Design, Synthesis, and Efficacy Testing of Nitroethylene- and 7-Nitrobenzoxadiazol-Based Flavodoxin Inhibitors against Helicobacter pylori Drug-Resistant Clinical Strains and in Helicobacter pylori-Infected Mice

Article abstract of DOI:10.1021/acs.jmedchem.9b00355

Helicobacter pylori (Hp) infection is the main cause of peptic ulcer and gastric cancer. Hp eradication rates have fallen due to increasing bacterial resistance to currently used broad-spectrum antimicrobials. We have designed, synthesized, and tested redox variants of nitroethylene- and 7-nitrobenzoxadiazole-based inhibitors of the essential Hp protein flavodoxin. Derivatives of the 7-nitrobenzoxadiazole lead, carrying reduced forms of the nitro group and/or oxidized forms of a sulfur atom, display high therapeutic indexes against several reference Hp strains. These inhibitors are effective against metronidazole-, clarithromycin-, and rifampicin-resistant Hp clinical isolates. Their toxicity for mice after oral administration is low, and, when administered individually at single daily doses for 8 days in a mice model of Hp infection, they decrease significantly Hp gastric colonization rates and are able to eradicate the infection in up to 60% of the mice. These flavodoxin inhibitors constitute a novel family of Hp-specific antimicrobials that may help fight the constant increase of Hp antimicrobial-resistant strains.

Full text of DOI:10.1021/acs.jmedchem.9b00355

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Article
Design, Synthesis and Efficacy testing of nitroethylene- and 7-
nitrobenzoxadiazol-based flavodoxin inhibitors against Helicobacter pylori
drug-resistant clinical strains and in Helicobacter pylori-infected mice
Sandra Salillas, Miriam Alías, Valerie Michel, Alejandro Mahía, Ainhoa Lucía,
Liliana Rodrigues, Jessica Bueno, Juan José Galano-Frutos, Hilde de Reuse, Adrián
Velázquez-Campoy, José Alberto Carrodeguas, Carlos Sostre, Javier Castillo, Jose A.
Ainsa, Maria Dolores Diaz-De-Villegas, Angel Lanas, Eliette Touati, and Javier Sancho
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00355 • Publication Date (Web): 07 Jun 2019
Downloaded from http://pubs.acs.org on June 9, 2019
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Design, Synthesis and Efficacy testing of
nitroethylene- and 7-nitrobenzoxadiazol-based
flavodoxin inhibitors against Helicobacter pylori
drug-resistant clinical strains and in Helicobacter
pylori-infected mice
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Sandra Salillas1,2,3, Miriam Alías , Valérie Michel , Alejandro Mahía1,2,3, Ainhoa
1,2
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Lucía3,5,7, Liliana Rodrigues3,5,7,9†, Jessica Bueno , Juan José Galano-Frutos1,2,3, Hilde De
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Reuse4, Adrián Velázquez-Campoy1,2,3,9, José Alberto Carrodeguas1,2,3, Carlos Sostres3,6,
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1,3,5,7
10
Javier Castillo , José Antonio Aínsa
, María Dolores Díaz-de-Villegas , Ángel
Lanas3,6,8, Eliette Touati , Javier Sancho
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1,2,3*
1Biocomputation and Complex Systems Physics Institute (BIFI)-Joint Units: BIFI-IQFR
CSIC) and GBsC-CSIC, University of Zaragoza, Zaragoza 50018, Spain
(
2Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias,
University of Zaragoza, Zaragoza 50009, Spain
3Aragon Health Research Institute (IIS Aragón), Zaragoza 50009, Spain
4Helicobacter Pathogenesis Unit, CNRS ERL6002, Department of Microbiology, Institut
Pasteur, 25-28 Rue du Dr. Roux, Paris 75724, France
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5Departamento de Microbiología, Medicina Preventiva y Salud Pública, Facultad de
Medicina, University of Zaragoza, Zaragoza 50009, Spain
6CIBER de Enfermedades Hepáticas y Digestivas-CIBEREHD, Instituto de Salud Carlos
III, Madrid 28029, Spain
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7CIBER de Enfermedades Respiratorias–CIBERES, Instituto de Salud Carlos III, Madrid
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8029, Spain
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Departamento de Medicina, Psiquiatría y Dermatología, University of Zaragoza, Zaragoza
0009, Spain
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9Fundación ARAID, Gobierno de Aragón, Zaragoza 50009, Spain
10Instituto de Síntesis Química y Catálisis Homogénea (ISQCH), CSIC - Departamento de
Química Orgánica, University of Zaragoza, Zaragoza 50009, Spain
†Present address: Global Health and Tropical Medicine, GHTM, Unit of Medical
Microbiology, Instituto de Higiene e Medicina Tropical, IHMT, Universidade Nova de
Lisboa, UNL, Lisbon 1349-008, Portugal
KEYWORDS
Novel antimicrobials; antimicrobial resistance; Helicobacter pylori; drug discovery;
flavodoxin
ABSTRACT
Helicobacter pylori (Hp) infection is the main cause of peptic ulcer and gastric cancer. Hp
eradication rates have fallen due to increasing bacterial resistance to currently used broad-
spectrum antimicrobials. We have designed, synthesized and tested redox variants of
nitroethylene- and 7-nitrobenzoxadiazole-based inhibitors of the essential Hp protein
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flavodoxin. Derivatives of the 7-nitrobenzoxadiazole lead, carrying reduced forms of the
nitro group and/or oxidized forms of a sulphur atom, display high therapeutic indexes
against several reference Hp strains. These inhibitors are effective against metronidazole-,
clarithromycin- and rifampicin-resistant Hp clinical isolates. Their toxicity for mice after
oral administration is low and, when administered individually at single daily doses for 8
days in a mice model of Hp infection, they decrease significantly Hp gastric colonization
rates and are able to eradicate the infection in up to 60% of the mice. These flavodoxin
inhibitors constitute a novel family of Hp-specific antimicrobials that may help fight the
constant increase of Hp antimicrobial-resistant strains.
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INTRODUCTION
Helicobacter pylori (Hp) is a Gram-negative bacterium that establishes life-long infections
in humans by colonising their gastric mucosa, usually during childhood.1,2 Hp infection is
the most common bacterial infection all over the world2–4 with a prevalence that varies
from 10 to 94 % in different countries.5–8 Hp transmission follows person-to-person, oral-
oral, faecal-oral, waterborne or iatrogenic routes.5,9 In most cases, Hp infection is
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asymptomatic, but it can progressively damage the gastric mucosa by inducing chronic
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gastritis and then diseases from peptic ulcer to gastric mucosa-associated lymphoid tissue
(
MALT) lymphoma and gastric adenocarcinoma.2,3,10 Hp is the main cause of gastric cancer
all over the world.10,11 Up to now, it is the only bacterium classified as a group I
carcinogen.4,5,9,11 Eradication of Hp infection is the gold standard in peptic ulcer disease
treatment and might prevent gastric cancer,11–13 the third most common cause of cancer
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death. Effective and affordable treatments are needed to reduce the impact of Hp infection
in those diseases. Moreover, Hp infection has been suggested to be involved in other extra-
gastrointestinal disorders11,15–17 and may modify the bioavailability and absorption of
essential nutrients and the plasma levels of metabolic hormones.15
Conventional therapy against Hp consists of empirical triple or, more often now, quadruple
treatments based on a proton pump inhibitor plus a combination of two or three broad
spectrum antimicrobials such as clarithromycin (Cla), amoxicillin, metronidazole (Mnz),
tetracycline and bismuth salts.11,17–19 Increasing Hp resistance to these common
antimicrobial drugs, especially to Cla and Mnz, is growing worldwide, reducing the
effectiveness of available therapies.11,18–21 Therefore, new specific treatments are
required,22 and eradication strategies need to be adapted geographically.11,18,23 Ongoing
research on vaccines against Hp has not been successful until now, probably due to Hp
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genetic variability and to the complex host immune response against the bacterium.9,24–26
Current alternatives proposed include the application of therapeutic regimes based on local
patterns of antimicrobial susceptibility and the use of personalized treatments consisting of
the pre-identification of Hp susceptibility/resistance to conventional antimicrobials by
molecular or by cultured-guided methods.12,18,20,27 The use of antimicrobial peptides and the
development of novel compounds acting on specific Hp targets have also been proposed.
Following the latter approach, specific Hp targets, essential for the bacterium and absent in
humans, have been identified.28–30 One such target is Hp flavodoxin (Hp-Fld), a small
acidic redox protein that contains one molecule of tightly bound flavin mononucleotide
cofactor.30–33
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Flavodoxins34,35 are bacterial proteins that take part in a variety of electron transfer
reactions. In particular, Hp-Fld accepts electrons from the pyruvate ferredoxin
oxidoreductase complex (PFOR), which catalyses the oxidative decarboxylation of
pyruvate, and transfers them to flavodoxin quinone reductase.30,36,37 Both Hp-Fld and Hp-
PFOR are essential for the bacterium survival30,38 and compounds interfering with this
pathway might be suited for Hp eradication therapies (Figure 1).30,39
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Figure 1. Graphical summary of the rational followed for the development of new
flavodoxin inhibitors against Helicobacter pylori. Hp colonizes the gastric mucosa of
over half of the world’s population, establishing a life-long infection if untreated. Growing
concern about the increasing Hp resistance to the conventional antibiotics used in therapy,
has led to the proposal of new targets for fighting this global health problem. One of them
is Hp flavodoxin, an electron transfer protein involved in a metabolic process essential for
bacterial viability. We have designed and synthesized redox variants of a 7-
nitrobenzoxadiazol Hp flavodoxin inhibitor and have tested them in Hp clinical isolates and
in Hp-infected mice.
Unlike most other flavodoxins, Hp-Fld contains a pocket near the active site where small
molecules could bind30,31 interfering with binding or electron transfer to Hp-Fld partner
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proteins. Because flavodoxins as such are not present in humans, and the Fld-like domain
found in human cytochrome P450 reductase lacks an equivalent pocket near its active site,
no side effects are anticipated for a therapy targeting Hp-Fld.39
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In earlier work we have characterized the biophysical properties of Hp-Fld31–33,39,40 and
have identified through experimental screening several small interacting molecules that
bind to it. Three of them (named compounds I, II and IV) inhibit Hp-Fld-mediated
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pyruvate decarboxylation and act as specific bactericidal agents for Hp. Based on those
three hits, 123 new derivative molecules were developed, some of which showed better
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bactericidal properties than the three original ones. Because the hits and many of the
improved derivatives contain a nitro group and Hp is known to activate nitro groups in
certain prodrugs through redox dependent reactions,20,43–46 we have synthesized new anti-
Hp compounds that contain partially or fully reduced forms of the nitro groups present in
the parent molecules, and additional redox variants modifying sulphur and vinyl groups of
the inhibitors. The efficacy of these compounds has been tested in reference Hp strains, Hp
clinical isolates, and in Hp-infected mice.
RESULTS AND DISCUSSION
Synthetic reactions. Compounds I-a, II-b, II-c, II-d, IV-c, IV-d, IV-e, IV-f and Mnz-a
could be readily synthesized using the routes described in Schemes 1-8.
The synthesis of compound I-a was performed by treatment of nitro compound I with iron
47
in acidic conditions (HCl) under ultrasonic activation, leading to the desired product in
short reaction times (10-40 min) (Scheme 1).
Scheme 1. Reduction of nitro-compound I to oxime I-a
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Cl
NO2
Cl
NOH
Fe, HCl
O
DCM/MeOH
O
Cl
Cl
F
F
I
I-a
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Reduction of nitrostyrene II with sodium borohydride in the presence of silica gel and in
a mixture of chloroform and 2-propanol led to the corresponding nitroalkane II-b (Scheme
2
). Further reduction by catalytic hydrogenation using palladium-on-carbon as the catalyst
led to aminoalkaneII-c.
Scheme 2. Reduction of nitrostyrene II to nitroalkane II-b and reduction of this last one to
aminoalkane II-c
F3C
NO2
F3C
NO2
F3C
NH2
NaBH , Silica Gel
H , Pd/C
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CHCl3, iPrOH
EtOH
CF3
CF3
II-b
CF3
II-c
II
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Oxime II-d could be obtained by a palladium-catalysed reduction with triisopropylsilane
of nitro compound II in a mixture of THF and water (Scheme 3).
Scheme 3. Reduction of nitrostyrene II to oxime II-d
F3C
NO2
F3C
NOH
i
Pd(OAc) / Pr SiH
2
3
THF, H O
2
CF3
CF3
II-d
II
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Reduction of nitro-compound IV with iron and hydrochloric acid, by using
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dichloromethane and methanol as solvents, afforded the desired product IV-c and also
some of the previously synthesized molecule IV-a (Scheme 4) from which it was separated
by column chromatography.
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Scheme 4. Reduction of nitro-compound IV to a mixture of amino-compound IV-a and
amino-chlorinated compound IV-c
S
S
S
N
N
Fe, HCl
N
N
N
N
OCH3
OCH3
+
OCH3
O
O
O
DCM/MeOH
Cl
NO2
NH2
NH2
IV
IV-a
IV-c
Amino-sulfoxide IV-d and amino-sulfone IV-f could be obtained by thio-ether oxidation of
molecules IV-a and IV, respectively, with hydrogen peroxide in the presence of glacial
acetic acid (Schemes 5 and 6). 50
Scheme 5. Oxidation of sulphur compound IV-a to sulfoxide IV-d
O
S
S
N
N
H O
N
N
OCH3
2
2
OCH3
O
O
HOAc
NH2
NH2
IV-a
IV-d
Scheme 6. Oxidation of sulphur compound IV to sulfoxide IV-f
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O
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S
N
N
H O
N
N
OCH3
2
2
OCH3
O
O
HOAc
NO2
NO2
IV
IV-f
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The amino-sulfone IV-e was synthesized by oxidation of compound IV-a using meta-
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chloroperbenzoic acid in ethyl acetate as the oxidant. Compound IV-e was obtained as a
mixture with compoundIV-d, from which it was separated by flash chromatography
(
Scheme 7).
Scheme 7. Oxidation of sulphur IV-a to a mixture of sulfoxide IV-d and sulfone IV-e
O
O
S
O
S
S
N
N
mCPBA
AcOEt
OCH3
N
N
N
OCH3
+
OCH3
O
O
O
N
NH2
NH2
NH2
IV-a
IV-d
IV-e
Commercially available metronidazole was transformed to Mnz-a by catalytic reduction
using palladium-on-carbon as the catalyst (Scheme 8).52 The resulting amino-imidazole
proved to be sensitive to oxygen, heat and acid and therefore of limited utility; nevertheless,
its toxicity and activity were determined.
Scheme 8. Reduction of metronidazole to amino-imidazole Mnz-a
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2
3
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8
9
NO2
NH2
H , Pd/C
2
N
N
OH
N
N
OH
THF
Mnz
Mnz-a
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
4
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4
4
4
5
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5
5
5
5
5
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5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Improving therapeutic indexes by modifying the oxidation state of chemical groups
present in the flavodoxin inhibitor molecules. Several new derivatives of compounds I,
II and IV (Figure 2) have been synthesized and tested.
Figure 2. Chemical structure of 19 flavodoxin inhibitors grouped by similarity to lead
compound (I, II, IV or Mnz). In each group the upper line provides the formula of the
lead and derivatives preliminary tested in vitro in previous work.41,42 The middle line gives
the formula of newly synthesized derivatives (see Schemes 1-8 and Synthesis details
below). The lower line for II-related compounds provides the formula of commercial
compounds not previously tested as antimicrobials against Hp.
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1
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1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
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3
3
3
3
4
4
4
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4
4
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5
5
5
6
All of them are less cytotoxic toward human HeLa cells than their corresponding parent
compounds (Table 1). In fact, most derivatives are not cytotoxic at concentrations as high
as 1 mM (Figure S2). The in vitro anti-Hp activities of these derivatives have been
evaluated using three Hp reference strains (Table 1). The specific impact on TIs brought
about by modifying the oxidation state of the nitrogen and sulphur atoms and the vinyl
group of the inhibitors has been evaluated from pair wise comparisons of the 19 compounds
analysed.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. In vitro activity of flavodoxin inhibitors I, II and IV and related
compounds against H. pylori reference strainsa
MCC50b
MICc
TId
Compound
Structure
(µM)
(µM)
(MCC /MIC)
5
0
HeLa
5
SS1
0.37
NCTC 26695 SS1
NCTC
11637
26695
14
1
1637
Cl
NO2
I
0.37
6.1
3.5
2.3
0.37
6.1
1.8
2.3
14
10
8.3
16
14
19
4.3
16
O
Cl
F
Cl
NOH
I-a
II
117
15
12
1.8
2.3
19
8.3
16
O
Cl
F
F3C
NO2
CF3
F
NO2
II-a
36
Cl
F
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F3C
F3C
F3C
NO2
>
1000
223
>249
236
212
132
>233
0.78
13.9
6.1
56
31
56
31
>4.5
~2.0
>2.1
>4.7
>7.6
~4.3
9.0
>18
>16
>8.5
>4.7
>15
~4.3
4.4
>18
>16
>4.2
>9.4
>15
~4.3
1.1
II-b
II-c
CF3
CF3
CF3
NH2
>500
>500
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NOH
II-d
II-e
II-f
59
118
106
66
NO2
>1000
>1000
>1000
7
212
66
NH2
NHOH
II-g
IV
>233
1.6
27.8
3.0
11.8
53
>233
6.3
27.8
3.0
5.9
26
S
N
OCH3
O
N
NO2
S
N
N
OCH3
O
IV-a
IV-b
IV-c
IV-d
>1000
>1000
>1000
>1000
>72
>36
>333
>85
>19
>36
>333
>170
>39
NH2
S
CH3
S
OCH3
H3C
H3C
>164
>170
>9.5
S
N
N
OCH3
O
5.9
Cl
NH2
S
O
N
N
OCH3
O
105
NH2
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
S
O
N
N
IV-e
IV-f
OCH3
>1000
179
100
12
100
6
100
6
>10
15
>10
30
>10
30
O
NH2
S
O
N
N
OCH3
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
NO2
NO2
Mnz
N
N
OH
>1000
>1000
2.9
187
227
12
>345
~2.2
>5.3
>4.4
>83
>2.2
NH2
OH
>453
453
Mnz-a
N
N
aParent compounds I, II and IV are highlighted in grey
b
The Minimal Cytotoxic Concentration (MCC ) tested in HeLa cells
50
cMinimal Inhibitory Concentration (MIC) tested in three different H. pylori reference
strains (NCTC 11637 (Mnz resistant), and 26695 and Sydney Strain 1 (Mnz susceptible))
d
Therapeutic Indexes (TI) have been calculated as MCC /MIC
5
0
Compounds I and II share the presence of a nitroethylene moiety and their derivatives will
be jointly discussed. In this family of compounds, reduction of the nitro groups to oxime
lowers similarly cytotoxicity and antimicrobial activity, barely changing the TIs for the
three Hp strains tested (Table 1). On the other hand, reduction of the ethylene group leads
to a drastic decrease of toxicity with a notable reduction of antimicrobial activity, which
results in a small increase of TI. Since reduction of the ethylene group lowers so much
toxicity, differences in TIs among the ethylene-reduced variants cannot be properly
evaluated. In Mnz, the reduction of the nitro group to amine clearly lowers the
antimicrobial activity, while possible changes in cytotoxicity cannot be evaluated as neither
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Mnz nor the amine variant are toxic at the higher concentration tested. Globally considered,
reduction of the nitro or the ethylene group in the leads carrying a nitroethylene moiety (I,
II and Mnz), decreases to a similar extent toxicity and antimicrobial activity. Only
4
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
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9
0
1
2
3
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8
9
0
1
2
3
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8
9
0
compounds II-a (previously tested by Galano et al., 2013 and here re-evaluated), II-b, II-
c and II-f slightly improve (about 2-fold) the TI of their parent compound.
Compound IV also contains a nitro group, which, in this case, is attached to a
benzoxadiazole ring. In this lead, reduction of the nitro group to amine lowers toxicity so
much (>100-fold) that the concomitant moderate decrease in activity observed (around 10-
fold) combines to a 10-fold higher TI in compound IV-a. The toxicity of compound IV is
also greatly reduced (25-fold) by oxidation of the sulphur atom to sulfoxide, which causes a
less pronounced decrease in activity and therefore also raises the TI. Oxidation of the
sulphur atom to sulfoxide in IV-a, compound IV-d, similarly causes a smaller decrease in
activity, while a possible further decrease in toxicity cannot be determined because IV-a is
already non-toxic at the higher concentration tested (1 mM). Further oxidation of the
sulphur atom in IV-a to sulfone, compound IV-e, lowers the activity. However,
chlorination of IV-a increases the antimicrobial activity 2-4 fold and does not introduce
toxicity, leading to the high TIs of compound IV-c (from >85 to >170, depending on the
Hp reference strain tested). We have also tested an additional IV-related compound: IV-b,
4
2
that was considered promising in previous assays . Unlike all other IV variants, IV-b lacks
the benzoxadiazol ring and, therefore, the nitro group. Its toxicity is very low (>1000 µM)
and its anti-Hp activity is similar to that of compound IV. Therefore, the TI of IV-b is quite
high (from 164 to 333 depending on the Hp strain).
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Efficacy of compounds against antimicrobial-resistant Hp-strains. Most compounds in
Table 1 display higher TI than Mnz for the Mnz-resistant strain NCTC 11637. In addition,
several nitro-reduced derivatives of compound IV (i.e IV-a, IV-c, IV-d) and compound
IV-b display, for the two Mnz-sensitive strains tested (strains SS1 and 26695), TIs close to
that of Mnz, or even higher. To explore the potential usefulness of these flavodoxin
inhibitors towards other Hp drug-resistant strains, we have determined the activity of 6
representative commonly used antimicrobials on six Hp strains obtained from patients who
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
53
have relapsed or are refractory to, at least, two conventional therapies . Three of these
strains (isolates 1-3) are Mnz-resistant, two are both Mnz- and Cla-resistant (isolates 4 and
6) and one is rifampicin-resistant (isolate 5) (Table 2).
Table 2. Antimicrobial resistance profiles of H. pylori drug-resistant clinical
isolates following the EUCAST criteria. (S = sensitive; R = resistant)
Compound
Isolate 1
Isolate 2
Isolate 3
Isolate 4
Isolate 5
Isolate 6
Amoxicillin
Clarithromycin
Tetracycline
Levofloxacin
Metronidazole
Rifampicin
S
S
S
S
R
S
S
S
S
S
R
S
S
S
S
S
R
S
S
R
S
S
S
S
S
S
R
S
R
S
S
S
R
S
R
S
Our analysis of the effect of the flavodoxin inhibitors on these clinical isolates (Table 3 and
Table S2) confirms the activity trends previously observed when evaluated against
reference strains (Table 1). While compounds II-b, II-d, II-e, IV-d, IV-e display low TIs,
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with the exception of II-e on the isolate 4 (a Cla- and Mnz- resistant strain), compounds
IV-a and IV-c show high TIs for most clinical strains. Indeed, according to EUCAST
criteria, both IV-c and IV-a are effective against two Mnz-resistant strains (isolates 1 and
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
) and against the two Cla- and Mnz-resistant strains (isolates 4 and 6). In addition, IV-c is
also effective against the rifampicin-resistant strain (isolate 5). Only one Mnz-resistant
strain (isolate 3) displayed low TIs for both IV-c and IV-a (Table 3).
Table 3. TI values (MCC /MIC) of some developed compounds against H. pylori
5
0
drug-resistant clinical isolates a
Compound
Isolate 1 Isolate 2
Isolate 3
Isolate 4
Isolate 5
Isolate 6
II-b
II-d
II-e
9.0
4.2
9.0
4.2
4.7
72
9.0
4.2
9.0
34
9.0
4.2
4.7
9.0
40
9.0
4.2
4.7
72
4.7
4.7
605
144
80
IV-a
IV-c
IV-d
IV-e
144
161
9.5
9.0
322
9.5
10.0
10.0
9.5
322
9.5
10.0
9.5
20.0
9.5
10.0
10.0
20.0
a
HeLa cells have been used to determine the Minimal Cytotoxic Concentration (MCC ),
5
0
as reported in Table 1. In bold, TI values indicative of effectivity, according to EUCAST
criteria.
Efficacy of compounds against Hp gastric colonization in mice. The anti-Hp activity of
these novel compounds has also been investigated in the mouse model of Hp infection.54,55
First, the toxicity of three representative inhibitors (II-a, IV and IV-a) at doses of 10, 20
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2
2
2
2
2
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2
3
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3
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6
and 40 mg/100 g bw has been evaluated. Daily administration of compound IV-a for 8 days
had no deleterious effects in stomach, liver, heart, lung and kidney at any dose, as
determined by histopathological examination (Figure S3) and analysis of their biochemical
parameters (data not shown). Compounds IV and II-a did not induce significant alterations
at any dose in liver, heart, lung or kidney either, but they did provoke histological
alterations in stomach when administered at the highest dose of 40 mg/100 g (compound
IV) or at 20 or 40 mg/100 g (compound II-a) (Figure S3). The histological alterations
found in stomach included hyperplasia, inflammation and necrosis of the epithelium and
the gastric mucosa. None of these compounds were associated with increased levels of
tested biochemical parameters when compared to animals which did not receive the
compounds. The highest dose of compound II-a was, however, clearly toxic since it
induced hair loss and weight loss (> 20%) in all mice treated. The lack of biochemical or
histological alterations associated to the administration of high doses of IV-a indicates that
reduction of the nitro group in compound IV to amine removes the toxicity exerted in the
stomach by compound IV when orally administered to mice at high doses. The possibility
that the histological alterations observed in stomach after administration of the highest dose
of compound IV were associated to chemical instability of the compound at gastric pH has
been tested (see “In vitro acid stability evaluation of compounds” section in Supplemental
file). Compound IV (and the three derivatives tested: IV-a, IV-b and IV-d) are highly stable
at pH 1.0. In the subsequent in vivo analysis on anti-Hp activity, the doses of compounds
IV and II-a have been kept below those that produce histological alterations in mice
stomachs.
0
1
2
3
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9
0
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0
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0
Four experiments have been conducted in mice infected with Hp SS1 to test the anti-Hp
activity of the leads (compounds I, II and IV) and of several of the best new derivatives:
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I-a, related to I; II-a, II-b, II-c and II-f, related to II; and IV-a, IV-b, IV-c and IV-d,
related to IV. The doses ranged from 0.1 to 20 mg/100 g bw. As expected, in the four
experiments, all control mice have provided a clean background for interpretation: all mice
5
1
1
1
1
1
1
1
1
1
1
2
2
2
2
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3
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8
9
0
in the infected non-treated groups have shown efficient stomach Hp colonization (10 to
1
06 CFU/g gastric tissue) and no Hp colonies have been found in any group corresponding
to non-infected mice. The efficacy of the different compounds on Hp gastric colonization
has been determined by comparing the CFU/g gastric tissue of each infected, treated-group
with the corresponding infected, non-treated control group. Olive oil (90-95%)/DMSO (10-
5
%), used as vehicle to solubilise compounds, has not influenced the Hp gastric
colonization (not shown). All the data gathered in the four experiments for the testing of
compounds of the I-II series are presented in Figure 3, and data for the IV series in Figure
4
. Data of gastric colonization for all compounds and doses tested are presented along with
those of the corresponding control groups in Figures 3A and 4A, while the percentages of
Hp–eradicated mice after treatment are shown in Figures 3B and 4B for the successful
cases.
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0
1
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9
0
Figure 3. Inhibition of Hp SS1 gastric colonization of mice by treatment with I and II-
related compounds (I, II, I-a, II-a, II-b, II-c and II-f). Four experiments have been
carried out in order to test seven compounds at four doses (0.1, 1, 5, 10 and 20 mg/100 g
body weight (bw)) as described in the methods section. All doses for each compound,
except for compound II-a, have been tested in the same experiment, so they share the same
control data. Two different experiments have been carried out in order to evaluate the effect
of compound II-a. Its dose 10mg/100g bw was tested in the same experiment as that of I-a,
II-b, II-c and II-f and this is why they share the same control. Gastric colonization was
measured at three weeks post infection. Olive oil and DMSO, which were used as vehicles
of the compounds, had no effect on the gastric colonization. A) Evaluation of Hp gastric
colonization by colony counting. The number of viable colonies (CFU/g gastric tissue) are
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represented for each compound dose tested. In all cases the CFU/g was related to that of the
corresponding control group. Mann-Whitney U and Unpaired t tests were used for
statistical analysis. Statistically significant inhibition of the gastric mucosa colonization by
Hp has only been observed for compounds I-a, II-a and II-c. The inhibitory effect does not
follow a clear dose dependency possibly related to the poor solubility of the compounds.
1
1
1
1
1
1
1
1
1
1
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p≤0.05; **p≤0.01; ***p≤0.001. B) Eradication rates in Hp-infected mice treated with the
indicated compounds able to eradicate the infection in some mice: I, II-b and II-c.
Percentage of colonized (Hp+) and non-colonized (Hp-) mice are represented by white and
grey bars, respectively.
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Figure 4. Inhibition of Hp SS1 gastric colonization in mice treated with IV-related
compounds (IV, IV-a, IV-b, IV-c and IV-d). Four experiments have been carried out to
test five compounds at five doses (0.1, 1, 5, 10 and 20 mg /100 g bw) as described in the
methods section. Doses 10 and 20 mg/100 g of all compounds have been tested in the same
experiment, so they share the same control data. All compounds were orogastrically
administered during eight days at one week post Hp infection. Gastric colonization was
measured at three weeks post infection. Olive oil and DMSO, which were used as vehicles
of the compounds, had no effect on the gastric colonization. A) Evaluation of Hp gastric
colonization inhibition by colony counting. The number of viable colonies (CFU/g gastric
tissue) is represented for each compound dose tested. In all cases the CFU/g was related to
that of the corresponding control group. Mann-Whitney U and Unpaired t tests were used
for statistical analysis. A statistically significant inhibition of the gastric mucosa
colonization has been observed for all compounds, except for IV-c, and especially for
compounds IV, IV-a and IV-d, which were able to reduce the colonization rate at almost
all doses. The inhibitory effect does not follow a clear dose dependency, possible related to
the poor solubility of the compounds. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. B) Eradication
rates in Hp-infected mice treated with the compounds able to eradicate the infection in
some mice: IV, IV-a, IV-b and IV-d. Percentages of colonized (Hp+) and non-colonized
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(Hp-) mice are represented by white and grey bars, respectively.
As reported in Figure 3A, parent compound I was able to eradicate the Hp infection in
some mice (1/5 and 1/7) at 0.1 mg and 1 mg/100 g bw daily doses, respectively, but the
overall reduction of the gastric colonization rate observed was not statistically significant.
The oxime variant I-a decreased significantly the colonization rate at 10 mg/100 g bw
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(
p=0,016), but did not eradicate Hp infection from any mouse. The performance of the II-
related derivatives was also limited. Parent compound II had no inhibitory effect on the
gastric colonization; however, the ethylene-reduced II-b eradicated Hp infection from some
mice (1/6) at a 20 mg/100 g bw dose; the ethylene-reduced and nitro reduced II-c amine
derivative also eradicated Hp infection from some mice (1/4) and significantly reduced the
colonization rate (p=0.024) at 20 mg/100 g dose. In addition, compound II-a reduced the
colonization (p=0.0009) at a 1 mg/100 g bw dose.
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The best performance comes from IV-related compounds, in agreement with in vitro
testing. Both the parent compound IV, the amine-derivatives IV-a and IV-d, and the non-
benzoxadiazole containing compound IV-b significantly lowered the colonization rate at
most of the doses tested (Figure 4A) and eradicated the infection in some mice (Figure 4B).
Compound IV lowered colonization at 0.1, 1, 5 and 10 mg/100 g bw doses (p=0.044;
0
.041; 0.048 and 0.0002, respectively) and eradicated infection in 43 % (3/7) and 17 %
3/18) of mice treated with 0.1 and 10 mg/100 g bw, respectively. Compound IV-a also
inhibited the gastric colonization at 1, 5 and 10 mg/100 g bw doses (p=0.0001; 0.0018 and
.0004, respectively) and eradicated the infection in 13 % (4/31) of treated mice at 1
(
0
mg/100 g bw. Compound IV-b, the derivative lacking the nitrobenzoxadiazole moiety,
inhibited gastric colonization (p=0.0277 and 0.0042) at 10 and 20 mg/100 g bw,
respectively, and eradicated the infection in 29 % (2/7) of mice at a 20 mg/100 g bw dose.
Finally, compound IV-d, the sulfoxide version of compound IV-a, decreased colonization
at 1, 10 and 20 mg/100 g bw doses (p=0.005, 0.0002 and 0.0122, respectively) and
eradicate the infection in 60 % (3/5) of animals at the higher dose and in 5 % (1/20) of mice
at 10 mg/100 g bw.
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Perspectives for novel antimicrobials based in flavodoxin inhibitors. In previous work
we discovered flavodoxin inhibitors I, II and IV41,42 and explored the feasibility of
4
2
improving their antibacterial activity by replacing some of their substituents. To try to
further improve their therapeutic indexes, we have synthesized new variants exhibiting
different redox forms of the nitro, ethylene or sulphur groups present in the original
inhibitors. Their potential for Hp eradication has been tested in the mice model of infection,
and their activity against drug-resistant clinical Hp isolates have been determined. In all
variants tested, partial or full reduction of the nitro group, reduction of the ethylene group,
or partial or full oxidation of the sulphur atom largely reduces their cytotoxicity towards
both HeLa cells and mice. While in the I and II series concomitant decreases in
antibacterial activity are similarly large and the TIs are not improved, within the IV series,
the activity decreases are only moderate and the resulting derivatives exhibit much larger
TIs for Hp reference strains than the parent compound (Table 1). In fact, some of these
novel flavodoxin inhibitors display TIs comparable to those of Mnz and are effective
against a variety of Hp clinical isolates resistant to common antimicrobials such as Cla,
Mnz or rifampicin. Importantly, these inhibitors, used as sole agents against Hp infection in
the mice model, can significantly lower the gastric Hp load (Figures 3A and 4A) and, in
some mice, they have eradicated the infection (Figures 3B and 4B). Thus, compound IV
and its derivatives constitute a promising new family of antimicrobial chemicals with
potential against the increase of Hp drug-resistant strains.
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While their usefulness in novel combination therapies may be already worth investigating
to provide alternatives for the existing drugs used against Hp infection, it is also clear that
these inhibitors need to be improved before they can be used as stand-alone eradication
drugs. Two research lines are being followed toward this end. On one hand, the mechanism
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of action of these inhibitors needs to be clarified. As they were discovered using a simple
binding assay to the target protein,41,56 a straightforward inhibitory mechanism was
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proposed consisting on the inhibitors sterically blocking the flavodoxin interaction with
physiological partner proteins.30,36,37 The fact that, besides the original inhibitors, all the
new derivatives herein tested bind to Hp flavodoxin (as determined by isothermal titration
calorimetry and thermal shift assay, not shown) lends support to this mechanism and
provides direct experimental evidence for in vitro target engagement. Work is in progress to
obtain structural information on flavodoxin/inhibitor complexes as this could facilitate their
rational redesign and improvement. In addition, the possibility that flavodoxin activates the
compounds needs to be considered as the inhibitors contain nitro groups whose reduction
could generate radical cytotoxic products ultimately responsible for the observed
antibacterial activity. In this respect, the antibacterial activity of Mnz towards Hp and other
microaerophilic pathogens is thought to be mediated by one such redox activation
mechanism.43,46,57–59 This alternative activating mechanism cannot be invoked, though, in
the case of compound IV-b, the analogue of IV lacking a nitro group. Interestingly, the fact
that inhibitors such as IV-a retain antibacterial activity when carrying an amine instead of
the reducible nitro group of lead compound IV indicates that, if radical species played a
role in the bactericidal activity, they would have to be generated in a reverse manner from
cellular oxidation of the amine. On the other hand, a second line for improvement is
suggested by the poor aqueous solubility of the inhibitors, reflected in their logS values
41
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(Table S1 and Figure S1), and possibly related to the toxicity observed in some of the mice
stomachs at the highest dose of the parent compound IV, which we interpret as a chemical
direct toxicity since no other alterations are observed. The solubility of the inhibitors needs
to be increased by a judicious incorporation of polar groups in order to increase their
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bioavailability and ensure a systemic effect. Work is in progress to synthesise and test more
soluble variants of compound IV-derived inhibitors. Finally, unlike broad-spectrum
antimicrobials targeting very fundamental cellular components such as DNA, ribosomes or
the cell wall, flavodoxin inhibitors target a protein that is not essential for all bacteria and
whose sequence conservation is not particularly high. Our preliminary analysis of the
inhibitors’ specificity revealed that compound IV was not effective against H. pylori distant
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bacteria such as E. coli or S. aureus. We have now confirmed the same is true for the
novel non-toxic inhibitors described here (not shown). These flavodoxin inhibitors thus
appear to be selective and may be less damaging to the commensal microbiota than broad-
spectrum antimicrobials.60
CONCLUSION
Several lead compounds previously shown to inhibit the flavodoxin from Hp have been
redesigned to improve their antimicrobial activity. By synthesizing new variants exhibiting
different redox forms of the nitro, ethylene or sulphur groups, a family of
nitrobenzoxadiazol-based flavodoxin inhibitors with low toxicity for HeLa cells and for
mice has been developed that exhibits high therapeutic indexes against reference Hp
strains. Importantly, some of these new inhibitors are effective against drug-resistant
clinical Hp isolates (including Cla-, Mnz- and rifampicin-resistant strains) and are able to
significantly reduce Hp gastric colonization in the mouse model of infection and to
eradicate the infection in some mice. After improvement of their pharmacokinetic
properties, these compounds could constitute a new, Hp-specific family of antimicrobials
that could be less damaging for the gut microbiota than currently used broad-spectrum
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antibiotics and might be used on their own or in new combinations with existing antibiotics
to fight Hp resistant strains.
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EXPERIMENTAL SECTION
General synthetic and analytic procedures. All reagents were of analytical grade and
were used as obtained from commercial sources. Compounds II-e, II-f and II-g are
commercially available and were acquired from FCH group, Acros Organics and
UkrOrgSyntez Ltd. (UORSY), respectively. Reactions were carried out using anhydrous
1
13
19
solvents. H NMR, C NMR and F NMR spectra were acquired at room temperature at
4
00, 100 and 376 MHz, respectively, using a 5 mm probe. Chemical shifts (δ) are reported
in parts per million from tetramethylsilane with the solvent resonance as the internal
standard. Coupling constants (J) are quoted in hertz. The splitting patterns are reported as s
(singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), m (multiplet), bs
(broad singlet). Attached proton test spectra were acquired to determine the types of carbon
signals, and Heteronuclear Multiple Bond Correlation spectra were acquired to confirm the
position of heteroatoms. Special precautions such as degassing of the sample were not
taken. All compounds used for biological assays are ≥ 95% purity based on NMR and
HPLC chromatography. These last experiments have been performed using a Waters HPLC
system equipped with a 600-E pump, a 2996 Photodiode Array detector, and a 2707
autosampler. The LC system was fitted with a C18 reversed phase column (VYDAC 238TP
C18 5 μm, 4.6 mm × 250 mm) and operated, unless otherwise stated, using a linear gradient
of buffer B (100% in 40 min) from 100% buffer A (buffer A consisting of 0.1% TFA in
H2O, buffer B consisting of 0.085% TFA in CH3CN/H2O, 95:5 v/v) at a flow rate of 1
mL/min. High-resolution mass measurements were made using a microTOF (time-of-flight)
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analyzer, and spectra were recorded from methanolic solutions using the positive
electrospray ionization mode (ESI+). Reactions were magnetically stirred and whenever
possible monitored by TLC. TLC was performed on precoated silica gel polyester plates
and products were visualized using ultraviolet light (254 nm), ninhydrin, potassium
permanganate and phosphomolybdic acid solution followed by heating. Column
chromatography was performed using silica gel (Kiesegel 60, 230−400 mesh).
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6,8-dichloro-2-(4-fluorophenyl)chroman-3-one oxime (I-a). Compound I (615 mg, 1.81
mmol) was dissolved in a mixture of dichloromethane (65 mL), conc. hydrochloric acid (4
mL) and methanol (29 mL). After the addition of iron powder (688 mg, 12.32 mmol), the
mixture was sonicated (JP Selecta ultrasonic bath, 40 kHz) at room temperature for 10-40
minutes. TLC monitoring was done till completion of the reaction. The reaction mixture
was filtered to remove excess iron, and the solvent was evaporated under reduced pressure
to dryness. The residue was dissolved in water (35 mL) and extracted with ethyl acetate
(3×50 mL). The organic layers were dried over anhydrous magnesium sulphate, filtered and
concentrated in vacuo. The crude product was then purified by flash chromatography
1
(
eluent: Hex/EtOAc: 8/2) to give 313 mg (53 % yield) of the desired product. H NMR
(400 MHz, DMSO-d ): δ 11.54 (s, 1H), 7.45 (d, J = 2.5 Hz, 1H), 7.37-7.31 (m, 2H), 7.30
6
(d, J = 2.5 Hz, 1H), 7.23-7.18 (m, 2H), 5.93 (s, 1H), 3.94 (d, J = 22.2 Hz, 1H), 3.68 (d, J =
1
9
13
2
2.2 Hz, 1H) ppm. F NMR (376 MHz, DMSO-d ): δ -113.87 ppm. C NMR (100 MHz,
6
DMSO-d ): δ 161.9 (d, J = 244.6 Hz), 150.9, 148.0, 133.2, 128.6 (d, J = 8.5 Hz), 128.2,
6
+
1
27.3, 125.5, 124.6, 122.4, 115.4 (d, J = 21.7 Hz), 77.2, 24.4 ppm. HRMS (ESI ) m/z
+
[
M+H] calc for C H Cl FNO 326.0146, found 326.0125.
1
5
11
2
2
1
-(2-nitroethyl)-3,5-bis(trifluoromethyl)benzene (II-b). To a vigorously stirred mixture
of compound II (522 mg, 1.83 mmol), silica gel (3.7 g, column chromatography grade,
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Sigma-Aldrich), 2-propanol (5.5 mL) and chloroform (30 mL), NaBH (284 mg, 7.5 mmol)
4
was added in about 40 mg portions over a period of 15 min at 25 ºC. The mixture was
stirred for additional 2 hours or until completion of the reaction. Excess NaBH was
4
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decomposed with diluted hydrochloric acid and the mixture was filtered. The filter was
extracted with dichloromethane (3×50 mL) and the combined filtrates were washed with
brine (50 mL), dried over anhydrous magnesium sulphate, filtered and evaporated in vacuo
to dryness to give 504 mg (96 % yield) of the desired product which was used in the next
1
step without further purification. H NMR (400 MHz, CDCl ): δ 7.81 (s, 1H), 7.70 (s, 2H),
3
1
9
4
.69 (t, J = 6.9 Hz, 2H), 3.46 (t, J = 6.9 Hz, 2H) ppm. F NMR (376 MHz, CDCl ): δ
3
1
3
−63.00 ppm. C NMR (100 MHz, CDCl ): δ 138.4, 132.5 (q, J = 33.5 Hz), 129.0 (q, J =
3
+
3
.6 Hz), 123.2 (q, J = 272.7 Hz), 121.8 (m, J = 3.8 Hz), 75.2, 32.8 ppm. HRMS (ESI ) m/z
+
[
M+H] calc for C H F NO 288.0454, found 288.0460.
1
0
8
6
2
2
-(3,5-bis(trifluoromethyl)phenyl)ethan-1-amine (II-c). A solution of II-b (57.4 mg, 0.2
mmol) in ethanol (10 mL) was hydrogenated under hydrogen atmosphere (1 atm) at room
temperature in the presence of 10 % palladium/carbon (10 mg). The reaction was stirred for
5
h or until complete as judged by TLC. Filtration of the catalyst and evaporation of the
1
solvent afforded 51.4 mg (≈ 100 %) of pure desired product. H NMR (400 MHz, CDCl ):
3
δ 7.74 (s, 1H), 7.66 (s, 2H), 3.05 (t, J = 6.8 Hz, 2H), 2.90 (t, J = 6.8 Hz, 2H), 1.96 (bs, 1H)
1
9
13
ppm. F NMR (376 MHz, CDCl ): δ −62.83 ppm. C NMR (100 MHz, CDCl ): δ 142.4,
3
3
131.8 (q, J = 33.2 Hz), 129.1 (q, J = 2.6 Hz), 123.5 (q, J = 272.2 Hz), 120.5 (m, J = 3.9 Hz),
+
+
4
3.0, 39.5 ppm. HRMS (ESI ) m/z [M+H] calc for C H F N 258.0712, found 258.0711.
10 9 6
2
-(3,5-bis(trifluoromethyl)phenyl)acetaldehyde oxime (II-d). A round-bottom flask was
charged with Pd(OAc) (10 mg, 0.04mmol), the nitroarene II (214 mg, 0.75 mmol) and
2
freshly distilled THF (4.5 mL). The flask was sealed and purged with Ar. Degassed H O
2
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