New promising compounds with in vitro nanomolar activity against Trypanosoma cruzi

Article abstract of DOI:10.1021/ml400039r

The antiparasitic activity of azole and new 4-aminopyridine derivatives has been investigated. The imidazoles 1 and 3-5 showed a potent in vitro antichagasic activity with IC₅₀ values in the low nanomolar concentration range. The (S)-1, (S)-3,

Full text of DOI:10.1021/ml400039r

Letter
pubs.acs.org/acsmedchemlett
New Promising Compounds with in Vitro Nanomolar Activity against
Trypanosoma cruzi
Laura Friggeri,† Luigi Scipione,† Roberta Costi,‡ Marcel Kaiser,§ Francesca Moraca,⊥ Claudio Zamperini,⊥
Bruno Botta,† Roberto Di Santo,‡ Daniela De Vita,*,† Reto Brun,*,§,∥ and Silvano Tortorella†
†Dipartimento di Chimica e Tecnologie del Farmaco, “Sapienza” Universita
̀
di Roma, P.le A. Moro 5, 00185 Rome, Italy
‡Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, “Sapienza” Universita
̀
di Roma,
P.le A. Moro 5, 00185 Rome, Italy
§Department Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, Socinstrasse 57, CH-4051 Basel,
Switzerland
∥University of Basel, Petersplatz 1, CH-4003 Basel, Switzerland
⊥Dipartimento di Biotecnologie, Chimica e Farmacia, Universita
̀
degli Studi di Siena, Via A. Moro 2, 53100 Siena, Italy
S
* Supporting Information
ABSTRACT: The antiparasitic activity of azole and new 4-aminopyridine derivatives has been
investigated. The imidazoles 1 and 3−5 showed a potent in vitro antichagasic activity with IC50 values
in the low nanomolar concentration range. The (S)-1, (S)-3, and (S)-5 enantiomers showed (up to) a
thousand-fold higher activity than the reference drug benznidazole and furthermore low cytotoxicity
on rat myogenic L6 cells.
KEYWORDS: Trypanosoma cruzi, CYP51, antiparasitic activity, Chagas disease, azole, aminopyridine
hagas disease (CD) is one of the most important
neglected tropical disorders, caused by the Kinetoplastid
ketoconazole, fluconazole, posaconazole, and voriconazole)
C
are known as CYP51Tc inhibitors due to the presence of an
imidazole or triazole ring able to coordinate the heme-iron.9,10
Moreover, recent studies demonstrated that N-heterocyclic
rings can be effectively replaced by 3-pyridyl or 4-pyridyl
moieties.11−13
We have selected eight compounds available from our
laboratory library: racemic 1-imidazolyl-2-phenylethanol de-
rivatives14 (1−5) (Figure 1) and new 4-aminopyridines (6−8)
(Scheme 1). Compounds 1−5 belonging to azole class have
been chosen for their structural similarity with CYP51Tc
inhibitors; the new 4-aminopyridines (6−8), second generation
parasite Trypanosoma cruzi,1 and approximately 10 million
people worldwide are estimated to be infected.2 CD is a
complex anthropozoonosis transmitted to humans by blood-
sucking insects of the Triatominae subfamily (mainly Triatoma
infestans, Rhodnius prolixus, and Triatoma dimidiatae).3
Currently, the treatment of CD is restricted to nifurtimox
and benznidazole used only in the acute phase. The clinical
efficacy in chronic patients is limited and controversial, and the
undesirable side effects of both drugs are a major drawback in
their use.4,5 To date, posaconazole and E1224, a pro-drug of
ravuconazole, are in phase 2 of clinical trials for chronic Chagas
disease treatment.6 However, the development of new effective
and safe drugs is urgent because of the lack of vaccines, the
inadequate chemotherapy, the large number of infected people,
and their migration toward nonendemic countries.
Similar to fungi and yeasts, Kinetoplastides are strictly
dependent on endogenously produced sterols, essential cellular
components, that modulate membrane fluidity/permeability
and also play multiple regulatory functions related to cell
division, growth, and developmental processes. Sterol 14α-
demethylase (CYP51) is an essential enzyme in sterol
biosynthesis and its inhibition causes the block of sterol
production and the accumulation of toxic methylated sterol
precursors followed by pathogen growth arrest and death.7,8
For these reasons, CYP51 of T. cruzi (CYP51Tc) is a highly
drug-targetable enzyme for the rational design of new
antitrypanosomal drugs. Currently, antifungal azoles (i.e.,
Figure 1. Compounds 1−5 described in this study.
Received: February 4, 2013
Accepted: May 7, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/ml400039r | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
a
showed that 1 and 3−5 possess high activity toward T. cruzi
with IC50 values from 5.0 to 36.0 nM; 3 and 4 turned out to be
the most active compounds with an IC50 of 5.0 nM.
Furthermore, 1 and 3−5 proved to be highly selective against
T. cruzi, as evidenced by low IC50 values compared to the other
studied parasites, characterized by low cytotoxicity with IC50
values ranging from 13.0 to 27.2 μM. The new 4-aminopyridine
derivatives 6−8 showed lower antiparasitic activity than the
imidazoles 1−5.
Scheme 1. Synthesis of Compounds 6−8
The high activity of the racemic azole derivatives prompted
us to define the differences in the antitrypanosomal activity of
the single enantiomers.
Compounds 1, 3, and 5 have been selected and pure (R)-1,
(S)-1, (R)-3, (S)-3, (R)-5, and (S)-5 have been prepared as
previously described for racemic compounds14 from the
corresponding enantiomeric 1-(phenyl)-2-(1H-imidazol-1-yl)-
ethanols obtained by modification of a reported procedure.16
The following enantiomeric purity was observed: (R)-1 98.0%
ee; (S)-1 98.0% ee; (R)-3 98.5% ee; (S)-3 98.5% ee; (R)-5
98.8% ee; (S)-5 98.8% ee (see Supporting Information).
The antiparasitic activity of pure enantiomers is reported in
Table 2 and clearly indicate that both the (R) and (S)
a
(a) TEA, triphosgene, anhydrous benzene, reflux, 5 h; (b)
metronidazole for compound 6, 12 h, r.t. (yield 68%); 2-
(morpholin-4-yl)ethanol for compounds 7 (yield 70%) and 8 (yield
60%), 12 h at r.t.
compounds, have been selected for the presence of the N-4-
pyridyl moiety able to interact with the CYP51Tc active site, as
previously reported.12 Compounds 1−8 have a nitrogen atom
for the heme-iron coordination and hydrophobic moieties able
to interact with lipophilic tunnel in the CYP51Tc active site.
Here, we report the evaluation of their antiparasitic activity.
Compounds 1−5 have been prepared as previously
reported.14 Compound 6 was obtained by reaction of
metronidazole with 3-bromo-4-aminopyridine previously acti-
vated with triphosgene, in anhydrous benzene; 7 and 8 were
similarly obtained by 2-(morpholin-4-yl)ethanol with 2,6-
dichloro-4-aminopyridine and 2-chloro-4-aminopyridine, re-
spectively (Scheme 1).
The biological activity of the selected compounds has been
evaluated as reported elsewhere15 against different parasites: T.
brucei rhodesiense STIB 900 trypomastigote stage; T. cruzi
Tulahuen C2C4 amastigote stage; L. donovani MHOM-ET-67/
L82 amastigote stage; P. falciparum K1 erythrocytic stages.
Furthermore, the cytotoxic activity against cultured rat L6
myogenic cells was determined (Table 1). The reported data
Table 2. In Vitro Activity of Compounds 1, 3 and 5 and
Their Pure Enantiomers against T. cruzi; the Cytotoxic
Activity on L6 Cells Is Also Given; Data Represent IC50
Values in μM with Standard Deviation (SD)
a
b
c
compd
Tc
SD
L6
SD
SI
1
0.0149
0.1927
0.0038
0.0052
0.1120
0.0018
0.0364
0.0972
0.0236
0.0109
0.1167
0.0030
0.0031
0.0469
0.0008
0.0170
0.0340
0.0143
23.59
27.50
18.24
12.97
13.96
13.44
27.25
15.38
31.65
5.92
4.89
1.82
0.73
1.77
2.63
3.33
4.83
5.00
1583
143
(R)-1
(S)-1
3
4800
2494
125
(R)-3
(S)-3
5
7467
749
(R)-5
(S)-5
158
1341
a
b
T. cruzi Tulahuen C2C4 amastigote stage. Rat myogenic L6 cells.
c
SI = selectivity index, IC50L6/IC50Tc.
enantiomers are highly active and that the biological activity is
mainly due to the (S) enantiomers, indeed (S)-1 and (S)-3
show IC50 values 50−60 times lower than the corresponding
(R) enantiomers. The carbamate (S)-3 was found to be the
most active compound with an IC50 of 1.8 nM vs T. cruzi,
resulting in a molar basis approximately a thousand-fold more
active than benznidazole. It is noteworthy that all compounds
showed a low toxicity toward L6 cells, including the most active
azole derivatives, 1 and 3−5, and the corresponding pure
enantiomers.
Furthermore, on the basis of the structural similarity between
the studied compounds and the current CYP51Tc inhibitors, a
docking study of compounds 1−8 against the energy
minimized structure of CYP51Tc (PDB ID: 2WX2), CYP51Tb
(PDB ID: 3GW9), and CYP51Li (PDB ID: 3L4D; L. infantum
belonging to L. donovani complex) was performed. Proteins
pretreatment was performed by means of the Protein
Preparation Wizard tool of Maestro 9.2 suite17 and the energy
minimization using Macromodel18 with the OPLS2005 force
field. Docking calculations were performed by means of
GOLD5.119 using the ASP scoring function because it was
the one that best reproduced the crystallographic pose of
Table 1. In Vitro Antiparasitic Activity and Cytotoxicity of
1−8 and Reference Drugs; Data Represent IC50 Values
Given in μM
a
b
c
d
e
f
compd
Tb
42.1
Tc
Ld
5.4
Pf
L6
23.6
SI
1
0.014
24.4
2.2
1686
7.5
2
293.8
18.2
17.3
7.0
36.7
1.8
182.5
13.0
3
0.005
0.005
0.036
48.4
2600
3360
756
4.6
4
19.1
15.0
11.4
85.1
122.7
190.4
MIL
4.6
16.8
5
34.0
1.5
27.2
6
138.9
117.4
230.3
MEL
60.2
23.1
79.8
CHL
222.1
79.3
7
133.1
175.3
BNZ
0.6
8
237.6
1.3
ref
drug
PDT
g
0.0075
1.671
0.366
0.328
0.001
a
b
T. brucei rhodesiense STIB 900 trypomastigote stage. T. cruzi
c
Tulahuen C2C4 amastigote stage. L. donovani MHOM-ET-67/L82
amastigote stage. P. falciparum K1 erythrocytic stages. Rat myogenic
L6 cells. SI = selectivity index, IC50L6/IC50Tc. MEL = melarsoprol;
BNZ = benznidazole; MIL = miltefosine; CHL = chloroquine; PDT =
podophyllotoxin.
d
e
f
g
B
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ACS Medicinal Chemistry Letters
Letter
cocrystallized ligands. In the CYP51Tc active site, 1, 3, and 5 are
mostly stabilized by the coordination of the nitrogen in position
3 of the imidazolic ring to heme-iron and by hydrophobic
interactions in the pocket formed by Val102, Tyr103, Met106,
Phe110, Tyr116, Leu127, Ala287, Ala288, Met289, Phe290,
Ala291, Met360, and Met460. Compound 2 belongs to the
same class of compounds 1, 3, and 5, coordinating the heme-
iron with the N3 of the imidazolic ring. The lack of its activity
could be ascribed to the loss of most of the hydrophobic
interactions that stabilize the other active molecules in the
pocket described just above. The difference in the activity
between (R) and (S) enantiomers of the most active
compounds could be ascribed to the better coordination of
the imidazolic ring to the heme-iron of (S) enantiomers than
that of (R) ones. From our docking calculations, the N3 of the
imidazolic ring in all (S) enantiomers coordinate the heme-iron
in about 1.9 Å distance, while in (R) enantiomers, the light
difference in the conformation and the consequent rotation of
the N3 cause a less favored coordination (e.g., 2.0 Å for
compound (R)-5, 2.2 Å for compound (R)-1, and 2.3 Å
compound (R)-3 approximately) (Figures 2−4). The high
Figure 4. Different conformations in the CYP51Tc active site of the
compound 3; (R)-3 shown in orange and (S)-3 in yellow.
inhibitory activity, probably because of the lack of the heme-
iron coordination (see Figure S4 in Supporting Information).
A preliminary study on hepatic metabolism has been carried
out on the most active (S)-1 and (S)-3. The results have
demonstred that (S)-3 is oxidized but not hydrolyzed by the
CYP-dependent metabolism. On the contrary, the ester moiety
of (S)-1 is hydrolyzed by the CYP enzyme family, and the
product is eliminated through the phase 1 metabolism. It has
been previously demonstrated that easy metabolized molecules
are not CYP inhibitors nor suicide substrate.21−23 Hence, those
compounds could be considered not to be toxic for the human
liver.
The reported results have evidenced that the azole
compounds 1 and 3−5 exert a potent in vitro antichagasic
activity showing IC50 values in the low nanomolar concen-
tration range.
The (S) enantiomers are more active than the corresponding
(R); in addition, (S)-3 showed an activity a thousand times
higher than the reference drug benznidazole. Furthermore, all
compounds showed low toxic effects on rat myogenic L6 cells.
Moreover, the docking studies are consistent with the
hypothesis of CYP51Tc inhibition, and they agree with the
higher activity of (S) enantiomers over (R) enantiomers and
the high selectivity vs T. cruzi. These molecules represent an
excellent starting point in the development of cheap, easy
workup and highly active antitrypanosomal drugs.
Figure 2. Binding mode of (S) enantiomers. The protein and the
heme prosthetic group (CYP51Tc) are represented in green and gray,
respectively; compounds 1, 3, and 5 in yellow. The distance between
the N3 of the imidazolic ring and the heme-iron is represented in black
dots.
ASSOCIATED CONTENT
* Supporting Information
All synthetic procedures, molecular docking, and biological
assay details. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*E-mail: daniela.devita@uniroma1.it (D.D.V.); reto.brun@
unibas.ch (R.B.).
■
Figure 3. Binding mode of (R) enantiomers. The protein and the
heme prosthetic group (CYP51Tc) are represented in green and gray,
respectively; compounds 1, 3, and 5 in orange. The distance between
the N3 of the imidazolic ring and the heme-iron is represented in black
dots.
Funding
This research was financially supported by grants from
Sapienza, Universita di Roma Progetti di Ricerca di Ateneo,
̀
̀
and by Ministero dell’Istruzione, dell’Universita e della Ricerca.
Notes
The authors declare no competing financial interest.
selectivity of compounds 1, 3, and 5 toward T. cruzi over T.
brucei and L. donovani is probably due to the active site volume
and surface area smaller in T. brucei and L. donovani than in T.
cruzi that could favor their access in the active site.20 The
aminopyridines 6−8 do not show an interesting CYP51Tc
ACKNOWLEDGMENTS
We thank Dr. Roberto Cirilli (Dipartimento del Farmaco,
■
Istituto Superiore di Sanita,
̀
Rome, Italy) for the chiral HPLC
C
dx.doi.org/10.1021/ml400039r | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
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(Dipartimento of “Chimica e Tecnologie del Farmaco”
Sapienza University of Rome, Italy) for obtaining the mass
spectra.
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