Structure-activity relationship of hybrids of Cinchona alkaloids and bile acids with in vitro antiplasmodial and antitrypanosomal activities

Article abstract of DOI:10.1016/j.ejmech.2015.05.044

In this work, a series of hybrid compounds were tested as antiparasitic substances. These hybrids were prepared from bile acids and a series of antiparasitic Cinchona alkaloids by the formation of a covalent C-C bond via a decarboxylative Barton-Zard reaction between the two entities. The bile acids showed only weak antiparasitic properties, but all the hybrids exhibited high in vitro activities (IC₅₀: 0.48-5.39 μM) against Trypanosoma brucei. These hybrids were more active than their respective parent alkaloids (up to a 135 fold increase in activity), and displayed good selectivity indices. Aditionally, all these compounds inhibited the in vitro growth of a chloroquine-sensitive strain of Plasmodium falciparum (3D7: IC₅₀: 36.1 nM to 8.72 μM), and the most active hybrids had IC₅₀s comparable to that of artemisinin (IC₅₀: 36 nM). Some structure-activity relationships among the group of 48 hybrids are discussed. The increase in antiparasitic activity may be explained by an improvement in bioavailability, since the more lipophilic derivatives showed the lowest IC₅₀s.

Full text of DOI:10.1016/j.ejmech.2015.05.044

European Journal of Medicinal Chemistry 100 (2015) 10e17
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Structure-activity relationship of hybrids of Cinchona alkaloids and
bile acids with in vitro antiplasmodial and antitrypanosomal activities
a
b
a
c
ꢀ
ꢀ
ꢀ ꢀ
Aurelie Leverrier , Joanne Bero , Julian Cabrera , Michel Frederich ,
b
a, *
€
Joelle Quetin-Leclercq , Jorge A. Palermo
UMYMFOR, Departamento de Química Organica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pab. 2
a
ꢀ
(1428) Buenos Aires, Argentina
b
ꢀ
Pharmacognosy Research Group, Louvain Drug Research Institute, Universite Catholique de Louvain, Avenue E. Mounier B1.72.03, B-1200 Brussels,
Belgium
c
ꢁ
^
ꢁ
Laboratory of Pharmacognosy, Drug Research Center (CIRM), University of Liege, Avenue de l'Hopital 1, B36, B-4000 Liege, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
In this work, a series of hybrid compounds were tested as antiparasitic substances. These hybrids were
prepared from bile acids and a series of antiparasitic Cinchona alkaloids by the formation of a covalent C
eC bond via a decarboxylative BartoneZard reaction between the two entities. The bile acids showed
only weak antiparasitic properties, but all the hybrids exhibited high in vitro activities (IC50: 0.48
Received 17 November 2014
Received in revised form
29 May 2015
Accepted 30 May 2015
Available online 1 June 2015
e5.39
mM) against Trypanosoma brucei. These hybrids were more active than their respective parent
alkaloids (up to a 135 fold increase in activity), and displayed good selectivity indices. Aditionally, all
these compounds inhibited the in vitro growth of a chloroquine-sensitive strain of Plasmodium falciparum
(3D7: IC50: 36.1 nM to 8.72 mM), and the most active hybrids had IC50s comparable to that of artemisinin
(IC50: 36 nM). Some structure-activity relationships among the group of 48 hybrids are discussed. The
increase in antiparasitic activity may be explained by an improvement in bioavailability, since the more
lipophilic derivatives showed the lowest IC50s.
Keywords:
Cinchona alkaloids
Bile acids
Hybrids
BartoneZard reaction
Antiparasitic activity
© 2015 Published by Elsevier Masson SAS.
1. Introduction
derivatives as a monotherapy to treat malaria, since this will lead
to an increase in drug resistance [1], and instead promotes the use
Infectious diseases, such as malaria and African sleeping sick-
ness, which are caused by the protozoan parasites Plasmodium sp.
and Trypanosoma sp. respectively, are still major global health
problems, especially in developing countries. According to the
World Health Organisation (WHO), in 2012 malaria caused an
estimated 627000 deaths, mostly among African children, and in
the same year, 7197 new cases of African trypanosomiasis were
reported [1]. Most of the drugs currently in use have the draw-
backs of their toxicities, increased resistance, and a variable effi-
cacy against different strains or species, making necessary the
search for new antiparasitic compounds [2e5]. Actually, there are
only a few efficient treatments for these diseases, and these are
often based in a combination of drugs. For example, the World
Health Organisation does not recommend the use of arteminisin
of a combination of drugs with different modes of action. In this
regard, the preparation of new bioactive substances by the use of
bioconjugation can be considered an alternative approach for the
discovery of new drugs. The synthesis of hybrids of bioactive
compounds with the combined properties of their individual
components has emerged as a fast growing methodology in me-
dicinal chemistry [6e8]. This strategy has been employed in the
search for new antimalarial compounds [9,10], and several ex-
amples of antiparasitic steroid-based hybrids have been reported
[11]. On the other hand, the efficacy of a drug is closely related to
its transport properties and bioavailability, which in turn are
related to other processes such as intestinal absorption, further
biotransformations, and the ease and mechanism of elimination
[12,13]. In this sense, bile acids are of great interest in drug dis-
covery because of their efficient transport system and their
properties as adsorption enhancers [14]. All these observations
led to the preparation of a new series of antiparasitic hybrids of
Cinchona alkaloids and bile acids [15]. The strategy was to
* Corresponding author.
E-mail address: palermo@qo.fcen.uba.ar (J.A. Palermo).
http://dx.doi.org/10.1016/j.ejmech.2015.05.044
0223-5234/© 2015 Published by Elsevier Masson SAS.

A. Leverrier et al. / European Journal of Medicinal Chemistry 100 (2015) 10e17
11
combine the antiparasitic properties of the natural Cinchona al-
kaloids, which are currently used to treat severe cases of malaria
[16], with the known properties of the bile acids as drug trans-
porters, especially taking into account that some bile acids also
have mild antiparasitic activity [17,18]. This strategy involved the
formation of a covalent CeC bond between the quinoline core of
the alkaloids and the side chain of a bile acid via a radical Bar-
toneZard decarboxylation reaction, constituting the first example
of this reaction using natural Cinchona alkaloids as substrates. The
BartoneZard reaction can be used for the formation of a new CeC
bond between a nucleophilic alkyl radical, obtained by decar-
boxylation of an O-acyl ester (Barton ester), and an electron-
deficient position of an aromatic substrate [19,20], such as the
C-20 position of the quinoline core of Cinchona alkaloids. In these
compounds C-40of the quinoline core is substituted, leaving C-20
as the only available position for a nucleophilic attack. From a
biological point of view, the incorporation of a norcholane moiety
to Cinchona alkaloids (quinine, quinidine, cinchonine and cin-
chonidine), had a positive effect on their in vitro antiparasitic
activity against Plasmodium falciparum and Trypanosoma brucei
[15]. In the first group of synthesized hybrids, the degree of
oxidation of the bile acid moiety had a marked influence on the
activity, since the compounds derived from chenodeoxycholic
acid were more active than those prepared from lithocholic acid.
For this reason, and as a continuation of this work, this promising
library of hybrids was expanded by the incorporation of four
additional bile acid moieties (cholic, deoxycholic, hyocholic and
hyodeoxycholic acids) in order to get a more complete picture of
the influence of the different hydroxy groups present on the ste-
roidal core. In the present work, 32 new hybrids were synthesised
(Fig. 1) to add to the series of the 16 previous derivatives prepared
from lithocholic and chenodeoxycholic acids [15]. The in vitro
antiparasitic activities of the hybrids 1e12aed, the four parent
alkaloids (QN ¼ quinine, QND ¼ quinidine, CN ¼ cinchonine,
CND ¼ cinchonidine) and the bile acids 1e12 were also evaluated
against P. falciparum and T. brucei brucei. The cytotoxicities of all
these compounds against the WI-38 cell line (human non cancer
fibroblast) were also determined. Based on previous disap-
pointing results [15], the activity of the new hybrids against
Leishmania mexicana mexicana was not tested. In this work, the
antiparasitic activity results of the complete series of 48 hybrids
are discussed, and some structure-activity relationships can now
be established.
2. Chemistry
Hybrids 3e6aed were prepared following the synthetic
pathway outlined in Scheme 1, using a BartoneZard decarboxyl-
ation reaction as described previously for the preparation of hy-
brids 1e2aed [15]. Briefly, the Barton esters of the peracetylated
bile acids 3e6, obtained respectively from bile acids 9e12, were
prepared by reaction with 2-mercaptopyridine-N-oxide and DCC
(N,N0-dicyclohexylcarbodiimide) in dry CH2Cl2 at 0 ꢀC. These esters
were then subjected to photolytic decarboxylation in the presence
of a large excess (10 eq.) of a protonated Cinchona alkaloid acting as
a radical trap, by irradiation with a 300 W tungsten lamp at 0 ꢀC in
CH2Cl2 under N2 atmosphere to yield the corresponding hybrids.
The purification of the final products was complicated by the large
excess of the free Cinchona alkaloids. For this reason, the yields in
Table 1 are of the final, purified compounds, and were calculated
after the 2 reactions and consecutive purification steps. In general,
the purification of the hybrids was achieved by normal-phase VLC,
to remove the less polar side-products, such as the thioether which
is the main side-product of the reaction [21], followed by reverse-
phase VLC to remove most of the large excess of the parent alka-
loid, and finally permeation through a Sephadex LH-20 column. The
removal of the acetate protecting groups was achieved by treat-
ment with 20% NaOH in MeOH under reflux, yielding quantitatively
the corresponding deacetylated hybrids (Scheme 1). Table 1 lists
the complete series of 48 hybrids, including those described herein
and in the previous work in order to get a full picture of some
structure-activity relationships [15].
3. Biological evaluation
The in vitro antiparasitic activities of the hybrids 3e6aed and
9e12aed as well as those of the parent free bile acids 7e12 and
acetylated bile acids 1e6, were evaluated against T. brucei brucei
bloodstream forms (Tbb), and also against
a chloroquine-
sensitive strain of Plasmodium falciparum (3D7). The cytotoxic-
ities of all these compounds were also evaluated against a human
normal fibroblast cell line (WI-38). Suramin, artemisinin and
campthotecin were used respectively as positive controls. In the
bioassays with T. brucei brucei and WI-38 cells, the hybrids were
11
10
3
OH
O
23''
R3
2
evaluated at a concentration range between 0.009
mg/ml and
12''
7
20 g/ml, and the corresponding IC50s (drug concentration
m
5
1''
H
9''
N
resulting in 50% inhibition of parasite or cell growth) were
calculated. In the case of P. falciparum, the hybrids were first
evaluated in duplicate at the same range of concentrations.
However, some of the compounds still showed a detectable
HO
6
1
9
H
H
R1
5'
4'
R'
HO 3''
6''
9'
H
R2
2'
N
10'
parasite growth inhibition at concentrations as low as 0.009
ml. In these cases, the evaluation was repeated (in triplicate) at
lower concentrations (between 4 g/ml and 1.8 ng/ml), in order
mg/
8'
Lithocholic acid (7) R1 = R2 = R3 = H
1'
Chenodeoxycholic acid (8) R1 = OH, R2 = R3 = H
Hyodeoxycholic acid (9) R2 = OH, R1 = R3 = H
Hyocholic acid (10) R1 = R2 = OH, R3 = H
Deoxycholic acid (11) R1 = R2 = H, R3 = OH
Cholic acid (12) R1 = R3 = OH, R2 = H
Quinine (QN) R' = OCH3, 9R
Quinidine (QND) R' = OCH3, 9S
Cinchonine (CN) R' = H, 9S
m
to confirm the IC50 values. The selectivity indices were calculated
for each parasite according to the formula: IC50(WI38)/IC50(par-
asite). Table 2 lists the in vitro biological activities of the com-
plete library of hybrids, which includes the 32 new compounds
described herein, as well as the 16 previously reported de-
rivatives [15] for a better understanding of the structure-activity
relationships among the compounds. The antiparasitic activities
and cytotoxicities of the parent bile acids 1e12 were also eval-
uated, and those results are displayed in Table 3. In the three
bioassays, the bile acids were only evaluated at 2 concentrations:
Cinchonidine (CND) R' = H, 9R
N
HO
R1
H
R2
H
R'
H
N
OR
H
20 and 100
20 g/ml, additional bioassays were performed at lower con-
centrations (100e0.5 g/ml) to determine the IC50s.
mg/ml. In the event of a larger than 50% inhibition at
R3
m
Fig. 1. Generic structure of the hybrids.
m

12
A. Leverrier et al. / European Journal of Medicinal Chemistry 100 (2015) 10e17
Scheme 1. Synthetic pathway for the preparation of the hybrids of Cinchona alkaloids and bile acids. Reagents and conditions: (i) 2-Mercaptopyridine-N-oxide (1.5 eq), DCC (1.5 eq),
CH2Cl2, 0 ꢀC; (ii) Cinchona alkaloids (10 eq), camphorsulfonic acid (20 eq), W-h
n; (iii) NaOH 20% in CH3OH, reflux.
4. Results and discussion
by spectroscopic methods. The molecular formulae were obtained
by ESIHRMS, and all the compounds were completely character-
ized by 1D and 2D NMR spectroscopy (See Supplementary
Information for NMR assignments). The formation of the new
CeC bond between C-20 of the quinoline nucleus and C-23 of the
4.1. Structural analysis of compounds 3e6aed and 9e12aed
The structures of the synthesized compounds were confirmed
Table 1
Structures of the synthesized hybrids and final purified yields.
Compound
R0
OH-9
R1
R2
R3
Yield (%)a
Deprotected compoundb
R ¼ COCH3
R ] H; R1, R2, R3 ] H or OHnd
1a
2a
3a
4a
5a
6a
1b
2b
3b
4b
5b
6b
1c
2c
3c
4c
5c
6c
1d
2d
3d
4d
5d
6d
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
H
H
H
H
R
R
R
R
R
R
S
S
S
S
S
S
S
S
S
S
S
S
R
R
R
R
R
R
H
OAc
H
OAc
H
OAc
H
OAc
H
OAc
H
OAc
H
OAc
H
OAc
H
OAc
H
OAc
H
OAc
H
H
H
OAc
OAc
H
H
H
H
OAc
OAc
H
H
H
H
H
H
H
OAc
OAc
H
H
H
21c
23c
23
19
30
26
13c
24c
36
27
21
16
23c
34c
49
22
23
14
19c
35c
41
2
7a
8a
9a
10a
11a
12a
7b
8b
9b
10b
11b
12b
7c
8c
9c
10c
11c
12c
7d
8d
9d
10d
11d
12d
H
OAc
OAc
H
H
H
H
OAc
OAc
H
H
H
H
OAc
OAc
H
H
H
H
OAc
OAc
H
OAc
OAc
H
H
H
H
24
18
OAc
H
a
The yields were calculated after the two steps of the reaction and the three steps of purification, and are based on the amount of starting bile acid peracetate.
Compounds obtained quantitatively after treatment of the corresponding peracetylated hybrids with NaOH in methanol under reflux. R1, R2, R3 ¼ H or OH.
Compounds obtained in our previous work, see Ref. [15].
b
c

A. Leverrier et al. / European Journal of Medicinal Chemistry 100 (2015) 10e17
13
Table 2
Biological evaluation of the parent alkaloids and hybrid compounds 1aede12aed.
Compound
Cytotoxicity WI-38
IC50 SD ( M)
Trypanosoma brucei brucei Tbb
Plasmodium falciparum 3D7
m
IC50 SD (
m
M)
Selectivity WI38/Tbb
Activity gain/alka
1.0
IC50 SD (nM)
632.7 348.8
Selectivity WI38/3D7
Activity gain/alka
QN
20.25 0.03
21.20 8.02
1.0
32.0
1.0
b
1aa
2aa
3a
4a
5a
23.67 1.78
5.19 1.76
5.86 1.79
3.76 0.08
4.41 0.38
4.47 0.27
>30.53
6.81 2.13
4.60 0.10
5.55 1.61
5.20 1.24
19.3 4.61
2.91 1.61
1.62 0.04
1.49 0.15
0.45 0.06
1.47 0.06
0.48 0.04
5.39 0.29
0.61 0.06
0.65 0.05
0.76 0.15
0.64 0.07
1.57 0.13
8.0
3.2
3.9
8.3
3.0
9.3
>5.7
11.1
7.0
7.3
8.7 4.7
2.7
5.6
0.1
0.7
1.4
11.3
3.2
14.6
0.1
1.4
2.8
2.3
2.6
0.3
13.1
14.3
46.8
14.4
44.1
3.9
34.7
32.4
27.8
33.3
13.5
920.5 255.6
459.6 437.2
56.0 19.8
13.0
67.2
22.0
103.0
>4.7
15.5
21.0
20.0
22.0
11.0
196.7 52.3
43.5 24.3
6a
b
7aa
8aa
9a
6.5 0.7
439.6 90.9
224.0 58.9
275.1 70.7
240.9 63.3
10a
11a
12a
7.3
8.2
12.3
b
1.82 0.75
QND
34.81 1.07
25.99 1.30
1.3
1.0
194.4 160.5
179.1
1.0
b
1ba
2ba
3b
4b
5b
11.18 2.81
4.11 1.47
4.49 0.68
3.29 0.58
5.33 0.88
4.66 1.16
3.95 2.63
2.91 1.25
2.20 0.63
4.75 0.27
4.30 1.45
11.82 1.04
4.26 0.37
0.58 0.01
1.47 0.17
0.56 0.23
0.69 0.19
0.53 0.09
1.21 0.29
0.55 0.04
0.89 0.29
0.77 0.21
0.61 0.03
1.21 0.47
2.6
7.1
3.1
5.8
7.7
8.7
3.3
5.3
2.5
6.2
7.1
9.8
6.1
1.2 0.2
9.7
8.1
0.2
0.4
1.3
2.7
1.0
2.3
0.2
0.9
1.6
1.1
0.7
0.3
44.6
17.7
46.2
37.5
48.8
21.5
47.1
29.2
33.9
42.7
21.6
508.6 102.0
154.6 41.3
73.2 20.8
195.0 49.1
83.8 46.2
29.0
45.0
27.0
56.0
3.8
14.0
18.0
27.9
15.0
17.0
6b
b
7ba
8ba
9b
1.0 0.0
207.2 14.9
123.5 57.4
170.2 79.9
281.2 112.5
700.1 358.0
10b
11b
12b
CN
>68.03
>68.03
n.d.
1.0
292.5 163.3
>222
1.0
b
1ca
2ca
3c
4c
5c
10.52 1.96
2.66 0.08
4.28 0.37
4.17 0.78
4.80 0.72
5.10 1.35
6.35 2.90
2.22 0.75
5.49 1.05
5.68 1.70
2.98 1.61
5.68 2.18
1.89 0.01
0.59 0.04
0.64 0.06
0.51 0.01
1.51 0.18
0.55 0.06
4.82 0.05
0.61 0.02
0.72 0.03
1.81 0.05
0.60 0.06
1.10 0.48
5.6
4.5
6.6
8.3
3.2
9.3
1.3
3.6
7.6
3.1
5.0
5.2
36.0
3.4 0.7
3.1
0.1
1.1
1.6
7.2
1.7
5.5
0.2
2.1
4.1
1.6
2.2
0.5
114.7
105.7
134.6
45.1
123.6
14.1
111.8
94.5
37.6
260.7 86.9
186.4 40.9
40.8 4.4
10.2
23.0
102.0
28.1
97.0
4.6
15.7
77.0
31.0
22.0
10.0
170.9 56.4
52.8 23.6
6c
b
7ca
8ca
9c
1.4 0.5
140.4 17.2
71.6 27.6
182.8 59.6
133.7 75.6
592.1 212.2
10c
11c
12c
113.1
61.8
CND
>68.03
62.18 4.18
n.d.
1.0
962.6 921.8
>71
1.0
b
1da
2da
3d
4d
5d
8.37 2.61
2.44 0.63
4.16 0.12
3.75 0.16
4.06 0.18
6.70 1.02
14.85 1.22
2.67 0.84
3.28 1.68
5.32 0.43
5.64 1.73
8.09 4.68
1.90 0.10
0.57 0.01
1.18 0.43
0.52 0.02
0.57 0.05
1.47 0.06
3.46 0.51
0.62 0.03
0.53 0.08
0.62 0.02
0.66 0.03
1.75 0.07
4.4
4.3
3.5
7.2
7.1
4.6
4.3
4.3
6.2
8.6
8.6
4.6
32.7
110.0
52.5
119.8
108.2
42.4
18.0
99.6
116.7
100.5
94.8
1.8 0.7
4.6
8.4
0.5
3.3
8.3
26.6
12.5
23.7
0.6
2.7
10.5
2.5
289.7 51.0
115.4 35.0
36.1 25.2
77.2 19.3
40.6 21.3
36.0
103.7
53.0
165.0
8.7
6d
b
7da
8da
9d
1.7 0.5
355.7 31.2
92.1 42.7
392.2 38.5
320.3 85.9
7.5
36.0
14.0
18.0
4.0
10d
11d
12d
3.0
0.5
b
35.6
1.8 0.7
Campthotecin
Suramine
Artemisinin
0.23 0.06
n.d.
n.a.
n.d.
0.03 0.01
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
36.2 14.2
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
SD ¼ standard deviation; selectivity ¼ IC50(WI38)/IC50(Tbb or 3D7); n.d. ¼ not determined; n.a. ¼ not active; activity gain/alka ¼ IC50 (alkaloid)/IC50 (compound).
a
Compounds reported in Ref. [15].
IC50(3D7) expressed in mM.
b
norcholane group could be rapidly confirmed by NMR analysis.
The NMR signals corresponding to H-20 (8.70 ppm) and CH-20
(typically at 147e150 ppm) of the Cinchona alkaloids were no
longer present in the spectra of the hybrids, while a new signal for
a quaternary carbon at 160 ppm was evident in the 13C NMR
spectra. Finally, HMBC correlations between H-30, H-2200, H-2300
and C-20, and between H-30 and C-2300, confirmed the newly
formed CeC bond and the structure of the compounds (S29
Supplementary Information).
The initial set of hybrids was obtained as their peracetylated
forms, as it was previously observed that the formation of the
Barton esters was not complete when the bile acids had free hy-
droxy groups [22]. The choice of acetate as protective group was
based on its simple protection/deprotection reactions and on the

14
A. Leverrier et al. / European Journal of Medicinal Chemistry 100 (2015) 10e17
Table 3
Biological evaluation of free and acetylated bile acids 1e12.
Bile acid
Cytotoxicity WI-38
% Inhibition
Trypanosoma brucei brucei Tbb
Plasmodium falciparum 3D7
IC50
SD
nd
16.8 1.8
nd
nd
nd
nd
nd
(
m
M)
% Inhibition
IC50
SD
nd
12.5 1.5
16.3 1.2
5.6 1.4
nd
nd
nd
nd
(
m
M)
% Inhibition
IC50
(mM)
100
m
g/ml
20
m
g/ml
100
mg/ml
20
mg/ml
100
m
g/ml
20
70
59 24
73
88
82 11
68 17
85 12
77 14
m
g/ml
8
SD
1
2
3
4
5
6
7
8
98
98
98
96
98
29 25
98
94
22 29
nd
50 22
nd
0
0
0
1
0
27
57
20
9
10
12
22
27
21
18
9
4
2
1
6
0
6
3
9
8
4
1
0
100
100
100
100
100
100
100
100
94
0
0
0
0
0
0
0
0
6
15 11
100
90
97
1
3
1
2
6
3
1
1
2
59.5 28.7
21.0 2.2
32.7 20.8
8.1 5.0
30.5 15.4
22.5 9.1
26.9 4.4
18.9 2.4
40.9 22.0
109.0 25.5
93.6 22.8
132.7 51.6
99
81
1
5
9
1
83 13
97
36
15
5
11
1
7
2
20
5
6
6
6
1
6
2
1
100
100
100
98
0
1
nd
nd
nd
nd
9
nd
nd
nd
nd
97
67
9
10
11
12
64 45
100
24
92 10
54 44
59 44
56 54
0
0
100
3
6
nd
83 13
increased bioavailability and reduced toxicity of some acetylated
compounds [23]. After deprotection under basic conditions, the
structures of the deacetylated hybrids were confirmed by the
disappearance of the acetyl singlets in the 1H NMR spectrum
(between 2.01 and 2.13 ppm), and of the C]O intense band in the
IR spectra (1730 cmꢁ1), along with the appearance of a wide band
at 3300 cmꢁ1 corresponding to the hydroxyl groups (see
Supplementary Information). In the case of compounds 11b, 11c,
12b and 12c, the NMR spectra had to be recorded in CD3OD, due to
the extensive broadening of some signals corresponding to the
quinoline protons, especially H-80, in the spectra recorded in
CDCl3 (S30 Supplementary Information). This broadening of the
quinoline signals is probably due to conformational reasons. This
behaviour was observed only in the spectra of the hybrids ob-
tained from quinidine and cinchonine, in which the hydroxylated
C-9 has an S configuration, bound to a norcholane moiety derived
from cholic or deoxycholic acid, both of which have a hydroxy
group at C-12. It is possible that in compounds 11b, 11c, 12b and
12c those two hydroxy groups may induce a conformation in
chloroform which favours proton exchange, and leads to signal
broadening.
against the WI-38 cell line with IC50 values between 2.20 and
23.7 M. In general, the cytotoxicity of the Cinchona alkaloids was
increased when a norcholane residue was attached at C-2, except
for hybrids 1a, 7a and 12a (IC50 ¼ 23.7, >30.5 and 19.3 M,
respectively) which exhibited IC50s comparable to that of quinine,
M). Generally speaking, the
m
m
their parent alkaloid (IC50 ¼ 20.3
m
hybrids with lithocholic or cholic moieties were the least cytotoxic
in this series (Table 2).
4.2.3. Antitrypanosomal activity
All the tested hybrids displayed good activities against
T. brucei, with IC50 values ranging from 0.48 to 5.39
mM and
selectivity indices from 1.3 to 12.3. Moreover, the activities of the
hybrids were higher than those of their corresponding parent
alkaloids, as determined by the ratio: IC50 (alka)/IC50 (compound).
This was especially evident in the cases of cinchonine and cin-
chonidine (ratios up to 100-fold and higher) (Table 2). The selec-
tivity indices (antitrypanosomal activity vs. cytotoxicity) were all
favourable, with the quinine derivative 12a showing the largest
ratio (12.3 times more active than cytotoxic, however with only a
13.5-fold increase in activity compared to quinine). Interestingly,
compound 12a was also one of the few hybrids that had a cyto-
toxic activity comparable to that of quinine. The absence of in vitro
antitrypanosomal activity for the bile acids 7e12 is remarkable
when compared to the activity of their corresponding hybrids, and
suggests that this increase in activity is not due simply to an ad-
ditive effect of the bile acids, but may also be related to an
improved penetration capacity of the drug. Interestingly, in the
case of the hybrids, the acetate groups did not have a marked
influence on the in vitro activity, as opposed to the trend observed
with the bile acids 1e12. In the previous work [15], it was
observed that the antitrypanosomal activity of the hybrids
increased with the presence of a second oxygenated group in the
bile acid moiety (IC50s (1aed, 7aed) > IC50s (2aed, 8aed)). This
trend was confirmed by the present results; however the
observed activities were comparable for all the dioxygenated bile
acid derivatives irrespectively of the positions of the hydroxy or
acetoxy groups. A third hydroxy group, as in cholic acid de-
rivatives, did not induce an additional increase in activity. The
structure of the alkaloid did not have a great influence in the
antitrypanosomal activity of the hybrids. All these observations
show that this activity is not as specific as expected within each
series of compounds. However, the present results show some
differences in the selectivity indices of the hybrids, mainly due to
their variable cytotoxicities (Table 2). Fourteen of the tested hy-
brids had good selectivity indices (SI > 7, SI: IC50WI38/IC50Tbb).
According to Pink et al. the criteria for antiparasitic hits are
4.2. Biological activities
4.2.1. Biological evaluation of bile acids 1e12
Although there are several examples of antiparasitic steroids [24],
to the best of our knowledge there are no previous reports of this
kind of activity for the bile acids. In order to test the possible in-
fluence of the norcholane moiety, compounds 1e12 were evaluated
against the human normal fibroblast cell line WI-38, T. brucei brucei
and P. falciparum (Table 3). None of the bile acids used in the present
work was significantly cytotoxic at 20
the peracetate of chenodeoxycholic acid 2, which was moderately
M). The results of
mg/ml, with the exception of
cytotoxic against the WI-38 cell line (IC50 ¼ 16.8
m
the in vitro bioassays revealed that the peracetylated derivatives
were generally more cytotoxic against WI-38 than the free bile acids.
Regarding the antitrypanosomal activity, the peracetylated bile acids
2e4 exhibited a moderate activity (IC50s between 5.6 and 16.3
mM),
while the other bile acids were inactive at 20 g/ml. These results
m
suggest that the acetate groups may have a positive effect on the
bioavailability of the compounds due to an increased lipophilicity.
Besides, the 12 bile acids had moderate in vitro antiplasmodial ac-
tivity (8.1 ꢂ IC50 ꢂ 132.7
mM), with the peracetylated hyocholic acid
4 standing out as the bile acid with the highest activity.
4.2.2. Cytotoxicity of the hybrids
Compounds 1e12aed showed moderate cytotoxic activity

A. Leverrier et al. / European Journal of Medicinal Chemistry 100 (2015) 10e17
15
IC50 < 1
promising hybrids would be compounds 6a, 6c, 8a, 12a and 12b
which have IC50s of 0.68, 0.55, 0.61, 1.57, 1.21 M (0.39, 0.43, 0.41,
1.08, and 0.83 g/ml, respectively) and selectivity indices: 9.3, 9.3,
m
g/ml and SI > 10 [25]. Following these rules, the most
moieties, which showed a moderate activity against P. falciparum
(Table 3).
m
m
11.1, 12.3 and 9.8 respectively. It is interesting to note that except
for 8a, all the antitrypanosomal “hits” of the series have a cholic
acid moiety which was also the bile acid with the lowest cyto-
toxicity (Table 3).
5. Conclusions
The synthesis and biological evaluation of 32 new hybrids of bile
acids and Cinchona alkaloids is reported. These hybrids differ in
their alkaloid moiety (quinine, quinidine, cinchonine or cinchoni-
dine) and the number and position of the oxygenated groups (hy-
droxy or acetate) of their norcholane fragment. All the hybrids
exhibited interesting in vitro antitrypanosomal and antiplasmodial
activities, but also low cytotoxicities against the normal cell line
WI-38. The activity of the hybrids against T. brucei brucei was
considerably improved compared to that of Cinchona alkaloids,
particularly when at least two oxygenated groups were present in
4.2.4. Antiplasmodial activity e SAR studies
Table 2 also shows the results of the in vitro antiplasmodial
evaluation of the 48 hybrids, all of which inhibited parasite growth
with IC50s ranging from 36.1 nM to 8.72 mM, and selectivity indices
ranging from 3 to 165. In Table 4, there is a comparison of the IC50
ranges of the different hybrids based on the structural features of
the bile acid moiety (number and position of the oxygenated sub-
stituents), while the IC50s of the individual compounds are listed in
Table 2. As observed previously [15], the antiplasmodial activity
increased with the number of oxygenated groups on the norcho-
lane moiety, and most of the compounds with at least two hydroxy
or acetate groups had good antiplasmodial activities. Those com-
pounds with oxygenated groups at positions 3, 6 (compounds 3aed
and 9aed) or 3, 12 (5aed; 11aed) had higher selectivity indices
(13 ꢂ SI ꢂ 77) compared with compounds 2aed and 8aed, which
have oxygenated groups at positions 3, 7 (6 ꢂ SI ꢂ 16). Furthermore,
those hybrids with oxygenated groups at positions 3, 6 or 3, 12 had
antiplasmodial activities which were comparable or higher than
those of their parent natural alkaloids (Tables 2 and 4), albeit with
lower selectivity indices. The most promising compounds were
those with three acetate groups, namely 4aed and 6aed, which
exhibited good in vitro activity against P. falciparum with
IC50s < 84 nM and selectivity index values higher than 45. In
particular, compounds 4c, 4d, 6a and 6d are respectively 7.2, 26.6,
14.6 and 23.7 times more active than their parent alkaloids, while
their IC50s (40.8, 36.1, 43.5 and 40.6 nM) and SI (102, 104, 103 and
165, respectively) are in the same range as that of artemisinin, the
reference compound used in the assays (IC50s ¼ 36.2 nM). Per-
acetylation of the norcholane moiety had a positive influence on
the antiplasmodial activity of those hybrids derived from hyocholic
and cholic acids, in an opposite sense as the effect generally
observed for the hybrids with one or two oxygenated groups. The
same trend was also observed for the parent bile acids (Table 3).
The lower activity of compounds 10aed and 12aed, compared to
4aed and 6aed, may be related to their reduced lipophilicity, due
to the presence of three hydroxy groups, which could lead to a
lower penetration capacity. All these observations suggest that the
improvement of the antiplasmodial activity observed for several
hybrids compared to the natural Cinchona alkaloids may be related
to their lipophilicity and an increased penetration capacity, with
perhaps a small additive antiparasitic effect of the bile acid
the norcholane moiety. Among them, the hybrids having
300 ,700 ,1200
-trioxygenated norcholane moiety (6a, 6b, 12a, 12b)
emerged as the most interesting trypanocidal “hits” with
IC50s ꢂ 1.5 M and selectivity indices > 9, due to their low
cytotoxicities.
a
a
a
a
m
Regarding the activity against P. falciparum, an increase in ac-
tivity was observed with the number of oxygenated groups of the
norcholane backbone, and some of the hybrids displayed better
activities than the natural Cinchona alkaloids. In particular, those
hybrids prepared from peracetylated cholic and hyocholic acids
were the most promising compounds of the series, with IC50s in the
same range as artemisinin.
In summary, the addition of a norcholane moiety to Cinchona
alkaloids had a favourable effect on the antiparasitic activity for
some of the compounds. This work produced several candidates,
namely compounds 4aed and 6aed, for further biological studies
such as in-vivo bioassays. These compounds can also be further
modified for structure-activity improvement. This increase in ac-
tivity may be related mostly to an enhanced bioavailability and cell
penetration capacity of the hybrids compared to the natural alka-
loids, since the bile acids per se did not display potent antiparasitic
activities. This hypothesis needs to be confirmed by additional
experiments. However, since the newly formed CeC bond con-
necting both parts in the hybrids is not hydrolysable, it is probable
that the bile acid moiety may also play a role in molecular recog-
nition, and, for this reason, compounds 1e12aed should not be
considered prodrugs. Also, in a future work, the hybrids will be
evaluated against other chloroquine-resistant strains of
P. falciparum, in order to estimate their effect on the resistance
phenomenon. In conclusion, in this work, the strategy of bio-
conjugation was used for the design and synthesis of promising
antiparasitic hybrids, with improved activity compared to the
parent alkaloids that can be possibly explained in terms of a better
bioavailability.
Table 4
Comparison of in vitro antiplasmodial activities.
Num oxygenated groups
1
Type
Positions
3
Compounds
IC50 (3D7)
Selectivity Index
Activity gain vs. natural alkaloid
OAc
OH
OAc
1aed
7aed
2aed
3aed
5aed
8aed
9aed
11aed
4aed
6aed
10aed
12aed
1.2e8.7
1.0e6.5
m
m
M
M
3.1e9.7
3.8 - > 8.7
5.6e10.2
No
No
2
3
3, 7
3, 6
3, 12
3, 7
3, 6
3, 12
3, 6, 7
3, 7, 12
3, 6, 7
3, 7, 12
260.7e920.5 nM
115.4e459.6 nM
77.2e196.7 nM
140.4e439.6 nM
71.6e224.0 nM
133.7e320.3 nM
36.1e73.2 nM
40.6e83.7 nM
170.2e392.2 nM
592.1 nMe1.8 mM
0.4e3.3
1.3e8.3
1.0e12.5
0.9e2.7
1.6e10.5
0.7e3.0
2.7e26.6
2.3e23.7
1.1e2.5
No
12.7e36.0
22.4e52.5
7.5e15.8
17.8e76.7
15.3e22.3
44.9e103.7
55.6e165.0
13.6e31.0
4.4e16.9
OH
OAc
OH

16
A. Leverrier et al. / European Journal of Medicinal Chemistry 100 (2015) 10e17
6. Experimental section
6.2.3. Compounds 1e2aed and 7e8aed
See Ref. [15].
6.1. General
6.2.4. Compounds 3e6aed and 9e12aed
Bile acids (cholic, deoxycholic, hyocholic and hyodeoxycholic
acids) and Cinchona alkaloids (quinine, quinidine, cinchonine,
cinchonidine) were obtained from commercial sources and used
as such or recrystallized prior to use when necessary. The per-
acetylated bile acids were obtained by standard procedures, using
Ac2O/DMAP/Pyridine. 2-Mercaptopyridine-N-oxide, N,N0-dicy-
clohexylcarbodiimide (DCC) and camphorsulfonic acid were pur-
chased from Aldrich. All the solvents were distilled prior to use,
and CH2Cl2 used for reactions was bidistilled from phosphorous
pentoxide. NMR spectra were recorded in CDCl3 or CD3OD on
Bruker AC-200 (200.13 MHz) and Bruker Avance II (500.13 MHz)
spectrometers, using the signals of the residual nondeuterated
solvent as internal reference (dH 7.26, dC 77.0 for chloroform and dH
3.31, dC 49.0 for methanol). All 2D NMR experiments (COSY, DEPT-
HSQC, HMBC, and NOESY) were performed using standard pulse
sequences. HRMS were recorded on a Bruker micrOTOF-Q II
spectrometer. IR spectra were obtained on an FT-IR Nicolet Magna
550 instrument, optical rotations on a Perkin Elmer Polarimeter
343 (at 589 nm), and melting points on a Fisher-Johns apparatus.
TLCs were carried out on Merck Sílicagel 60 F254 plates. TLC plates
were sprayed with 2% vanillin in concentrated H2SO4 or with
Dragendorff's spray reagent (Aldrich). Merck Silicagel (230e400
mesh) and RP-18 (Aldrich) stationary phases were used for Vac-
uum Column Chromatography. Sephadex LH-20 (GE Healthcare)
was used for exclusion chromatography.
See Supplementary Information.
6.3. Biological assays
6.3.1. Parasites, cells and media
Trypanosoma brucei brucei (strain Lister 427) bloodstream forms
were cultured in vitro in HMI9 medium containing 10% heat-
inactivated foetal bovine serum [26]. Parasites were incubated in
a humidified atmosphere with 5% CO2 at 37 ꢀC.
P. falciparum (3D7, was originally isolated from a patient living
near Schipol airport, The Netherlands) asexual erythrocytic stages
were cultivated continuously in vitro according to the procedure
described by Trager and Jensen (1976) at 37 ꢀC and under an at-
mosphere of 5% CO2, 5% O2 and 90% N2 [27]. The host cells were
human red blood cells (A or O Rhþ). The culture medium was RPMI
1640 (Gibco) containing 32 mM NaHCO3, 25 mM HEPES and L-
glutamine. The medium was supplemented with 1.76 g/l glucose
(SigmaeAldrich), 44 mg/ml hypoxanthin (SigmaeAldrich), 100 mg/
l gentamycin (Gibco) and 10% human pooled serum (A or O Rhþ).
Parasites were subcultured every 3e4 days with initial conditions
of 0.5% parasitaemia and 1% haematocrit.
The human non-cancer fibroblast cell line WI38, was cultivated
in vitro in DMEM medium (Gibco) containing 4 mM L-glutamine,
1 mM sodium pyruvate supplemented with 10% heat-inactivated
foetal bovine serum (Gibco) and penicillinestreptomycin (100 UI/
ml to 100 mg/ml). Cells were incubated in a humidified atmosphere
6.2. Chemistry
with 5% CO2 at 37 ꢀC.
6.2.1. General procedure for the formation of hybrids 3e6aed
In a flask protected from light with aluminium foil, the per-
acetate of a bile acid (3e6, around 0.2e0.3 mmol) and 2-
mercaptopyridine-N-oxide (1,5 equivalents) were dissolved in
4 ml of dry CH2Cl2, and the solution was cooled to 0 ꢀC. Then,
1.5 eq.eq. of DCC were added, the solution was stirred at 0 ꢀC for
2e3 h, and the reaction was monitored by TLC. The solution was
filtered through a cotton plug, to remove dicyclohexylurea, into a
flask containing a Cinchona alkaloid (QN, QND, CN or CND, 10 eq.)
and camphorsulfonic acid (20 eq.) dissolved in 4 ml of dry CH2Cl2.
The solution was cooled to 0 ꢀC, placed under N2, and subsequently
irradiated using a single 300 W tungsten lamp during 1h150. The
reaction was quenched with an aqueous solution of NaOH (2 N) and
extracted 3 times with CH2Cl2. The organic layers were combined,
washed with water, dried over Na2SO4 and evaporated yielding a
residue. The latter was quickly separated in 4 fractions by VLC on
silica, eluting with cyclohexane/EtOAc/methanol 1/1/0, 0/1/0, 0/95/
5 and 0/9/1 respectively. Fractions 3 and 4 were pooled together to
be further purified by VLC on reversed-phase (Rp-18), eluting with
mixtures of H2O/MeOH from 1/0 to 0/1, increasing the proportion of
methanol by steps of 10%. Fractions eluted with 100% MeOH, con-
tained the hybrid, together with a small amount of the parent
alkaloid. These fractions were taken to dryness and submitted to a
final purification by Sephadex LH-20 (MeOH) to yield the desired
hybrids 3e6aed.
6.3.2. In vitro antitrypanosomal activity
The in vitro tests were performed in 96-wells microtiter plates as
described by Hoet et al. (2004) [28]. Suramin (a commercial anti-
trypanosomal drug) was used as positive control. First, stock so-
lutions of the compounds were prepared in DMSO at 4 mg/ml for
the hybrids and for suramin, and at 10 mg/ml for the bile acids. The
highest concentration of solvent to which the parasites were
exposed was 0.5%, which was shown to have no measurable effect
on parasite viability. Bile acids 1e12 were tested at 20 and 100 mg/
ml in duplicate (six wells/concentration) and in addition bile acids
2e5 were tested in triplicate in an eight serial three-fold dilutions
(final concentration range: 100e0.05 mg/ml, two wells/concentra-
tion). Suramin and hybrids 3e6aed and 9e12aed were tested in
eight serial three-fold dilutions (final concentration range:
20e0.009 mg/ml, two wells/concentration), at least in triplicate.
6.3.3. In vitro antiplasmodial activity
Parasite viability was measured using parasite lactate dehy-
drogenase (pLDH) activity according to the methods described by
Makler et al. [29]. The in vitro tests were performed in 96-wells
microtiter plates as described by Murebwayire et al. [30]. The
parasitaemia and the haematocrit were 2% and 1%, respectively.
Artemisinin (Sigma) was used as positive control in all experiments
with an initial concentration of 100 ng/ml. First stock solutions of
the compounds were prepared in DMSO at 4 mg/ml for the hybrids
and 10 mg/ml for the bile acids. The highest concentration of DMSO
to which the parasites were exposed was 1%, which was shown to
have no measurable effect on parasite viability. Bile acids 1e12
6.2.2. Deacetylation of hybrids 3e6aed
A solution of NaOH in methanol (20%) was added to compounds
3e6aed to reach a concentration of 1 mg/ml, and the solution was
then refluxed overnight. The solvent was removed under reduced
pressure and the residue dissolved in H2O before extraction with
dichloromethane. Evaporation of the organic layer yielded quanti-
tatively the deacetylated compound (9e12aed).
were tested at 20 and 100
tration) and then in duplicate in an eight serial three-fold dilutions
(final concentration range: 100e0.05 g/ml, two wells/concentra-
tion). Hybrids 3e6aed and 9e12aed were tested in two eight serial
three-fold dilutions (final concentration range: 20e0.009 g/ml
mg/ml in duplicate (four wells/concen-
m
m

A. Leverrier et al. / European Journal of Medicinal Chemistry 100 (2015) 10e17
Chem. 83 (2014) 274e283.
17
and 4000e1.8 ng/ml, two wells/concentration), in duplicate or
[3] M.V. de Oliveira Cardoso, L. Rabelo Pessoa de Siqueira, E. Barbosa da Silva,
L. Bandeira Costa, M. Zaldini Hernandes, M. Montenegro Rabello, R. Salgado
triplicate for each range of concentrations.
~
^
Ferreira, L. Faria da Cruz, D. Rodrigo Magalhaes Moreira, V. Rego Alves Pereira,
M.C. Accioly Brelaz de Castro, P.V. Bernhardt, A.C. Lima Leite, Eur. J. Med.
Chem. 86 (2014) 48e59.
6.3.4. Cytotoxicity assay
The cytotoxicity of compounds on WI38 cells was evaluated as
described by Stevigny et al. [31], in 96-well microtiter plates, using
the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (Sigma)) colorimetric method based
on the cleavage of the reagent by mitochondrial dehydrogenases in
viable cells [32]. Camptothecin was used as positive cytotoxic
reference compound. First stock solutions of the compounds were
prepared in DMSO at 4 mg/ml for the hybrids and camptothecin
and at 10 mg/ml for the bile acids. The highest concentration of
solvent to which the cells were exposed was 1%, which was shown
to be non-toxic. Bile acids 1e12 were tested at 20 and 100 mg/ml in
duplicate (four wells/concentration) and in addition compound 2
was tested in triplicate in a eight serial three-fold dilutions (final
mg/ml, two wells/concentration).
The solutions of hybrids 3e6aed and 9e12aed and of campto-
thecin were tested in eight serial threefold dilutions with a final
ꢀ
[4] M.P. Carrasco, A.S. Newton, L. Gonçalves, A. Gois, M. Machado, J. Gut,
€
F. Nogueira, T. Hanscheid, R.C. Guedes, D.J.V.A. dos Santo, P.J. Rosenthal,
R. Moreira, Eur. J. Med. Chem. 80 (2014) 523e534.
[5] R.S. Rao Vidadala, K.K. Ojo, S.M. Johnson, Z. Zhang, S.E. Leonard, A. Mitra,
R. Choi, M.C. Reid, K.R. Keyloun, A.M.W. Fox, M. Kennedy, T. Silver-Brace,
J.C.C. Hume, S. Kappe, C.L.M.J. Verlinde, E. Fan, E.A. Merritt, W.C. Van Voorhis,
D.J. Maly, Eur. J. Med. Chem. 74 (2014) 562e573.
[6] B. Meunier, Acc. Chem. Res. 41 (2008) 69e77.
[7] G. Mehta, V. Singh, Chem. Soc. Rev. 31 (2002) 324e334.
[8] L.F. Tietze, H.P. Bell, S. Chandrasekhar, Angew. Chem. Int. Ed. 42 (2003)
3996e4028.
[9] F.W. Muregi, A. Ishih, Drug Dev. Res. 71 (2010) 20e32.
[10] R.A. Massarico Serafim, J.E. Gonçalves, F. Pereira de Souza, A.P. de Melo
Loureiro, S. Storpirtis, R. Krogh, A. Defini Andricopulo, L.C. Dias, E.I. Ferreira,
Eur. J. Med. Chem. 82 (2014) 418e425.
[11] As examples see (a) L.M.R. Antinarelli, A.M.L. Carmo, F.R. Pavan, C.Q.F. Leite,
A.D. Da Silva, E.S. Coimbra, D.B. Salunke, Org. Med. Chem. Lett. 2 (2012) 2e16;
(b) R.C.N.R. Corrales, N.B. de Souza, L.S. Pinheiro, C. Abramo, E.S. Coimbra,
A.D. Da Silva, Biomed. Pharmacother. 65 (2011) 198e203.
concentration range: 100e0.05
[12] G.A. Kullak, M.B. Becker, Drug Metab. Rev. 35 (2003) 305e317.
[13] R.H. Ho, R.B. Kim, Clin. Pharmacol. Ther. 78 (2005) 260e277.
[14] A. Enhsen, W. Kramer, G. Wess, Drug Discov. Today. Rev. 3 (1998) 409e418.
concentration range of 20e0.009 mg/ml (two wells/concentration),
at least in triplicate.
ꢀ
ꢀ
[15] A. Leverrier, J. Bero, M. Frederich, J. Quetin-Leclercq, J. Palermo, Eur. J. Med.
Chem. 66 (2013) 355e363.
[16] S. Hoet, F. Opperdoes, R. Brun, V.J. Quetin-Leclercq, Nat. Prod. Rep. 21 (2004)
353e364.
[17] E. Sievanen, Molecules 12 (2007) 1859e1889.
Acknowledgement
€
Research at the University of Buenos Aires was supported by
Grants from CONICET (PIP No. 516-2009), UBACYT
(20020100100123, Prog. 2011-2014), and ANPCYT (PICT 2010-
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