of leishmanicidal and tubercular activities using steroids linked to
aminoquinoline. Org Med Chem Lett 2012;2:16.
22] Chong J, Poutaraud A, Hugueney P. Metabolism and roles of stilbenes in plants.
Plant Sci 2009;177:143–55.
23] Shen T, Wang XN, Lou HX. Natural stilbenes: an overview. Nat Prod Rep
2009;26:916–35.
24] Lu J, Li C, Chai Y-F, Yang D-Y, Sun C-R. The antioxidant effect of imine
resveratrol analogues. Bioorg Med Chem Lett 2012;22:5744–7.
25] Santos JAd, Lima RM, Pereira TV, Carmo AMRd, Raposo NRB, Silva ADd.
Antioxidant activity of thio-schiff bases. Lett Drug Des Discov 2013;10:
557–60.
26] Tamizh MM, Kesavan D, Sivakumar PM, Mereiter K, Deepa M, Kirchner K, et al.
Antibacterial activities of 4-substituted-2-[(E)-{(1S,2R)/(1R,2S)-1-hydroxy-1-
phenylpropan-2-ylimino}methyl]phenol. Chem Biol Drug Des 2012;79:
[
2
g have approximately three times the SPF of the azastilbene.
[
[
[
[
This suggests that the non steroidal portion may play a significant
role in the final photoprotection observed for these molecules used
as single UV filter in the products tested.
Thus, the compounds which stood out are likely to be further
used as ingredients in sunscreen formulations. Studies arising from
the relationship between the properties of the analogues and their
chemical structure, along with new molecular changes on them,
may contribute to the development of more effective bioactive
molecules.
[
177–85.
[
[
27] Shakeel AS, Kalyane NV, Karjgi SR, Md. Liyakat A. Synthesis and antibacterial
activity of new schiff bases. Int J Pharm Life Sci 2010;1:246–9.
28] Kumar S, Niranjan MS, Chaluvaraju KC, Jamakhandi CM, Kadadevar D.
Synthesis and antimicrobial study of some schiff bases of sulfonamides. J
Curr Pharm Res 2010;1:39–42.
29] da Silva CM, da Silva DL, Modolo LV, Alves RB, de Resende MA, Martins CVB,
et al. Schiff bases: a short review of their antimicrobial activities. J Adv Res
2011;2:1–8.
30] Silva CMd, Silva DLd, Martins CVB, Resende MAd, Dias ES, Magalhães TFF, et al.
Synthesis of aryl aldimines and their activity against fungi of clinical interest.
Chem Biol Drug Des 2011;78:810–5.
31] Fujita Y, Islam R, Sakai K, Kaneda H, Kudo K, Tamura D, et al. Aza-derivatives of
resveratrol are potent macrophage migration inhibitory factor inhibitors.
Invest New Drugs 2012;30:1878–86.
Acknowledgements
The authors would like to thank the Brazilian agencies CAPES,
CNPq and FAPEMIG.
[
[
[
[
[
[
[
References
[
[
[
[
1] Virtanen E, Kolehmainen E. Use of bile acids in pharmacological and
supramolecular applications. Eur J Org Chem 2004;2004:3385–99.
2] Mukhopadhyay S, Maitra U. Chemistry and biology of bile acids. Curr Sci
2
004;87:1666–83.
3] Hofmann AF, Hagey LR. Bile acids: chemistry, pathochemistry, biology,
pathobiology, and therapeutics. Cell Mol Life Sci 2008;65:2461–83.
4] Hazra B, Pore V, Dey S, Datta S, Darokar M, Saikia D, et al. Bile acid amides
derived from chiral amino alcohols: novel antimicrobials and antifungals.
Bioorg Med Chem Lett 2004;14:773–7.
32] Paula DTSd, Carvalho GSGd, Almeida AC, Lourenço M, Silva ADd, Coimbra ES.
Synthesis, cytotoxicity and antileishmanial activity of aza-stilbene derivatives.
Med J Chem 2013;2:493–502.
33] Lima LL, Lima RM, SilvaCarmo AFd, Carmo A, et al. Azastilbene analogs as
tyrosinase inhibitors: new molecules with depigmenting potential. Sci World J
[
[
5] Kakati D, Sarma RK, Saikia R, Barua NC, Sarma JC. Rapid microwave assisted
synthesis and antimicrobial bioevaluation of novel steroidal chalcones.
Steroids 2013;78:321–6.
6] Willemen HM, de Smet LCPM, Koudijs A, Stuart MCA, Heikamp-de Jong IGAM,
Marcelis ATM, et al. Micelle formation and antimicrobial activity of cholic acid
derivatives with three permanent ionic head groups. Angew Chem Int Ed
2013;2013:7.
34] Polonini HC, Lima LL, Gonçalves KM, do Carmo AMR, da Silva AD, Raposo NRB.
Photoprotective activity of resveratrol analogues. Bioorg Med Chem
2
013;21:964–8.
35] Misra S, Pandeya K, Tiwari A, Ali A, Saradamani T, Agawane S, et al.
Antihyperglycemic, -glucosidase inhibitory and DPPH free radical
a
2
002;41:4275–7.
scavenging activity of 5-bromosalicylaldehyde and schiff bases. Med Chem
Res 2011;20:1431–7.
36] Sreejayan N, Rao MN. Free radical scavenging activity of curcuminoids.
Arzneimittelforschung 1996;46:169–72.
[
[
7] Salunke DB, Hazra BG, Pore VS, Bhat MK, Nahar PB, Deshpande MV. New
steroidal dimers with antifungal and antiproliferative activity. J Med Chem
[
[
2
004;47:1591–4.
8] Visbal G, San-Blas G, Maldonado A, Álvarez-Aular Á, Capparelli MV, Murgich J.
Synthesis, in vitro antifungal activity and mechanism of action of four sterol
hydrazone analogues against the dimorphic fungus Paracoccidioides
brasiliensis. Steroids 2011;76:1069–81.
37] Koleva II, van, Beek TA, Linssen JPH, Groot Ad, Evstatieva LN. Screening of plant
extracts for antioxidant activity:
a comparative study on three testing
methods. Phytochem Anal 2002;13:8–17.
[
38] Chun S-S, Vattem DA, Lin Y-T, Shetty K. Phenolic antioxidants from clonal
oregano (Origanum vulgare) with antimicrobial activity against Helicobacter
pylori. Process Biochem 2005;40:809–16.
39] Barreiros ALBS, David JM, David JP. Estresse oxidativo: relação entre geração de
espécies reativas e defesa do organismo. Quim Nova 2006;29:113–23.
40] Rosa CSd, Hoelzel SC, Viera VB, Barreto PM, Beirão LH. Atividade antioxidante
do ácido hialurônico extraído da crista de frango. Ciência Rural 2008;38:
[
9] Scozzafava A, Supuran CT. Carbonic anhydrase inhibitors. Preparation of potent
sulfonamides inhibitors incorporating bile acid tails. Bioorg Med Chem Lett
2
002;12:1551–7.
[
[
[
[
10] Bülbül M, Saraço g˘ lu N, I_rfan Küfrevio g˘ lu Ö, Çiftçi M. Bile acid derivatives of 5-
amino-1,3,4-thiadiazole-2-sulfonamide as new carbonic anhydrase inhibitors:
synthesis and investigation of inhibition effects. Bioorg Med Chem 2002;10:
2
561–7.
2593–698.
11] Brossard D, El Kihel L, Clément M, Sebbahi W, Khalid M, Roussakis C, et al.
Synthesis of bile acid derivatives and in vitro cytotoxic activity with pro-
apoptotic process on multiple myeloma (KMS-11), glioblastoma multiforme
[
[
41] Paulai FR, Serrano SHP, Tavares LC. Aspectos mecanísticos da bioatividade e
toxicidade de nitrocompostos. Quim Nova 2009;32:1013–20.
42] Velasco MVR, Balogh TS, Pedriali CA, Sarruf FD, Pinto CASDO, Kaneko TM, et al.
Novas metodologias analíticas para avaliação da eficácia fotoprotetora
(
GBM), and colonic carcinoma (HCT-116) human cell lines. Eur J Med Chem
2
010;45:2912–8.
[
[
12] Mrózek L, Dvo rˇ áková L, Mandelová Z, Rárová L, Rˇ ezá cˇ ová A, Pla cˇ ek L, et al.
(
in vitro) – revisão. 2011.
43] Wang SQ, Osterwalder U, Jung K. Ex vivo evaluation of radical sun protection
factor in popular sunscreens with antioxidants. Am Acad Dermatol
011;65:525–30.
[
[
[
Investigation of new acyloxy derivatives of cholic acid and their esters as drug
absorption modifiers. Steroids 2011;76:1082–97.
13] Virtanen E, Tamminen J, Linnanto J, Mätttäri P, Vainiotalo P, Kolehmainen E.
Synthesis, 1 H, 13 C, 15 N, and 113 Cd NMR, ESI-TOF MS, semiempirical MO
J
2
44] Food and Drug Administration CP, 310 and 352. Sunscreen drug products for
over the counter human use; final rules and proposed rules. Silver Spring;
(
PM3), ab initio/HF and cation/anion binding studies of N-deoxycholyl-l-
2011.
tryptophan. J Inclusion Phenom 2002;43:319–27.
45] Springsteen A, Yurek R, Frazier M, Carr KF. In vitro measurement of sun
protection factor of sunscreens by diffuse transmittance. Anal Chim Acta
[
[
14] Swaan PW, Hillgren KM, Szoka FC, Øie S. Enhanced transepithelial transport of
peptides by conjugation to cholic acid. Bioconjug Chem 1997;8:520–5.
15] Rasras AJM, Al-Tel TH, Al-Aboudi AF, Al-Qawasmeh RA. Synthesis and
antimicrobial activity of cholic acid hydrazone analogues. Eur J Med Chem
1999;380:155–64.
[
[
46] Fourtanier A, Mexoryr SX. Protects against solar-simulated UVR-induced
photocarcinogenesis in mice. Photochem Photobiol 1996;64:688–93.
47] Ltd., BtC. The revised guidelines to the practical measurement of UVA/UVB
ratios according to the Boots star rating system. Nottingham, England: The
Boots Co. PLC; 2004.
2
010;45:2307–13.
[
16] Kannan A, De Clercq E, Pannecouque C, Witvrouw M, Hartman TL, Turpin JA,
et al. Synthesis and anti-HIV activity of
Tetrahedron 2001;57:9385–91.
a bile acid analog of cosalane.