New 8-Nitroquinolinone Derivative Displaying Submicromolar in Vitro Activities against Both Trypanosoma brucei and cruzi

Article abstract of DOI:10.1021/acsmedchemlett.9b00566

An antikinetoplastid pharmacomodulation study was conducted at position 6 of the 8-nitroquinolin-2(1H)-one pharmacophore. Fifteen new derivatives were synthesized and evaluated in vitro against L. infantum, T. brucei brucei, and T. cruzi, in parallel with a cytotoxicity assay on the human HepG2 cell line. A potent and selective 6-bromo-substituted antitrypanosomal derivative 12 was revealed, presenting EC₅₀ values of 12 and 500 nM on T. b. brucei trypomastigotes and T. cruzi amastigotes respectively, in comparison with four reference drugs (30 nM ≤ EC₅₀ ≤ 13 μM). Moreover, compound 12 was not genotoxic in the comet assay and showed high in vitro microsomal stability (half life >40 min) as well as favorable pharmacokinetic behavior in the mouse after oral administration. Finally, molecule 12 (E° = -0.37 V/NHE) was shown to be bioactivated by type 1 nitroreductases, in both Leishmania and Trypanosoma, and appears to be a good candidate to search for novel antitrypanosomal lead compounds.

Full text of DOI:10.1021/acsmedchemlett.9b00566

Subscriber access provided by Nottingham Trent University
Letter
New 8-nitroquinolinone derivative displaying submicromolar
in vitro activities against both Trypanosoma brucei and cruzi
Julien Pedron, Clotilde Boudot, Jean-Yves Brossas, Emilie Pinault, Sandra Bourgeade-
Delmas, Alix Sournia-Saquet, Elisa Boutet-Robinet, Alexandre Destere, Antoine Tronnet,
Justine Bergé, Colin Bonduelle, Céline Deraeve, Genevieve Pratviel, Jean-Luc Stigliani, Luc
Paris, Dominique Mazier, Sophie Corvaisier, Marc Since, Aurélie Malzert-Freon, Susan Wyllie,
Rachel Milne, Alan H. Fairlamb, Alexis Valentin, Bertrand Courtioux, and Pierre Verhaeghe
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00566 • Publication Date (Web): 06 Feb 2020
Downloaded from pubs.acs.org on February 9, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 20
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 2 of 20
1
2
3
4
5
6
New 8-nitroquinolinone derivative displaying submicromolar in vitro
activities against both Trypanosoma brucei and cruzi
7
8
9
Julien Pedron1#, Clotilde Boudot2#, Jean-Yves Brossas3, Emilie Pinault4, Sandra Bourgeade-Delmas5,
Alix Sournia-Saquet1, Elisa Boutet-Robinet6, Alexandre Destere7, Antoine Tronnet1, Justine Bergé1,
Colin Bonduelle1, Céline Deraeve1, Geneviève Pratviel1, Jean-Luc Stigliani1, Luc Paris3, Dominique
Mazier8, Sophie Corvaisier9, Marc Since9, Aurélie Malzert-Fréon9, Susan Wyllie10, Rachel Milne10, Alan
H. Fairlamb10, Alexis Valentin5, Bertrand Courtioux2 and Pierre Verhaeghe1*
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1 LCC-CNRS, Université de Toulouse, CNRS, UPS, Toulouse, France.
2
Université de Limoges, UMR INSERM 1094, Faculté de Pharmacie, 2 rue du Dr Marcland, 87025
Limoges, France
3 AP-HP, Groupe Hospitalier Pitié-Salpêtrière, Service de Parasitologie Mycologie, Paris, France.
4 Université de Limoges, BISCEm Mass Spectrometry Platform, CBRS, 2 rue du Pr. Descottes, F-87025
Limoges, France
5 UMR 152 PharmaDev, Université de Toulouse, IRD, UPS, Toulouse, France.
6 Toxalim, Université de Toulouse, INRA, ENVT, INP-Purpan, UPS, Toulouse, France
7 Department of Pharmacology, Toxicology and Pharmacovigilance, CHU Limoges, France, INSERM,
UMR 1248, University of Limoges, France
8 CIMI-Paris, Sorbonne Université 91 boulevard de l'Hôpital 75013 Paris, France.
9 Centre d’Etudes et de Recherche sur le Médicament de Normandie (CERMN), Normandie Univ, Caen,
France
10 University of Dundee, School of Life Sciences, Division of Biological Chemistry and Drug Discovery,
Dow Street, Dundee DD1 5EH, Scotland, United Kingdom
#Co-first authors
*Email: pierre.verhaeghe@lcc-toulouse.fr
ACS Paragon Plus Environment

Page 3 of 20
ACS Medicinal Chemistry Letters
1
2
3
4
Abstract
5
6
7
8
9
An antikinetoplastid pharmacomodulation study was conducted at position 6 of the 8-nitroquinolin-
2(1H)-one pharmacophore. Fifteen new derivatives were synthesized and evaluated in vitro against L.
infantum, T. brucei brucei and T. cruzi, in parallel with a cytotoxicity assay on the human HepG2 cell
line. A potent and selective 6-bromo-substituted antitrypanosomal derivative 12 was revealed,
presenting EC50 values of 12 nM and 500 nM on T. b. brucei trypomastigotes and T. cruzi amastigotes
respectively, in comparison with four reference drugs (30 nM ≤ EC50 ≤ 13 µM). Moreover, compound
12 was not genotoxic in the comet assay, showed a high in vitro microsomal stability (half-life > 40
min) as well as a favorable pharmacokinetic behaviour in mouse after oral administration. Finally,
molecule 12 (E° = -0.37 V/NHE) was shown to be bioactivated by type 1 nitroreductases, both in
Leishmania and Trypanosoma and appears as a good candidate to search for novel antitrypanosomal
lead compounds.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Keywords
Trypanosoma brucei brucei / Trypanosoma cruzi / 8-nitroquinolin-2(1H)-ones / Redox potentials /
NTR1
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 4 of 20
1
2
3
4
5
6
7
8
9
Human African trypanosomiasis (HAT),[1] Chagas diseases (CD)[2] and visceral leishmaniasis
(VL)[3] are infectious diseases caused by unicellular flagellated kinetoplastid parasites belonging to the
Trypanosoma and Leishmania genera. From an epidemiological point of view, the World Health
Organization (WHO) estimates that millions of people are at risk of contracting HAT,CD and VL. These
three diseases are also estimated to be responsible for about 30,000 annual deaths, with the caveat that
these numbers may be underestimated due to the difficulty in accessing some rural areas and the
unspecific symptoms during the early stage of these infections.[4] Although these diseases are lethal if
untreated, there are very few efficient and safe drugs available, each of them presenting various
drawbacks for the patient (toxicity, emergence of resistances, mode of administration, treatment cost).
Thus, due to the lack of consideration and investments from the pharmaceutical industry, the WHO
classified HAT, CD and VL among “neglected tropical diseases” (NTDs). [5]
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
O
O
CH3
N
N
S
O
O2N
O2N
N
O2N
H
N
N
CH3
N
N
O
S
H3C
Nifurtimox
Antitrypanosomal drug (HAT & CD)
Benznidazole
Antitrypanosomal drug (CD)
Fexinidazole
Antitrypanosomal drug (HAT)
Phase II against CD
O2N
N
O
N
N
O
F
DNDI-0690
Phase I against VL
Figure 1. Structures of nitroaromatic drugs displaying antikinetoplastid activity.
In this context, nitroaromatic derivatives play a key role in the fight against kinetoplastids
(Figure 1). Beyond Nifurtimox and Benznidazole (antitrypanosomal drugs), nitroaromatics are fully part
of the research efforts of the Drugs for Neglected Diseases initiative (DNDi). Thus, among few novel
chemical entities in clinical trials against these infections[6], the development of fexinidazole (an orally
available 5-nitroimidazole derivative that received an agreement from the EMA in the treatment of HAT
ACS Paragon Plus Environment

Page 5 of 20
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
in 2018 and that is in phase 2 against CD)[7-9], delamanid (a derivative marketed against tuberculosis and
that was studied against VL)[10] and DNDi-0690 (a 2-nitroimidazooxazine derivating from delamanid
and that has recently entered phase I clinical trials against VL) illustrate well the strong potential of
nitroaromatic molecules in the search for novel antitrypanosomatids.[11]
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The antiparasitic mechanism of action of these molecules involves parasitic nitroreductases
(NTRs). NTRs contain a flavin as cofactor and catalyse the one- or two-electron reduction of nitrodrugs
into cytotoxic electrophilic metabolites such as nitroso and hydroxylamine derivatives, able to form
[12]
covalent adducts with DNA or proteins.
Two NTRs were found in Leishmania: a mitochondrial
[13]
NTR1 catalysing a two-electron reduction
and a cytosolic NTR2 catalyzing a one-electron
reduction.[14] In Trypanosoma, only a mitochondrial type 1 NTR is expressed.[15,16] It is important to note
that these enzymes are absent from mammalian cells, an important feature for developing selective
antikinetoplastid molecules. Moreover, depending on the chemical structure of nitrated derivatives, the
selectivity for NTRs could be modulated. For instance, fexinidazole is selectively bioactivated by NTR1,
both in Leishmania and Trypanosoma meanwhile delamanid is only bioactivated by NTR2 in
Leishmania. [14]
In this context, by studying the antiparasitic potential of the 8-nitroquinoline scaffold,[17] our
research team reported a new antileishmanial pharmacophore: 8-nitroquinolin-2(1H)-one[18]. After
pharmacomodulation studies at position 4,[19,20] an electrochemistry-guided work highlighted the
[21]
importance of an intramolecular hydrogen bond between the nitro group and the lactam function.
This work also led to the identification of a new 3-bromosubstituted antikinetoplastid hit compound
derivative (Hit 1, Figure 2), which is bioactivated by type 1 NTRs, both in L. donovani and T. b.
brucei.[21] Recently, by introducing a p-carboxyphenyl functionality at position 3 of the pharmacophore,
we reported a new selective antitrypanosomal hit compound (Hit 2, Figure 2).[22]
COOH
Br
N
O
N
O
N
H
O
H
H
NO2
NO2
NO2
Pharmacophore
Hit 1
Hit 2
EC50 L.infantum axe. ama. = 15.5 µM
EC50 T. b brucei trypo. = 23.4 µM
CC50 HepG2 = 164 µM
7.1 µM
1.9 µM
92 µM
> 100 µM
1.5 µM
120 µM
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 6 of 20
1
2
3
4
5
6
Figure 2. Antitrypanosomatid profile of previously reported 8-nitroquinolin-2(1H)-one hit compounds.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
We present herein a medicinal chemistry work that greatly improved the antitrypanosomal
activity of the current series via the synthesis of novel derivatives bearing electron withdrawing groups
at position 6 of the quinolinone scaffold.
In order to increase the oxidant character of the series and facilitate its bioactivation by
nitroreductases, fifteen derivatives (1-15) were prepared, bearing various electron withdrawing groups
(EWG) at position 6. As presented in Scheme 1, 8 compounds bearing a trifluoromethyl group at position
6 of the quinolinone scaffold were synthesized according to a previously reported protocol. [23] Briefly,
3-ethoxyacryloyl chloride was synthesized in two steps by the saponification of commercial 3,3’-
diethoxyethylpropionate followed by a reaction with thionyl chloride. This acyl chloride was then
reacted with p-trifluoromethylaniline to afford, after a cyclization step in H2SO4 (98%), compound 1 in
42% yield (a). Molecule 1 was then either nitrated at position 8, using classical conditions, to afford
compound 2 in 89% yield (b), or selectively halogenated at position 3, to generate derivatives 3 or 4 in
48 and 54% yields, respectively (c and d). The O-methylation of compound 2 was performed with a
protocol using a mixture of methyl iodide and sodium hydride, to prepare derivative 5 in 69% yield (e).
Nitrated molecule 2 could also undergo chlorination at position 3, affording compound 6 in 88% yield
(f), or react with NaBrO3 in refluxing HBr, according to a previously reported protocol,[24] to afford
halogenated derivative 7 in 94% yield (g). The nitro group of compound 7 was finally reduced to afford
the amino-derivative 8, considered as a negative control, using SnCl2, in 68% yield (h).
Scheme 1. Synthesis of compounds 1-8 from p-trifluoromethylaniline.
ACS Paragon Plus Environment

Page 7 of 20
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
F3C
Cl
O
F3C
Cl
O
N
H
N
H
NO2
3
2
6
F3C
c
f
48%
F3C
88%
F3C
NH2
O
F3C
e
a
b
+
EtO
89%
69%
N
OCH3
42%
N
O
N
H
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
H
NO2
NO2
1
5
g
d
54%
94%
Cl
F3C
Br
O
F3C
Br
F3C
Br
O
h
68%
N
H
N
H
O
N
H
NO2
NH2
7
8
4
The synthesis pathway used for preparing derivatives bearing a halogen atom at position 6 of
the quinolinone ring is presented in Scheme 2. It starts from 8-nitroquinolin-2(1H)-one [21] which was
reacted with bromine in refluxing acetic acid, saturated with sodium acetate, leading to a mixture of both
6-bromo and 3,6-dibromo derivatives 9 and 10 in 9 and 33% yields, respectively (a). Dichloro analogue
11 was also prepared from 8-nitroquinolin-2(1H)-one by reacting with 5 equiv. of sodium chlorate in
refluxing hydrochloric acid for 24 h (b). By using the same reaction, decreasing the amount of sodium
chlorate to 3 equiv. and limiting the reaction time to 45 min, 3-chloro-8-nitroquinolin-2(1H)-one was
formed (c). [21] This compound could be brominated at position 6 by reacting with an excess of NaBrO3
in refluxing HBr, leading to 12 in good yield (d). Taking into account that we previously reported the
benefit of introducing a p-carboxyphenyl moiety at position 3 of the scaffold toward antitrypanosomal
[22]
activity,
we finally engaged dibromo-derivative 10 into a Suzuki-Miyaura reaction with p-
(methoxycarbonyl)phenylboronic acid and then saponified the resulting ester coupling product to afford
carboxylic acid 13 (e). Finally, from brominated compound 12, derivatives 14 and 15 were obtained
through a Sonogashira coupling reaction (f).
Scheme 2. Synthesis of compounds 9-15.
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 8 of 20
1
2
3
4
5
6
7
8
9
Br
Cl
Br
Br
O
N
H
O
N
H
NO2
NO2
NO2
NO2
9
10
33%
55%
9%
e
COOH
a
Cl
Br
b
N
O
N
H
O
N
H
O
56%
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
H
NO2
NO2
11
13
c
62%
R
Cl
O
Br
Cl
Cl
O
d
f
N
H
N
O
N
79%
H
H
NO2
NO2
A
12
14
15
, R= CH2OH, 15%
, R= (CH2)3OH, 36%
Electrochemistry. Since the compounds were expected to be nitroreductase substrates, we
studied the influence of the introduction of EWG groups at position 6 of the pharmacophore toward
.-
redox potentials. Thus, redox potentials (couple R-NO2/RNO2 ) of the synthesized compounds were
measured by cyclic voltammetry in DMSO (one-electron reversible reduction). Redox potentials ranged
between -0.36 and -0.75 V/NHE (Figure 3). In comparison with the original pharmacophore, the
introduction of a bromine atom at position 6 (compound 9) led to a significant increase of the E° value
from -0.54 V to -0.40 V. The same effect was observed when a bromine atom was introduced at position
6 of hit 2 (compound 13), with an increase of the E° value of + 0.16 V. It is to note that introducing a
bromine atom has a higher influence on the redox potential at position 6 (compound 9) compared to
position 3 (hit 1): + 0.14 versus + 0.09 V. The influence of the CF3 group toward the redox potential of
the scaffold was the same as that of the bromine atom: + 0.15 V (comparison between the
pharmacophore and compound 2). Then, when two bromine atoms were combined at positions 3 and 6
(compound 10), a slight increase of + 0.03 V was observed (no additive effect) in comparison with 6-
bromo-derivative 9. Surprisingly, by comparing compounds 7 and 10-12, there was almost no effect of
the variation of the EWG at positions 3 or 6 toward the redox potential values, all compounds ranging
from - 0.36 to - 0.37 V. Finally, by comparing compound 2 with its O-methylated derivative 5, it
appeared that the redox potential value was drastically lower (- 0.36 V shift). This shift is not only due
to the donating mesomeric effect of the methoxy group, but also to the disappearance of the
intramolecular hydrogen bond between the nitro group and the lactam function. [21]
ACS Paragon Plus Environment

Page 9 of 20
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3 Redox potentials of synthesized nitroaromatic compounds determined by cyclic voltammetry.
Compound evaluation. The cytotoxicity of all compounds was first assessed on the human HepG2 cell
line using doxorubicin as a positive control (Table 1). Compared to this reference drug (CC50 = 0.2 µM),
the series showed little (CC50 = 17 µM) to no (CC50 > 100 µM) cytotoxicity. Non nitrated molecules 1, 3
and 4, O-methylated derivative 5 and 8-amino-derivative 8, considered as negative controls, displayed
high CC50 values, above 100 µM. The results show that the introduction of a bromine atom or a CF3 group
at position 6 of the nitrated scaffold leads to derivatives (2 and 9) that are 4 to 5 times more cytotoxic (CC50
= 28-41 µM) than the 8-nitroquinolinone pharmacophore (CC50 = 164 µM). When introducing an
additional halogen atom at position 3 of the scaffold (6, 7, 10-12), the cytotoxicity remains close to the
ones of 6-monosubstituted derivatives (CC50 = 17-41 µM). Nevertheless, introducing a p-carboxyphenyl
functionality at position 3 of the scaffold (compound 13), results in a 3-fold decrease of the cytotoxicity
(CC50 = 60 µM), in comparison with the 3,6-dibromo derivative 10 (CC50 = 17 µM).
All synthesized molecules were screened in vitro for their activity against Leishmania infantum
axenic amastigotes and their EC50 were compared to the ones of three reference drugs (amphotericin B,
miltefosine and fexinidazole) and antikinetoplastid hit 1 (Table 1). Among tested compounds, only
compound 7 (E° = - 0.36 V) showed a weak antileishmanial activity, displaying an EC50 value of 3.9 µM
in addition to a selectivity index superior to 10. Derivatives of 7 without a nitro group at position 8 of the
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 10 of 20
1
2
3
4
5
6
7
8
9
scaffold (1, 3, 4 and 8) were inactive toward L. infantum, as well as the O-methylated analogue 5 (E° = -
0.75 V), confirming both the key role of the nitro group toward antiparasitic activity and the importance
of the interaction between the nitro group and the lactam function to confer suitable redox potentials for
displaying antileishmanial properties in this chemical series.[21] Note that, contrary to hit 2, which was not
active against L. infantum (EC50 > 100 µM), the 6-bromo derivative 13 was active (EC50 = 8 µM).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 1. Antikinetoplastid activities and cytotoxicity of compounds 1-15.
ACS Paragon Plus Environment

Page 11 of 20
ACS Medicinal Chemistry Letters
1
2
3
4
Cytotoxicity
HepG2
5
6
L. infantum
axenic amastigote trypomastigotes
T. brucei brucei
CC50 (µM)
&
Cpd
Structure
7
8
9
EC50 (µM)
EC50 (µM)
Selectivity
index regarding
T. b. brucei
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
164 +/-28
Phar.
Hit 1
15.5 +/-0.5
7.1 +/-1.5
23.4 +/-5.7
1.9 +/-0.4
N
H
O
7
NO2
NO2
NO2
Br
O
92 +/-13
N
H
48
Cl
O
85 +/-4
71
A[27]
16 +/-0.9
> 100a
1.2 +/-0.2
1.5 +/- 0.2
N
H
COOH
120+/-7
Hit 2
80
N
O
H
NO2
F3C
> 100a
ND
1
2
> 100a
> 50a
N
H
O
F3C
41.3 +/- 3.9
18.5 +/- 3.9
1.7 +/- 0.9
N
H
O
24
NO2
F3C
F3C
Cl
> 100a
ND
3
4
> 100a
> 100a
> 50a
> 50a
N
O
H
Br
> 100a
ND
N
H
O
F3C
> 100a
>5
5
6
> 100a
9.2 +/- 1.5
3.9 +/- 1.5
> 100a
20.9 +/- 14.5
0.57 +/- 0.25
0.2 +/- 0.09
8.7 +/- 3.2
N
OCH3
Cl
NO2
F3C
F3C
F3C
Br
31.4 +/- 3.3
N
H
O
55
NO2
NO2
NH2
NO2
NO2
NO2
Br
41.3 +/- 3.9
7
N
H
O
206
Br
> 100a
>11
8
N
H
O
27.9 +/- 6.0
9
11.6 +/- 2.5
6.5 +/- 1.8
6.2 +/- 0.6
0.07 +/- 0.01
0.05 +/- 0.008
0.1 +/- 0.04
N
H
O
Br
O
399
Br
16.6 +/- 1.2
10
11
N
H
332
Cl
Cl
O
24.3 +/- 0.9
N
H
243
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 12 of 20
1
2
3
4
5
6
7
8
9
Br
Cl
O
18.1 +/- 2.5
12
13
6.7 +/- 0.8
8.0 +/- 1.5
0.012 +/- 0.002
N
1508
H
NO2
COOH
Br
60.4 +/- 14.4
0.2 +/- 0.02
302
N
H
O
NO2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
HO
Cl
O
30.9 +/- 2.1
14
15
8.9 +/- 0.6
1.0 +/- 0.09
0.85 +/- 0.1
31
N
H
NO2
HO
Cl
O
32.7 +/- 0.4
10.1 +/- 1.0
38
N
H
NO2
Doxorubicinb
-
-
-
-
0.2 +/-0.02
5.5 +/-0.25
85 +/-8.8
> 200a
Amphotericin Bc
Miltefosinec
0.06 +/-0.001
0.8 +/-0.2
Fexinidazolec,d,e
3.4 +/-0.8
0.4 +/- 0.2
0.03 +/- 0.009
13.3 +/- 2.1
>500
> 100a
Suramind
-
-
>3333
> 100a
Eflornithined
>8
aThe EC50 or CC50 value was not reached at the highest tested concentration; bDoxorubicin was used as a cytotoxic reference drug;
cAmphotericin B, Miltefosine and Fexinidazole were used as antileishmanial reference drugs ; dFexinidazole, Suramin and Eflornithine were
used as anti-Trypanosoma brucei reference drugs
All molecules were also screened in vitro for their antitrypanosomal activity against
Trypanosoma brucei brucei trypomastigotes and their EC50 were compared to the ones of 3 references
drugs: suramin, eflornithine, fexinidazole and to antitrypanosomal hit 2. Among the fifteen tested
molecules, 7, 9, 10, 11, 12 and 13 displayed both submicromolar activity (12 ≤ EC50 ≤ 200 nM) and
good selectivity indices (SI > 200), in comparison with suramin (EC50 = 30 nM), eflornithine (EC50 =
13 µM), fexinidazole (EC50 = 400 nM) and hit 2 (EC50 = 1.5 µM). Molecule 12 was the most active and
selective in the series: EC50 = 12 nM & SI = 1508. Compounds without a nitro group at position 8 of the
scaffold appeared to be inactive (compounds 1, 3 and 4) and amino-derivative 8 being quite less active
(EC50 = 9 µM). It is important to note that the introduction of a bromine atom at position 6 of the scaffold
seems crucial to afford highly active antitrypanosomal molecules, compound 9 being 334 times more
active than the 8-nitroquinolin-2(1H)-one pharmacophore and 27 times more active than its 3-bromo-
position isomer (hit 1). This key effect of the bromine atom at position 6 of the scaffold also appears
when comparing the activities of 3-chloro-6-bromo hit-compound 12 with its 3-chloro-analogue A: 12
is 100 times more active than A. When comparing molecules 2 and 9, it appeared that the CF3 group
ACS Paragon Plus Environment

Page 13 of 20
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
does not provide the same level of improvement in activity. Introducing an additional halogen atom at
position 3 of hit molecule 9, leading to dibromo-derivative 10 and dihalo-derivative 12 afforded the
most active compounds in the series (EC50 = 12 – 50 nM). Comparison between compound 12 and 3,6-
dichlorosubstituted derivative 11 showed that the replacement of the bromine atom by a chlorine atom
at position 6 was not in favor of antitrypanosomal activity. During pharmacomodulation studies at
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
position 3 of the scaffold, hit 2 was identified as a promising antitrypanosomal hit with an EC50 of 1.5
[22]
µM.
When a bromine atom is introduced at position 6 of hit 2, leading to compound 13, the
antitrypanosomal activity is improved, reaching an EC50 value of 200 nM.
Thus, the molecule presenting the best antitrypanosomal profile in the series, regarding both
activity and selectivity, was compound 12. To better evaluate its antitrypanosomal potential, an in vitro
evaluation against intracellular T. cruzi amastigotes was carried out, using benznidazole and
fexinidazole as positive controls (Table 1). Quite interestingly, despite being about 10 times more
cytotoxic than reference drugs, molecule 12 displayed a high activity (EC50 = 0.5 µM) against T. cruzi,
identical to the one of benznidazole (EC50 = 0.5 µM) and better than the one of fexinidazole (EC50 = 3
µM).
To investigate deeper the potential of compounds 7, 12 and 13, as the most active antikinetoplastid
compounds of the studied series, an in vitro pharmacokinetic evaluation was performed, including
microsomal stability assay (female mouse microsomes), human albumin binding assay and a PAMPA
assay using a blood-brain barrier model (Table 2). All compounds presented a good microsomal stability
(T1/2 > 40 min) and a strong binding to human albumin (≥ 99%). However, the development of
antitrypanosomal agents requires that molecules cross the BBB, as the second stage of HAT is
meningoencephalitic. A PAMPA assay on a BBB model was then performed.[25, 26] Compounds 7 and 12
diffused through the lipid bilayer with Pe values significantly higher than the positive control (Pe = 450.3
and 405.1 nm/s, respectively), whereas compound 13 displayed a low Pe value (Pe = 12.7 nm/s), indicating
a limited crossing of the BBB, hindering further development for 13. This last result is not surprising as
electrostatic repulsions between negatively charged carboxylic acid group of this molecule and the
negatively-charged membrane constituents are probably responsible for its low permeability across the
BBB in the PAMPA model. Moreover, 13 has a molecular weight approaching 400 g/mol which could
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 14 of 20
1
2
3
4
5
6
7
8
9
also contribute to limiting its diffusion across the BBB. [27] Unfortunately, the methylic ester precursor or
13 could not be evaluated in vitro because of a lack of aqueous solubility. The ability of hit-compound 12
to cross the BBB in the PAMPA assay is in accordance with its CNS MPO score[28] that has a calculated
value of 4.35. Finally, the aqueous solubility of hit-compound 12 was measured with a thermodynamic
solubility assay at physiological pH and was determined at 21.7 µM (Table 2).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 2. In vitro pharmacokinetic, physicochemical and toxicological properties of compounds 7, 12 and 13.
COOH
F3C
Br
O
Br
Cl
O
Br
N
N
N
O
H
H
12
H
13
7
NO2
NO2
NO2
Microsomal stability (min)
Albumin binding assay (%)
BBB PAMPA assay (nm/s)
cLogD* at pH = 7.4
> 40
> 40
99.0
> 40
> 99
99.4
450.3 ± 170.5
3.24
405.1 ± 69.3
2.97
12.7 ± 0.9
0.44
Thermodynamic solubility at
pH = 7.4 (µM)
-
-
21.7 +/-4.1
Negative
-
-
Comet assay at 1 and 10 µM
*Calculated with MarvinSketch® (ChemAxon)
To check the antikinetoplastid mechanism of bioactivation of compounds 7, 12 and 13, their
activities were evaluated in vitro toward Leishmania donovani promastigote strains overexpressing
leishmanial nitroreductases (NTR1 or NTR2). EC50 values were determined and compared to the ones
obtained on a wild-type strain, to evaluate the bioactivation of these nitroaromatic compounds (Table 3).
As reported for hit 1,[21] compounds 7 and 12 are selectively bioactivated by leishmanial NTR1 with EC50
values being 3.5 and 16 times lower on the strain overexpressing the NTR than on the wild type. Contrary
to 7 and 12, compound 13 did not demonstrate significant bioactivation, neither by NTR1 nor by NTR2 in
Leishmania. The same experiments were carried out with compounds 7, 12 and 13 on T. brucei brucei
strains overexpressing the type 1 trypanosomal NTR, comparing the EC50 values with the ones determined
on the wild-type strain (Table 3). All tested compounds were bioactivated by type 1 nitroreductase of T. b.
ACS Paragon Plus Environment

Page 15 of 20
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
brucei, being respectively 7, 15 and 40 times more active against the strain overexpressing the NTR than
against the wild type. It is interesting to note that hit 2 (E° = - 0.56 V) did not seem to be bioactivated by
T. b. brucei NTR whereas its 6-brominated derivative 13 (E° = - 0.40 V) was. Finally, hit-compound 12
also appeared to be 6 times more active than nifurtimox against T. brucei brucei, underlining the potential
of this molecule as an antitrypanosomal candidate.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 3. Evaluation of the bioactivation of compounds 7, 12 and 13 by leishmanial and trypanosomal NTRs.
Even though many safe nitroaromatic molecules are today on the market (for example
antiprotozoal 5-nitroimidazoles such as metronidazole, several antihypertensive dihydropyridines like
nicardipine, some benzodiazepines like loprazolam, anti-Parkinson catechol O-methyltransferase
(COMT) inhibitors like entacapone, antiandrogens like flutamide, anti-inflammatory nimesulide,
immuno-modulating azathioprine, anticoagulant acenocoumarol, anthelminthic niclosamide…) or in
clinical trials (for instance delamanid), drugs including a nitroaromatic moiety suffer from a bad
reputation, as possible mutagenic or genotoxic molecules, which narrows their pharmaceutical
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 16 of 20
1
2
3
4
5
6
7
8
9
development. [29] The Ames test is the most commonly used assay to evaluate the mutagenic potential
of compounds, using Salmonella typhimurium strains. However, these bacteria possess NTR making the
Ames test generally unsuitable for the evaluation of most of nitroaromatic compounds, genotoxicity
being attributed to the reduced metabolites that are bioactivated by these bacterial enzymes (absent in
human cells). [30] Thus, it is accepted than the comet assay or the micronucleus assay are more pertinent
methods to evaluate the genotoxic potential of nitroaromatic compounds. For example, fexinidazole is
positive in the Ames test, but negative in the micronucleus assay. [31] A comet assay was then performed
on the HepG2 cell line with hit compound 12, using methyl methanesulfonate (MMS) as a positive
control (see Suppl. info). Compound 12 was not genotoxic after 2 or 72 h of incubation, neither at 1 nor
10 µM, concentrations chosen to be lower than its CC50 on HepG2 cell line (CC50 = 18 µM).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Regarding tolerance in mouse, a once daily oral dose of compound 12, at 25 or 50 mg/kg, was
administrated to eight mice (4 mice in each group). These two doses were well tolerated with just a little
apathy during 1 h for all mice receiving the dose of 50 mg/kg. Administration of a repeated dose of 25
mg/Kg once daily for 5 days was well tolerated without any adverse effect noted. The No Observed
Adverse Event Level (NOAEL) [31] in mice was then set at 25 mg/kg/day. After euthanasia, no lesions
were found on the different organs (kidney, liver, brain, heart and lung).
Finally, the in vivo pharmacokinetic parameters were determined in mouse, after oral
administration at 25 mg/kg in a mixture of 5% Tween 80 and 95% of 0.5% carboxymethylcellulose in
[32]
water, by using the QuEChERS method.
Monolix Lixoft software was used to analyze data by
[33]
noncompartmental model to fit pharmacokinetics parameters.
As presented in the supporting
information file (figure S17), hit-compound 12 showed good pharmacokinetic properties: 12 is highly
and rapidly absorbed after oral administration and shows good exposure. Its plasmatic half-life (2.9 h)
is higher than the one of the drug fexinidazole (0.8 h). [31]
CONCLUSION
Via an antitrypanosomal pharmacomodulation study of the 8-nitroquinolin-2(1H)-one scaffold, by
introducing EWG at positions 3 and 6 of the quinolinone ring, a novel potent antitrypanosomal hit-compound
ACS Paragon Plus Environment

Page 17 of 20
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
(12) was identified, displaying high activities, toward T. b. brucei (EC50 = 12 nM) and T. cruzi (EC50 = 0.5
µM), in comparison with eflornithine (EC50 = 13 µM), suramin (EC50 = 30 nM), benznidazole (EC50 = 0.5
µM) and fexinidazole (EC50 = 0.4 and 3.0 µM). Compound 12 displays good in vitro pharmacokinetic
parameters (good microsomal stability and BBB permeable), is rapidly absorbed after oral administration
and well tolerated in mouse at 25 mg/Kg. Nitroaromatic hit compound 12 is selectively bioactivated by type
1 NTR, both in Leishmania and Trypanosoma. An electrochemistry study showed the strong influence of the
introduction of an EWG in the benzenic part of the pharmacophore toward the redox potentials and the
corresponding antitrypanosomal activities. Finally, the lack of genotoxicity of compound 12 in the comet
assay is an additional favorable element so as to consider further development. Compound 12 meets all
criteria for an antitrypanosomal hit as defined by Katsuno and co-workers.[34] In vivo studies on mice
model of trypanosomiasis are now planned to investigate the potential of this compound to become an
antitrypanosomal lead.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
AUTHOR INFORMATION
CORRESPONDING AUTHOR
*E-mail: pierre.verhaeghe@lcc-toulouse.fr. Phone: +33 561 333 236.
ACKNOWLEDGMENT
The Paul Sabatier University and the Région Occitanie are acknowledged for financial support. A. H.
Fairlamb, S. Wyllie and R. Milne are supported by funding from the Wellcome Trust (WT105021). C.
Piveteau from Institut Pasteur de Lille, C. Bijani from Laboratoire de Chimie de Coordination de
Toulouse, C. Claparols, N. Martins-Froment, V. Bourdon and E. Leroy from the Institut de Chimie de
Toulouse are also acknowledged for their support. We also thank Dr J-B. Woillard for the in vivo
pharmacokinetic analysis, Mr F-L. Sauvage for his help in mass spectrometry analysis and Dr Ismail
Belfquih for his assistance in the culture of Vero cells and T. cruzi.
ASSOCIATED CONTENT
SUPPORTING INFORMATION
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 18 of 20
1
2
3
4
5
6
Material and methods, additional analysis of hit compounds 7, 12 and 13 as well as electrochemistry
data, in vitro PK data and in vivo PK data are provided.
7
8
9
REFERENCES
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1. Büscher, P., Cecchi, G., Jamonneau, V., and Priotto, G. Human african trypanosomiasis. Lancet 2017,
390, 2397-2405.
2. Pérez-Molina, J. A., and Molina, I. Chagas disease. Lancet 2017, 391, 82-94.
3. Burza, S., Croft, S. L., Boelaert, M. Leishmaniasis. Lancet 2018, 392, 951-970.
4.
World
Health
Organization,
neglected
tropical
diseases:
https://www.who.int/neglected_diseases/diseases/en/ (accessed November 1st 2019).
5. Molineux, D., Savioli, L., and Engels, D. Neglected tropical diseases: progress towards addressing
the chronic pandemic. Lancet 2017, 389, 312-325.
6. Drugs for neglected diseases initiative (DNDi), DNDi R&D Portfolio June 2019:
https://www.dndi.org/diseases-projects/portfolio/ (accessed November 1st 2019).
7. Wyllie, S., Patterson, S., Stojanovski, L., Simeons, F. R. C., Norval, S., Kime, R., Read, K. D., and
Fairlamb, A. H. The anti-trypanosome drug fexinidazole shows potential for treating visceral
leishmaniasis. Sci. Transl. Med. 2012, 4, 119re1.
8. Patterson, S., and Fairlamb, A. H. Current and future prospects of nitro-compounds as drugs for
trypanosomiasis and leishmaniasis. Curr. Med. Chem., 2019, 26, 4454-4475.
9. Mesu, V. K. B. K., Kalonji, W. M., Bardonneau, C., Mordt, O. V., Blesson, S., Simon, F., Delhomme,
S., Bernhard, S., Kuziena, W., Lubaki, J. F., Vuvu, S. L., Ngima, P. N., Mbembo, H. M., Ilunga, M.,
Bonama, A. K., Heradi, J. A., Solomo, J. L. L., Mandula, G., Badibabi, L. K., Dama, F. R., Lukula, P.
K., Tete, D. N., Lumbala, C., Scherrer, B., Strub-Wourgaft, N., and Tarral, A. Oral fexinidazole for late-
stage African Trypanosoma brucei gambiense trypanosomiasis: a pivotal multicentre, randomised, non-
inferiority trial, Lancet 2018, 391, 144-154.
10. Patterson, S., Wyllie, S., Norval, S., Stojanovski, L., Simeons, F. R. C., Auer, J. L., Osuna-Cabello,
Read, K. D., and Fairlamb, A. H. The anti-tubercular drug delamanid as a potential oral treatment for
visceral leishmaniasis. eLife, 2016, 5:e09744.
ACS Paragon Plus Environment

Page 19 of 20
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
11. Ang, C. W., Jarrad, A. M., Cooper, M. A., and Blaskovich, M. A. T. Nitroimidazoles: Molecular
Fireworks That Combat a Broad Spectrum of Infectious Diseases. J. Med. Chem. 2017, 60, 7636-7657.
12. Patterson, S., and Wyllie, S. Nitro drugs for the treatment of trypanosomatid diseases: past, present,
and future prospects. Trends Parasitol. 2014, 30, 289-298.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13. Wyllie, S., Patterson, S., and Fairlamb, A. H. Assessing the essentiality of leishmania donovani
nitroreductase and its role in nitro drug activation. Antimicrob. Agents Chemother. 2013, 57, 901-906.
14. Wyllie, S., Roberts, A. J., Norval, S., Patterson, S., Foth, B. J., Berriman, M., Read, K. D. and
Fairlamb, A. H. Activation of bicyclic nitro-drugs by a novel nitroreductase (NTR2) in Leishmania.
PLoS Pathog. 2016, 12:e1005971.
15. Hall, B. S., Bot, C., and Wilkinson, S. R. Nifurtimox activation by trypanosomal type I
nitroreductases generates cytotoxic nitrile metabolites. J. Biol. Chem. 2011, 286, 13088-13095.
16. Hall, B. S., and Wilkinson, S. R. Activation of Benznidazole by trypanosomal type I nitroreductases
results in glyoxal formation. Antimicrob. Agents Chemother. 2012, 56, 115-123.
17. Verhaeghe, P., Rathelot, P., Rault, S. and Vanelle, P. Convenient preparation of original vinylic
chlorides with antiparasitic potential in quinolone series. Lett. Org. Chem. 2006, 3, 891-897.
18. Paloque, L., Verhaeghe, P., Casanova, M., Castera-Ducros, C., Dumètre, A., Mbatchi, L., Hutter, S.,
Kraiem-M’Rabet, M., Laget, M., Remusat, V., Rault, S., Rathelot, P., Azas, N., and Vanelle, P.
Discovery of a new antileishmanial hit in 8-nitroquinoline series. Eur. J. Med. Chem. 2012, 54, 75-86.
19. Kieffer, C., Cohen, A., Verhaeghe, P., Hutter, S., Castera-Ducros, C., Laget, M., Remusat, V.,
Kraiem-M’Rabet, M., Rault, S., Rathelot, P., Azas, N., and Vanelle, P. Looking for new antileishmanial
derivatives in 8-nitroquinolin-2(1H)-one series. Eur. J. Med. Chem. 2015, 92, 282-294.
20. Kieffer, C., Cohen, A., Verhaeghe, P., Paloque, L., Hutter, S., Castera-Ducros, C., Laget, M., Rault,
S., Valentin, A., Rathelot, P., Azas, N., and Vanelle, P. Antileishmanial pharmacomodulation in 8-
nitroquinolin-2(1H)-one series. Bioorg. Med. Chem. 2015, 23, 2377-2386.
21. Pedron, J., Boudot, C., Hutter, S., Bourgeade-Delmas, S., Stigliani, J-L., Sournia-Saquet, A.,
Moreau, A., Boutet-Robinet, E., Paloque, L., Mothes, E., Laget, M., Vendier, L., Pratviel, G., Wyllie,
S., Fairlamb, A. H., Azas, N., Courtioux, B., Valentin, A., and Verhaeghe, P. Novel 8-nitroquinolin-
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 20 of 20
1
2
3
4
5
6
2(1H)-ones as NTR-bioactivated antikinetoplastid molecules: synthesis, electrochemical and SAR
study. Eur. J. Med. Chem. 2018, 155, 135-152.
7
8
9
22. Pedron, J., Boudot, C., Bourgeade-Delmas, S., Sournia-Saquet, A., Paloque, L., Rastegari, M.,
Abdoulaye, M., El-Kashef, H., Bonduelle, C., Pratviel, G., Wyllie, S., Fairlamb, A. H., Courtioux, B.,
Verhaeghe P., and Valentin A. Antitrypanosomal pharmacomodulation at position 3 of the 8-
nitroquinolin-2(1H)-one scaffold using pallado-catalyzed cross coupling reactions., ChemMedChem
2018, 13, 2217-2228.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
23. Zaragoza, F., Stephensen, H., Peschke, B., and Rimvall, K. 2-(4-alkylpiperazin-1-yl)quinolines as a
new class of imidazole-free histamine H3 receptor antagonists. J. Med. Chem. 2005, 48, 306-311.
24. O’Brien, N. J., Brzozowski, M., Wilson, D. J. D., Deady, L. W., and Abbott, B. M. Synthesis and
biological evaluation of substituted 3-anilinoquinolin-2(1H)-ones as PDK1 inhibitors. Bioorg. Med.
Chem. 2014, 22, 3781-3790.
25. Kerns, D. L., Bezar, I. F., Petusky, S. L. and Huang, Y. Comparison of blood-brain barrier
permeability assays: in situ brain perfusion, MDR1-MDCKII and PAMPA-BBB. J. Pharm. Sci. 2009,
98, 1980-1991.
26. Jourdan, J. P., Since, M., El Kihel, L., Lecoutey, C., Corvaisier, S., Legay, R., Sopkova-de Oliveira
Santos, J., Cresteil, T., Malzert-Fréon, A., Rochais, C. and Dallemagne P. Benzylphenylpyrrolizinones
with anti-amyloid and radical scavenging effects, potentially useful in Alzheimer’s disease treatment.
ChemMedChem 2017, 12, 913–916.
27. Van De Waterbeemd, H., Camenisch, G., Folkers, G., Chretien, J. R. and Raevski, O. A. Estimation
of blood-brain barrier crossing of drugs using molecular size and shape, and H-bonding descriptors. J.
Drug Target 1998, 6, 151-156.
28. Wager, T. T., Hou, X., Verhoest, P. R., Villalobos, A. Moving beyond rules: the development of a
central nervous system multiparameter optimization (CNS MPO) approach to enable alignment of
druglike properties, ACS Chem. Neurosci. 2010, 1, 435-449.
29. Nepali, K., Lee, H., Y., Liou, J. P. Nitro-Group-Containing Drugs J. Med. Chem., 2019, 62, 2851-
2893.
ACS Paragon Plus Environment

Page 21 of 20
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
30. Rosenkranz, E. J., McCoy, E. C., Mermelstein, R., and Rosenkranz, H. S. Evidence for the existence
of distinct nitroreductases in Salmonella typhimurium: roles in mutagenesis, Carcinogenesis 1982, 3,
121-123.
9
31. Torreele, E., Bourdin Trunz, B., Tweats, D., Kaiser, M., Brun, R., Mazué, G., Bray, M. A., and
Pécoul, B. Fexinidazole - a new oral nitroimidazole drug candidate entering clinical development for
the treatment of sleeping sickness, PLoS Neglected Trop. Dis. 2010, 4, e923.
32. Westland J., Dorman F., “QuEChERS extraction of benzodiazepines in biological matrices”, Journal
of Pharmaceutical Analysis, 2013, 3, 509-517.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
33.Pkanalix® data processing and calculation rules: http://pkanalix.lixoft.com/calculation-rules/
(accessed September 15th 2019).
34. Katsuno, K., Burrows, J. N., Duncan, K., Hooft van Huijsduijnen, R., Kaneko, T., Kita, K.,
Mowbray, C. E., Schmatz, D., Warner, P., and Slingsby, B. T. Hit and lead criteria in drug discovery
for infectious diseases of the developing worlds. Nat. Rev. Drug. Discov. 2015, 14, 751-758.
ACS Paragon Plus Environment