WO2020079307A1

WO2020079307A1 - 4-phenyl dihydropyridine derivatives for the treatment and/or prevention of an infection or disease caused by helicobacter

Info

Publication number: WO2020079307A1
Application number: PCT/ES2019/070710
Authority: WO
Inventors: Andrés GONZÁLEZ RODRÍGUEZ; Ángel LANAS ARBELOA; Sandra SALILLAS BERGES; Adrián VELÁZQUEZ CAMPOY; Javier Sancho Sanz
Original assignee: Fundacion Instituto De Investigacion Sanitaria Aragon; Universidad De Zaragoza; Fundación Agencia Aragonesa Para La Investigación Y El Desarrollo (Araid)
Priority date: 2018-10-19
Filing date: 2019-10-18
Publication date: 2020-04-23
Also published as: ES2754900A1

Abstract

The invention relates to 4-phenyl dihydropyridine derivatives of formula I, and to pharmaceutical compositions of same, wherein the meaning of R₁, R₂, R₃ and A is that indicated in the description, for the treatment and/or prevention of an infection or disease caused by said infection, the infection being caused by the bacteria Helicobacter.

Description

4-Phenyldihydropyridine derivatives for the treatment v / or prevention of an infection or disease caused by Helicobacter

The present invention relates to 4-phenyldihydropyridine derivatives of formula I or to pharmaceutical compositions thereof for use in the treatment and / or prevention of an infection or disease caused by said infection, where the infection is caused by bacteria of the genus Helicobacter, preferably by the species Helicobacter pylori.

BACKGROUND OF THE INVENTION

Helicobacter pylori is considered the most prevalent pathogen in humans. A recent study suggests that more than half of the world's population is infected. The prevalence of infection usually varies according to socio-economic conditions, ranging from almost 20% in high-income countries like Switzerland or Sweden, to almost 90% of the infected population in countries like Nigeria. The prevalence of Helicobacter pylori infection in Spain is currently estimated at 54.9% of the population (Hooi JKY, et al. Global prevalence of Helicobacter pylori infection: systematic review and meta-analysis. Gastroenterology 2017; 153 ( 2): 420-429).

Helicobacter pylori is a microaerophilic Gram-negative bacterium of the Epsilonproteobacteria class, to which other pathogens of clinical relevance such as Campylobacter jejuni belong. The transmission mechanism of Helicobacter pylori infection is not perfectly elucidated. It is possible that the bacteria is transmitted from person to person through saliva or via the faecal-oral route, after eating contaminated food or water. The microorganism crosses the mucous layer that protects the stomach with the help of flagella and adheres to the gastric epithelial cells using adhesins. The bacterium produces a variety of enzymes such as urease, which allow it to neutralize gastric acidity and create a neutral microenvironment around it. Helicobacter pylori synthesizes large amounts of the urease enzyme that accumulates in the cytoplasm, periplasmic space and cell surface. The enzyme catalyzes the hydrolysis of urea present in the stomach to ammonia and carbon dioxide. Ammonium ions are capable of neutralizing heartburn, but they also cause damage to stomach epithelial cells causing necrosis and favoring various gastric pathologies. Other enzymes and cytotoxins produced by Helicobacter pylori such as VacA and CagA are also associated with damage to the gastric mucosa and with the oncogenic potential of this microorganism (Kusters JG, et al. Pathogenesis of Helicobacter pylori Infection. Clin Microbiol Rev 2006; 19 (3) : 449-490).

The current eradicating treatment of Helicobacter pylori infection consists of the combination of at least two antibiotics, associated with a proton pump inhibitor (PPI). The most widely used regimen currently and accepted as "first line" by the American College of Gastroenterology and the latest Maastricht consensus is the administration of Amoxicillin (1 g every 12 hours) together with Clarithromycin (500 mg every 12 hours) and one PPI at standard dose every 12 hours, or substituting Amoxicillin for Metronidazole (400 mg every 12 hours). This treatment is known as the "triple therapy" and usually requires 10 to 14 days, reaching eradication rates of 70-85%. However, in recent years this first-line therapy has significantly decreased its eradication rates, down to levels as low as 50% of treated cases. The root cause of this loss of efficacy lies in the increasing development of antimicrobial resistance by the microorganism (Yang JC, et al. Treatment of Helicobacter pylori infection: current status and future concepts. World J Gastroenterol 2014; 20 (18): 5283 -5293; and Safavi M., et al. Treatment of Helicobacter pylori infection: current and future insights. World J Clin Cases 2016; 4 (1): 5-19).

In Helicobacter pylori, the HsrA protein constitutes a response regulator that acts as an activator of transcription. HsrA regulates the expression of a large number of genes and operons involved in a variety of essential physiological processes of the microorganism, synchronizing metabolic functions and virulence with the availability of nutrients, cell division, and redox homeostasis. HsrA is an essential protein for the viability of Helicobacter pylori, gene deletion or inhibition of the biological activity of the protein imply cell death. HsrA is a ~ 25 kDa protein, with two defined structural and functional domains: an N-terminal domain with regulatory function and a C-terminal DNA binding domain, both domains are connected by a short flexible amino acid sequence without secondary structure defined. The protein dimerizes through its N-terminal domain forming a stable dimer both in vitro and in vivo. The regulator recognizes and binds to the promoters of its target genes covering a region of about 30 bp close but not overlapping to element -10 of the promoter. The binding of the regulator to its target promoters could favor the contact of RNA polymerase with DNA and positively influence the recruitment of the enzyme, playing an activating role for transcription. The HsrA protein has been cloned and extensively characterized in the laboratory, its crystallographic structure is known and relatively simple tests are available to evaluate its biological activity and the inhibition of this activity by potential inhibitors. HsrA is a typical protein of epsilonbacteria and there is no counterpart or protein of similar sequence in humans, which minimizes the risk of cross-reactivity and undesirable side effects of potential HsrA inhibitors on the normal microbiota or on human biomolecules (Delany I., et al. Growth phase-dependent regulation of target gene promoters for binding of the essential orphan response regulator HP1043. J Bacteriol 2002; 184 (17): 4800-4810; Schár J., et al. Phosphorylation-independent activity of atypical response regulators of Helicobacter pylori. J Bacteriol 2005; 187 (9): 3100-3109; Hong E., et al. Structure of an atypical orphan response regulator protein supports a new phosphorylation-independent regulatory mechanism. J Biol Chem 2007; 282 (28) : 20667-20675; Olekhnovich IN, et al. Mutations to essential orphan response regulator HP1043 of Helicobacter pylori result in growth-stage regulatory defects. Infect Immun 2013; 81 (5): 1439-49; and Olekhno vich IN, et al. Response to metronidazole and oxidative stress is mediated through homeostatic regulator HsrA (HP1043) in Helicobacter pylori. J Bacteriol 2014; 196 (4): 729-39).

Therefore, it would be desirable to have new inhibitory drugs against the transcriptional regulator HsrA of Helicobacter pylori.

DESCRIPTION OF THE INVENTION

In a first aspect, the present invention relates to a compound of formula I for use in the treatment and / or prevention of an infection caused by a bacterium of the genus Helicobacter or for use in the treatment and / or prevention of a disease caused by an infection caused by a bacterium of the genus Helicobacter:

I

where:

Ri is C _1-4 alkyl, -NH ₂ or -CN, where C _1-4 alkyl is optionally substituted by a group -OCONH ₂ , -OR ₅ or -SR ₅ ;

R ₂ is C _1-4 alkyl optionally substituted by a group -OR ₅ or -SR ₅ ;

R ₃ is C _1-4 alkyl or Cyi, where C _1-4 alkyl is optionally substituted by a group

R ₇ ;

A is a group of formula II, III, IV, V or VI:

V VI

R ₄ is -N0 ₂ , halogen, or -OCF ₃ ;

R ₅ is C _1-4 alkyl optionally substituted by a group -NH ₂ or -OR ₆ ;

Re is C _1-4 alkyl optionally substituted by a group -NH ₂ ;

R ₇ is -OC _1-4 alkyl, -NR ₈ Rg, Cy ₂ , -COC _1-4 alkyl or C _2-4 alkenyl, where C _2-4 alkenyl is optionally substituted by a phenyl group;

Cyi is a 4-6 membered saturated heterocycle attached to the rest of the molecule through of an available C or N atom, containing 1 or 2 N heteroatoms, where Cyi is optionally substituted by a Ci _-4 alkyl group, and where the Ci _-4 alkyl group is optionally substituted by one or two phenyl groups;

Cy ₂ is a phenyl or a saturated or aromatic 5- or 6-membered heterocycle containing from 1 to 3 heteroatoms selected from N and O, where Cy ₂ is attached to the rest of the molecule through an N or C atom, when Cy ₂ is a saturated heterocycle may optionally be fused to a piperidine ring forming a spiranic ring, and where Cy ₂ is optionally substituted by one or more Ri ₀ groups;

R ₈ is Ci- ₄ alkyl;

R ₉ is Ci- ₄ alkyl optionally substituted by one or more phenyl groups, and where each phenyl group is optionally substituted by a halogen group;

each R _i or independently is vinyl, phenyl or Cy ₃ , where the phenyl group is substituted by an Rn group;

R1 1 is halogen; and

Cy ₃ is a 5- or 6-membered saturated heterocycle, containing 1 or 2 N hetero atoms, that binds to the rest of the molecule through an available N or C atom and is optionally substituted by a Ci _-4 group alkyl, where the Ci _-4 alkyl group is optionally substituted by one or two phenyl groups.

In another embodiment the invention relates to the compound of formula I according to the use defined above, where the bacterium of the Helicobacter genus is of the Helicobacter pylori species.

In another embodiment the invention refers to the compound of formula I according to the use defined above, where Ri is methyl, isopropyl, -CH ₂ OR ₅ , -CH ₂ SR ₅ , -CH ₂ OCONH ₂ , -NH ₂ O -CN.

In another embodiment the invention refers to the compound of formula I according to the use defined above, where Ri is methyl, isopropyl, -CH ₂ OCH ₂ CH ₂ NH ₂ , -CH ₂ OCH ₂ CH ₂ OR ₆ , -CH ₂ SCH ₂ CH ₂ NH ₂ , -CH ₂ SCH ₂ CH ₂ 0 Re, -CH ₂ OCONH ₂ , -NH ₂ O -CN.

In another embodiment the invention refers to the compound of formula I according to the use defined above, where Ri is methyl, isopropyl, -CH ₂ OCH ₂ CH ₂ NH ₂ , -CH ₂ OCH ₂ CH ₂ OCH ₂ CH ₂ NH ₂ , - CH ₂ SCH ₂ CH ₂ NH ₂ , -CH ₂ S CH ₂ CH ₂ O CH ₂ CH ₂ NH ₂ , -CH ₂ OCONH ₂ , -NH ₂ O -CN. In another embodiment the invention refers to the compound of formula I according to the use defined above, where Ri is C _1-4 alkyl, and preferably where Ri is methyl. In another embodiment the invention refers to the compound of formula I according to the use defined above, where R ₂ is C _1-4 alkyl.

In another embodiment the invention refers to the compound of formula I according to the use defined above, where R ₂ is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl or tert-butyl, preferably where R ₂ is methyl, ethyl or isopropyl, and more preferably where R ₂ is methyl.

In another embodiment the invention refers to the compound of formula I according to the use defined above, where R ₃ is Ci _-4 alkyl optionally substituted by a group R ₇ .

In another embodiment the invention relates to the compound of formula I according to the use defined above, where R ₄ is -N0 ₂ , -Cl or -OCF ₃ , and preferably -N0 ₂ or -Cl. In another embodiment the invention refers to the compound of formula I according to the use defined above, where A is a group of formula II or III.

In another embodiment the invention refers to the compound of formula I according to the use defined above, where R ₇ is -OC _1-4 alkyl, -NR ₈ Rg, Cy ₂ or -COC _1-4 alkyl.

In another embodiment the invention refers to the compound of formula I according to the use defined above, where R ₃ is methyl.

In another embodiment the invention is related to the compound of formula I according to the use defined above, where R ₉ is Ci _-4 alkyl substituted by one or two phenyl groups, and where each phenyl group is optionally substituted by a halogen group.

In another embodiment the invention refers to the compound of formula I according to the use defined above, where Rg is -CH ₂ Ph or -CH ₂ CH ₂ C (Ph) (4-F-Ph).

In another embodiment the invention refers to the compound of formula I according to the use defined above, where Cyi is a 4- to 6-membered, preferably 4- or 5-membered saturated heterocycle, linked to the rest of the molecule through an available C atom, containing 1 N heteroatom, where Cyi is optionally substituted by a Ci _-4 alkyl group, and where the Ci _-4 alkyl group is optionally substituted by one or two phenyl groups.

In another embodiment the invention refers to the compound of formula I according to the use defined above, where Cyi is a group of formula Vil or VIII:

Vil VIII

In another embodiment the invention refers to the compound of formula I according to the use defined above, where Cy ₂ is a phenyl optionally substituted by one or more R _i o groups. In another embodiment the invention refers to the compound of formula I according to the use defined above, where Rn is F or Cl, and preferably Cl.

In another embodiment the invention refers to the compound of formula I according to the use defined above, where Cy ₃ is 1-vinylpiperazine or pyrrolidine.

In another embodiment the invention refers to the compound of formula I according to the use defined above, where the compound of formula I is selected from:

Amlodipine, Arandipine, Azelnidipine,

Barnidipine, Benidipine, Cilnidipine,

Clevidipine, Cronidipine, Darodipine,

Dexniguldipine, Elnadipine, Felodipine,

Furnidipino, Iganidipino, Isradipino,

Lemildipine, Lercanidipine, Levamlodipine,

Levniguldipine, Manidipine, Nicardipine,

Nifedipine, Niguldipine, Niludipine,

Nilvadipine, Nimodipine, Nisoldipine,

Nitrendipine, Olradipine, Palonidipine,

Pranidipine, Sornidipine, Teludipine,

Tiamdipine, Thrombodipine, and Vatanidipine.

Nicardipine, Nisoldipine, Nimodipine,

Felodipine, Nitrendipine, Lercanidipine,

Amlodipine, and Nifedipine. In another embodiment the invention refers to the compound of formula I according to the use defined above, where the compound of formula I is selected from:

Nicardipine, Nisoldipine, Nimodipine,

Nitrendipine, Lercanidipine, and Nifedipine.

In another embodiment the invention relates to the compound of formula I according to the use defined above, where the compound of formula I is selected from:

Nimodipine, and Nitrendipine. In another embodiment the invention is concerned with the compound of formula I according to the use defined above, where the disease caused by an infection is a gastrointestinal pathology, and preferably where the gastrointestinal pathology is selected from acute gastritis, chronic gastritis, duodenitis, functional dyspepsia, gastric ulcer, duodenal ulcer, gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma (MALT lymphoma).

The present invention also relates to a pharmaceutical composition comprising at least one compound of the invention (or a pharmaceutically acceptable salt or solvate thereof) and one or more pharmaceutically acceptable excipients. The excipients must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not being harmful to whoever takes said composition.

Thus, another aspect of the present invention relates to a pharmaceutical composition. comprising at least one compound of formula I defined above, for use in the treatment and / or prevention of an infection caused by a bacterium of the genus Helicobacter or for use in the treatment and / or prevention of a disease caused by an infection caused by a bacterium of the genus Helicobacter, and preferably where the bacterium of the genus Helicobacter is of the species Helicobacter pylori.

In another embodiment the invention relates to the pharmaceutical composition defined above, where the disease caused by an infection is a gastrointestinal pathology, and preferably where the gastrointestinal pathology is selected from acute gastritis, chronic gastritis, duodenitis, functional dyspepsia, gastric ulcer, ulcer duodenal, gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma (MALT lymphoma). In the above definitions, the term Ci - ₄ alkyl as a group or part of a group, means an alkyl group of straight or branched chain containing from 1 to 4 carbon atoms and includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl and tert-butyl. A halogen radical or its abbreviation halo means fluoro, chloro, bromo or iodo.

The term "optionally substituted by one or more" means the possibility of a group to be substituted by one or more, preferably by 1, 2, 3 or 4 substituents, more preferably by 1, 2 or 3 substituents and even more preferably by 1 or 2 substituents, provided that said group has enough available positions that can be substituted. If present, said substituents can be the same or different and can be located on any available position.

Throughout the present description, the term "treatment" refers to eliminating, eradicating, totally eradicating, reducing or diminishing the cause or effects of a disease. For the purposes of this invention, treatment includes, but is not limited to, alleviating, decreasing, or eliminating one or more symptoms of the disease; reduce the degree of disease, stabilize (that is, do not worsen) the state of the disease, delay or slow down the progression of the disease, alleviate or improve the state of the disease and remit (either total or partial). Examples include, but are not limited to, "eradicating infection,""total eradication of the microorganism" and "Decreased colonization of the microorganism".

As used in the present invention, the term "prevention" refers to preventing the onset of the disease from occurring in a patient who is predisposed or has risk factors, but who does not yet have symptoms of the disease. Prevention also includes preventing the recurrence of a disease in a subject who has previously suffered from said disease.

The compounds of the present invention contain one or more basic nitrogens and could therefore form salts with acids, both organic and inorganic. Some compounds of the present invention could contain one or more acidic protons and therefore could also form salts with bases.

There is no limitation on the type of salt that can be used, provided that when used for therapeutic purposes they are pharmaceutically acceptable. Pharmaceutically acceptable salts are understood as those salts which, in medical judgment, are suitable for use in contact with the tissues of humans or other mammals without causing undue toxicity, irritation, allergic response or the like. Pharmaceutically acceptable salts are widely known to any person skilled in the art.

The compounds of formula I and their salts may differ in certain physical properties, but are equivalent for the purposes of the invention. All salts of the compounds of formula I are included within the scope of the invention.

The compounds of the present invention can form complexes with solvents in which they are reacted or precipitated or crystallized. These complexes are known as solvates. As used herein, the term solvate refers to a complex variable stoichiometry consisting of a solute (a compound of formula I or a salt thereof) and a solvent. Examples of solvents include pharmaceutically acceptable solvents such as water, ethanol, and the like. A complex with water is known as a hydrate. Solvates of the compounds of the invention (or their salts), including hydrates, are included within the scope of the invention.

The compounds of formula I can exist in different physical forms, that is to say in amorphous and crystalline forms. Also, the compounds of the present invention may have the ability to crystallize in more than one way, a characteristic that it is known as polymorphism. Polymorphs can be differentiated by various physical properties well known to those skilled in the art such as their X-ray diffractograms, melting points or solubility. All physical forms of the compounds of formula I, including all of their polymorphic forms ("polymorphs"), are included within the scope of the present invention.

Some compounds of the present invention could exist in the form of various diastereoisomers and / or various optical isomers. Diastereoisomers can be separated by conventional techniques such as chromatography or fractional crystallization. The optical isomers can be resolved by using conventional optical resolution techniques, to give the optically pure isomers. This resolution can be carried out on the synthetic intermediates that are chiral or on the products of formula I. The optically pure isomers can also be obtained individually using enantiospecific syntheses. The present invention covers both the individual isomers and their mixtures (for example racemic mixtures or mixtures of diastereoisomers), whether obtained by synthesis or by physically mixing them.

The compounds of the present invention can be administered in the form of any pharmaceutical formulation, the nature of which, as is well known, will depend on the nature of the active ingredient and its route of administration. In principle, any route of administration can be used, for example oral, parenteral, nasal, ocular, rectal, and topical. Throughout the description and claims, the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge in part from the description and in part from the practice of the invention. The following figures and examples are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

Fig. 1. Inhibition of HsrA DNA binding activity by compounds of formula I. (A) Under appropriate in vitro conditions, as described in the procedure, the recombinant HsrA protein specifically recognizes and binds to DNA sequences of their target promoters. The binding of the protein to the target DNA determines the formation of a stable DNA-protein complex, with a molecular size greater than that of free DNA, experiencing a delay in electrophoretic mobility. The specific binding of HsrA to its target promoter PporGDAB and the formation of a HsrA-DNA complex detectable by the EMSA technique is directly proportional to the concentration of the recombinant protein and cannot be inhibited by the presence of a non-specific competitor DNA fragment. . Free target DNA disappears in the presence of increasing concentrations of the recombinant protein to form a stable DNA-protein complex of higher molecular weight. However, the concentration of competitor DNA remains unchanged, since the protein does not recognize or bind to this non-specific sequence. (B) Compounds of formula I inhibit the binding of the recombinant HsrA protein to its target promoters, preventing the formation of HsrA-DNA complexes. The inhibition capacity of the compounds of formula I on the activity of HsrA is directly proportional to the concentration of the inhibitor. As seen in Figure 1B, compounds of formula I completely inhibit the activity of the recombinant HsrA protein.

Fig. 2. Study of the antimicrobial effect of Nimodipine and Nitrendipine in vivo against infection with Helicobacter pylori. A. Quantitative data of bacterial counts expressed in CFU / mg of stomach. B. Quantitative PCR results presented as Helicobacter / 10,000 murine cells ratio. The results are presented in box diagrams. Each box represents 50% of the values around the median (horizontal dividing line of the boxes). The lines extending vertically from the box (whiskers) represent the minimum and maximum of all data. ^* P <0.05 or ^** P <0.01 between infected untreated animals (Hp + Om) and infected and treated animals (Hp + Nimodipine / Nitrendipine + Om).

EXAMPLES

Next, the invention will be illustrated by tests carried out by the inventors, which shows the effectiveness of the product of the invention.

Process

Cloning of the HsrA response regulator gene

The genomic DNA of Helicobacter pylori strain 26695 (ATCC 700392) was purified using the phenol-chloroform method. The biomass obtained from 100 mL of Helicobacter pylori 26695 culture in brain heart infusion broth (BHI) supplemented with 4% fetal bovine serum (SFB) for 48 h under microaerobic conditions was resuspended in 400 pL of 10 mM Tris-buffer. CI (pH 8) and 1 mM EDTA. To this cell suspension, 1% SDS and 10 pL of proteinase K (10 mg / mL) were added and incubated for 1 h at 55 ^e C. After this time, the sample was washed with 1 volume (same volume of the sample). ) of phenol, then with 1 volume of phenol-chloroform (1: 1) and finally two washes with 1 volume of chloroform were performed. Genomic DNA was precipitated overnight at -20 ^e C with 2 volumes of cold absolute ethanol and 0.1 volume of 3M sodium acetate (pH 5.2), the precipitate was washed with 70% ethanol and finally resuspended in 10mM Tris-CI buffer (pH 8) and 1mM EDTA.

The entire sequence of the hsrA gene (hp1043) was amplified by PCR from genomic DNA purified from Helicobacter pylori strain 26695 (ATCC 700392) using the 5 ^' -GGAATT CCAT AT GCGCGTT CT ACT GATT G-3 ^' primers (SEQ ID NO: 1) and 5 ^' - CCCAAGCTTTTACT CTT CACACGCCGG-3 ^' (SEQ ID NO: 2) and the enzyme Pfu DNA polymerase high fidelity (Agilent). The resulting PCR product was digested with the restriction enzymes Ndel and Hindlll and cloned between the same restriction sites of the expression vector pET-28a (Novagen). The final vector was partially sequenced to check the integrity of the gene.

Recombinant HsrA expression and purification

Competent E. coli BL21 (DE3) cells were transformed with the vector pET-28a containing the Helicobacter pylori ATCC 700392 hsrA gene. This vector allows the recombinant protein to be expressed as a fusion protein to a 6 residue peptide of histidines in its N-terminal end. The transformed E. coli cells were cultured in Luria-Bertani (LB) medium supplemented with 50 pg / mL of kanamycin under conditions of vigorous shaking and 37 ^e C until reaching an optical density of 0.8 measured at a wavelength of 600 nm. Expression of the recombinant HsrA protein was induced by adding 1mM IPTG to the culture medium, after which the cells were allowed to grow under the same temperature and shaking conditions for 6 hours. After this time, the culture was centrifuged at 8,000 rpm for 10 min at 4 ^and C and the obtained cell biomass was washed with cold PBS pH 7.4.

Recombinant HsrA protein is expressed in soluble form in the E. coli BL21 cytoplasm (DE3). For protein purification, cells were resuspended in 50mM Tris-CI buffer (pH 8), with 500mM NaCI, 10mM imidazole and 1mM PMSF. Cell disruption was performed using 10 45-second ultrasonic cycles in an ice bath, with 30-second rests between each cycle. Cell debris was removed by centrifugation at 18,000 rpm for 20 min at 4 ^° C. The recombinant histidine-tailed protein contained in the supernatant was purified by immobilized metal affinity chromatography (IMAC) using the Chelating Sepharose Fast Flow (GE) matrix. Flealthcare) loaded with Ni ²⁺ ions, previously balanced with the buffer used in cell lysis. Recombinant matrix bound protein was eluted using an imidazole gradient (0.01 to 1M) and dialyzed against 50mM T ris-HCI buffer (pH 8), 300mM NaCI and 10% glycerol. Protein concentration was determined using the commercial BCA ™ Protein Assay kit (Thermo Fisher Scientific). The poly-histidine tail was removed from the N-terminus of recombinant HsrA by treatment with thrombin (GE Flealthcare), using 10 U of enzyme for each milligram of recombinant protein. After enzymatic restriction, the recombinant HsrA protein was obtained in the dead volume from a second purification step by IMAC-Ni ²⁺ . The thus purified protein was stored at -20 ^e C.

Biological activity and inhibition assays

The HsrA protein is a response regulator with DNA binding activity, which acts as a transcriptional regulator. The biological activity of this protein is determined by the electrophoretic change of mobility test (EMSA). In this assay, the transcriptional regulator is contacted with a DNA fragment corresponding to the promoter region of its target genes. If the protein is active, it will specifically bind to its target promoters and form a protein-DNA complex that will experience a delay in electrophoretic mobility relative to free DNA, given the larger molecular size of the complex. As a target promoter region of HsrA, a sequence of 288 bp corresponding to the promoter of the operon by GDAB was amplified by PCR from the genomic DNA of Helicobacter pylori ATCC 700392 using as primers 5 ^' - CCCCACACTTGCCCCATACAGAC-3 ^' (SEQ ID NO: 3) and 5 ^' - GCAT GCCAT CT AATTT GAAACAT GG-3 ^' (SEQ ID NO: 4). The synthesized promoter was purified using the commercial ¡Ilustra ™ GFX PCR DNA and Gel Band Purification kit (GE Healthcare) and its concentration was quantified using a NanoVue Plus spectrophotometer (GE Healthcare). For assays of biological activity of freshly purified recombinant HsrA protein, increasing concentrations of the protein, from 2 to 7 mM, were mixed with 120 ng of DNA in 10 mM bis-Tris buffer (pH 7.5), 40 mM KCI, 100 pg / mL BSA, 1 mM DTT and 5% (v / v) glycerol, in a final volume of reaction mixture of 20 mI_. As nonspecific competitor DNA, 120 ng of a 121 bp fragment corresponding to a portion of the coding sequence of the pkn22 (alr2502) gene of the cyanobacterium Anabaena sp. Was added to all the mixtures. PCC 7120. The mixture of protein and DNA ^and incubated at 25 C for 30 minutes and subsequently separated by nondenaturing polyacrylamide gel electrophoresis using 6% Tris buffer 25 mM and 190 mM glycine for electrophoretic chamber. Polyacrylamide gels were stained with SYBR® Safe DNA gel stain (Invitrogen) and processed with Gel Doc 2000 Image Analyzer (Bio-Rad).

For assays of inhibition of biological activity, 6 mM of the recombinant HsrA protein were mixed with 2; one ; 0.5 and 0.1 mM of the compound of formula I described in the present invention. Since the analyzed compounds were 100% dissolved in DMSO, the protein in the presence of the same amount of DMSO was included as a negative inhibition control, replacing the compounds. The protein and inhibitor mixture in 10 mM bis-Tris buffer (pH 7.5), 40 mM KCI, 100 pg / mL BSA, 1 mM DTT and 5% (v / v) glycerol was incubated for 10 min at 25 ° C. After this time, 120 ng of target promoter (porGDAB) and 120 ng of competitor DNA (pkn22) were added to each mixture. The rest of the procedure was carried out under the same conditions of the activity assay described above. Each test was performed in triplicate and the inhibitory activity of at least 6 different compounds with formula I described in the present invention was analyzed.

Antibacterial activity against Helicobacter pylori of HsrA inhibitors

The antibacterial activity of 6 inhibitory compounds of formula I of the present invention was tested against two different strains of Helicobacter pylori, the strain AT CC 700392 and the strain resistant to clarithromycin ATCC 700684. Both strains were grown on blood agar (base) N ^e 2 (OXOID) supplemented with 8% of defibrillated horse blood (OXOID) in a humid microaerobic atmosphere (85% N ₂ , 10% C0 ₂ , 5% 0 ₂ ) at 37 ° C. The minimum inhibitory concentration (MIC) of each compound was determined by the microdilution method using flat bottom 96-well polystyrene plates. For the tests, the bacteria were subcultured in brain heart infusion broth (BHI) supplemented with 4% fetal bovine serum (SFB) for 48 h under microaerobic conditions. At the end of this time, the cultures were diluted with BHI + 4% SFB broth to an optical density of 0.01 measured at a wavelength of 600 nm. For the microdilution test on plates, 100 pL of the Thus adjusted bacterial cultures were dispensed into each of the wells of the plate, except for the first column of the plate, which received 200 µl of bacterial suspension with 5 ml of the inhibitory compound of formula I, at a concentration of 10.24 mg / ml_ in 100% DMSO. Each of the compounds of formula I tested was added to the first well of each row of the plate. Serial double dilutions of each compound were performed, evaluating the antibacterial activity of a concentration range between 256 and 0.125 pg / mL of each of the compounds. Positive and negative growth controls were incorporated in all plates with BHI + 4% SFB broth without the presence of inhibitors, inoculated and not inoculated with bacteria. Controls with DMSO and clarithromycin were also incorporated. All plates were incubated under microaerobic conditions at 37 ^e C and visually examined after 48 h of incubation. The MIC value of each compound was defined as the lowest concentration of the compound that completely inhibited the visible growth of the bacteria after 48 h.

The minimum bactericidal concentration (MBC) of each of the 6 compounds of formula I of the present invention was also determined. After completion of the MIC assay, 10 pL aliquots of two dilutions before and two after the MIC value were withdrawn and plated on N ^e 2 (OXOID) blood (base) agar supplemented with 8% defibrillated horse blood ( OXOID) in a humid microaerobic atmosphere (85% N ₂ , 10% C0 ₂ , 5% 0 ₂ ). Agar plates were incubated at 37 ° C for 48 hr. The MBC value of each compound was defined as the minimum concentration of the compound that prevented the growth of ³99.9% of the Helicobacter pylori cells subcultured on this inhibitor-free medium. Each experiment was performed in triplicate, twice, to confirm the results.

Energetic binding of inhibitors to HsrA

The specific binding of 6 inhibitory compounds of formula I of the present invention to the essential response regulator HsrA of Helicobacter pylori was confirmed by isothermal titration calorimetry (ITC). This technique not only measures the affinity of each compound for the target protein, but also determines the contribution of the enthalpic and entropic components, as well as the stoichiometry of the binding.

The binding experiments were carried out on the MicroCal Auto-iTC200 high sensitivity microcalorimeter (Malvern Instruments). A solution of 20mM HsrA in 50mM Tris-CI buffer (pH 8), 150mM NaCI, 10% glycerol and 1% DMSO located in the Measurement cell was titrated with a 200 mM solution of the corresponding inhibitory compound dissolved in the same buffer as above, located in the injection syringe.

In vivo antimicrobial activity of compounds of formula I against Helicobacter pylori infection in mice.

The antibacterial activity against Helicobacter pylori of 2 compounds of formula I of the present invention was tested in the mouse model C57BL / 6. The study was carried out in facilities with biosafety level A2 for laboratory animals of the University of Bordeaux (France) and approved by a local Ethics Committee of the University of Bordeaux, in accordance with the Regulations of the French Ministry of Agriculture on the care laboratory animals and the French Genetic Engineering Committee. Female C57BL / 6 mice (free of specific pathogens, SPF) 6 weeks old were kept in heated conditions and placed in polycarbonate boxes for one week before the start of the experiments. Two groups of 9 mice were fasted to facilitate bacterial colonization and then force fed with a dose of 10 ⁸ CFU of Helicobacter pylori strain PMSS1 for 3 consecutive days. Bacterial inocula were prepared fresh in Brucella broth medium from cultures grown for 48 h at 35 ^e C on Wilkins Chalgren agar plates enriched with 10% (v / v) human blood and supplemented with a mixture of antibiotics (10 pg / mL of vancomycin, 5 pg / mL of trimethoprim, 1 pg / mL of amphotericin B, and 2 pg / mL of cefsulodin) under microaerobiosis conditions (85% N ₂ , 10% C0 ₂ , 5% 0 ₂ ). After 4 weeks of inoculation, the mice in each group were orally treated with 100 mg / kg / day of Nimodipine or Nintrendipine (commercial formulation in oral tablets, STADA SL) in combination with omeprazole (140 mg / kg / day) , daily for 7 days. Since both drugs are highly sensitive to light, all manipulations were performed away from light. Two additional groups of 10 mice were used as controls, a control group of uninfected animals and another control group of untreated infected animals. Both control groups received 140 mg / kg / day of omeprazole during the 7 days of treatment.

One month after the end of the treatment, all the animals were sacrificed by cervical dislocation and their stomachs were conveniently isolated (cut in the esophagus and duodenum) and washed with sterile saline. The organs were first cut along the axis of the greater curvature and then along the axis minor curvature. Next, the right half of the stomach, once freed from the cardia, was cut into two equal fragments. One of these fragments was taken for bacteriological cultures and molecular studies, while the second fragment was kept at -80 ^e C for complementary experiments. The fragments for bacteriological and molecular studies were weighed, crushed using a sterile mortar and resuspended in 200 pL of sterile phosphate buffered saline. For the count of Helicobacter pylori colony forming units (CFUs) that were colonizing the stomach of the animals of each study group, serial dilutions of the stomach cell suspension were performed and plated on enriched Wilkins Chalgren agar. with 10% (v / v) of human blood and supplemented with a mixture of antibiotics (10 pg / mL of vancomycin, 5 pg / mL of trimethoprim, 1 pg / mL of amphotericin B and 2 pg / mL of cefsulodin) under conditions microaerobiosis (85% N ₂ , 10% C0 ₂ , 5% 0 ₂ ). Plates so seeded were incubated under microaerobiosis conditions (85% N ₂ , 10% C0 ₂ , 5% 0 ₂ ) for at least 5 days. The identity of the Helicobacter pylori colonies was confirmed according to the phenotypic and biochemical characteristics of the microorganism (morphology, urease test, oxidase test). Colony count was performed in two independent experiments and the results were expressed as CFU / mg stomach. The efficacy of Nimodipine and Nitrendipine in eradicating Helicobacter pylori gastric colonization in the mouse model was also determined by quantitative PCR (qPCR). Previously homogenized stomach samples were treated with the Arrow system (Nordiag) for DNA extraction. A qPCR based on fluorescence resonance energy transfer (FRET) was performed on the DNA sample extracted from the stomach biopsy, taking as a target the gene for the 23S ribosomal RNA (rRNA) of Helicobacter pylori (oligonucleotides: 5'- AGGTTAAGAGGAT GCGT CAGT C-3 '[HPY-S] (SEQ ID NO: 5) and 5'-

CGCAT GAT ATT CCCATT AGCAGT -3 '[HPY-A] (SEQ ID NO: 6)) and the gene for the enzyme glyceraldehyde-3P dehydrogenase (oligonucleotides: 5 ^' - CT GCAGGTT CT CCACACCT ATG-3 ^' [mGapdh1 -F] (SEQ ID NO: 7) and 5 ^' -

GAATTT GCCGT GAGT GGAGT C-3 ^' [mGapdh1 -R] (SEQ ID NO: 8)). Each target was tested in duplicate on all samples. A standard curve was prepared using serial dilutions of DNA extracted from calibrated Helicobacter pylori SS1 bacterial suspensions with known CFU / mL values. For the amplifications, LightCycler ^® 480 SYBR ^® Green I Master Mix (Roche Diagnostics), compatible with the LightCycler ^® 480 thermal cycler (Roche Diagnostics), was used, following the instructions manufacturer. In addition, SYBR® Premix Ex Taq ™ Mix (Tli RNaseH Plus) (Takara) compatible with the CFX96 ™ (Bio-Rad) thermal cycler available on the TBMCore real-time PCR platform from the University of Bordeaux was used, according to the instructions of the maker. PCR started with a 3 min denaturation step at 95 ° C, followed by 40 cycles with 2 steps: denaturation at qd'Ό for 5 sec and hybridization of the oligonucleotides at 60 ° C for 30 sec. After each cycle, fluorescence was measured in order to quantify the newly synthesized DNA. A the end of the procedure , a melting curve was generated by slowly raising the temperature from 65 to 95 ^and C and continuous fluorescence measurement. The generation of this melting curve allowed the verification of a specific peak at the expected melting temperature for each product, demonstrating the specificity of the PCR. The final results were expressed as the ratio (ratio) bacteria / murine cells. DNA extracted from the murine epithelial cell line m-ICcl2 available in the laboratory was used to express the results as a bacteria / murine cell ratio. The detection limit of the method was approximately 0.001 bacteria / murine cell.

The results obtained by both quantification methods (CFU / mg of stomach and qPCR) in the groups of treated mice were compared with those obtained in the group of untreated infected mice. Statistical analysis was performed using the non-parametric Mann-Whitney test. The differences between the samples were considered significant for a p value <0.05. Statistical analysis was performed using GraphPad Prism 6.0 (GraphPad Software, Inc.).

Results a) Inhibition of HsrA activity

The compounds of formula I inhibit the biological function of the essential protein HsrA of Helicobacter pylori. The HsrA protein is a response regulator with DNA binding activity. This protein acts as a transcriptional regulator in the cell, specifically binds to the promoter region of target genes and regulates the transcription of these genes. The compounds of formula I are shown to inhibit the binding of HsrA to their target promoters, as demonstrated in Figure 1. b) Antimicrobial activity of HsrA inhibitors on Helicobacter pylori The use of HsrA as a therapeutic target may be effective for the treatment of infectious diseases caused by Helicobacter pylori and associated pathologies, given that inhibitors of the biological activity of this protein demonstrate a high bactericidal activity on strains of this pathogen. The HsrA protein is essential for the viability of the microorganism, so the inhibition of its biological activity leads to the death of the pathogen and potentially to the eradication of the infection. Table 1 describes the minimum bactericidal concentration (MBC) values of various compounds of formula I against two strains of Helicobacter pylori.

Table 1. Bactericidal activity of compounds of formula I on Helicobacter pylori

c) Affinity and stoichiometry of binding between compounds of formula I and HsrA

The specific binding of the compounds of formula I to the HsrA protein was confirmed by Isothermal Titration Calorimetry (ITC). As can be seen in Table 2, all the compounds tested bind to the protein with an affinity in the micromolar order. In all cases, the binding stoichiometry was 1: 1; that is, one molecule of inhibitor for each molecule of monomeric protein.

Table 2. HsrA / Inhibitor Binding Affinity

d) In vivo evaluation of the anti-Helicobacter pylori activity of compounds of formula I in mice. The compounds of formula I of the present invention tested in preclinical efficacy studies in the mouse model demonstrated significant antimicrobial activity in vivo against Helicobacter pylori. Both Nimodipine + omeprazole therapy and Nitrendipine + omeprazole therapy resulted in a significant decrease in colonization by Helicobacter pylori of the gastric mucosa of the animal model, compared to untreated infected animals, who were given only omeprazole ( figure 2). The decrease in bacterial load in the stomach biopsies of the animals treated with the compounds of formula I was observed both in the studies of the count of CFU / mg of the stomach, and in the qPCR analyzes (Helicobacter cells / 10,000 murine cells). ).

Claims

1 Compound of formula I for use in the treatment and / or prevention of an infection caused by a bacterium of the genus Helicobacter or for use in the treatment and / or prevention of a disease caused by an infection caused by a bacterium of the genus Helicobacter :

I

where:

Ri is Ci- ₄ alkyl, -NH ₂ or -CN, where Ci _-4 alkyl is optionally substituted by a group -OCONH2, -OR ₅ or -SR ₅ ;

R ₂ is C _1-4 alkyl optionally substituted by a group -OR ₅ or -SR ₅ ;

R ₃ is C1-4alkyl or Cyi, where C1-4alkyl is optionally substituted by a group

R ₇ ;

A is a group of formula II, III, IV, V or VI:

V VI

R ₄ is -NO2, halogen, or -OCF ₃ ;

R ₅ is C1 -4 alkyl optionally substituted by a group -NH ₂ or -OR ₆ ;

Re is C1-4alkyl optionally substituted by a group -NH ₂ ;

R ₇ is -OC1 -4 alkyl, -NR ₈ R _g , Cy ₂ , -COC1 -4 alkyl or C _2-4 alkenyl, where C _2-4 alkenyl is optionally substituted by a phenyl group;

Cyi is a 4-6 membered saturated heterocycle attached to the rest of the molecule through an available C or N atom, containing 1 or 2 N heteroatoms, where Cyi is optionally substituted by a C1-4alkyl group, and where the C1-4alkyl group is optionally substituted by one or two phenyl groups;

Cy ₂ is a phenyl or a saturated or aromatic 5- or 6-membered heterocycle containing from 1 to 3 heteroatoms selected from N and O, where Cy ₂ is attached to the rest of the molecule through an N or C atom, when Cy ₂ is a saturated heterocycle may optionally be fused to a piperidine ring forming a spiranic ring, and where Cy ₂ is optionally substituted by one or more R10 groups;

R ₈ is C1 -4 alkyl;

R ₉ is C1-4alkyl optionally substituted by one or more phenyl groups, and where each phenyl group is optionally substituted by a halogen group;

each R10 independently is vinyl, phenyl or Cy ₃ , where the phenyl group is substituted by an Rn group;

R1 1 is halogen; and Cy ₃ is a 5- or 6-membered saturated heterocycle, containing 1 or 2 N hetero atoms, that binds to the rest of the molecule through an available N or C atom and is optionally substituted by a Ci _-4 group alkyl, where the Ci _-4 alkyl group is optionally substituted by one or two phenyl groups.

2. The compound of formula I according to the use of claim 1, wherein the bacterium of the genus Helicobacter is of the species Helicobacter pylori.

3. The compound of formula I according to the use of any of claims 1 or 2, where Ri is methyl, isopropyl, -CH ₂ OR ₅ , -CH ₂ SR ₅ , -CH ₂ OCONH ₂ , -NH ₂ or - CN.

4. The compound of formula I according to the use of any of claims 1 to 3, where R ₂ is Ci _-4 alkyl, and preferably where R ₂ is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl or tert-butyl, preferably where R ₂ is methyl, ethyl or isopropyl, and more preferably where R ₂ is methyl.

5. The compound of formula I according to the use of any of claims 1 to 4, wherein R ₃ is Ci _-4 alkyl optionally substituted by a group R ₇ .

6. The compound of formula I according to the use of any of claims 1 to 5, where R ₄ is -N0 ₂ , -Cl or -OCF ₃ , and preferably -N0 ₂ or -Cl.

7.- The compound of formula I according to the use of any of claims 1 to 6, where A is a group of formula II or III.

8. The compound of formula I according to the use of any of claims 1 to 7, where R ₈ is methyl.

9. The compound of formula I according to the use of any of claims 1 to 8, where R ₉ is Ci _-4 alkyl substituted by one or two phenyl groups, and where each phenyl group is optionally substituted by a halogen group.

10. The compound of formula I according to the use of any of claims 1 to 9, wherein Cyi is a group of formula Vil or VIII:

Vil VIII

January 1 .- The compound of formula I according to the use of any of claims 1 to 10, wherein Cy ₂ is phenyl optionally substituted by one or more groups R _Y.

12. The compound of formula I according to the use of claim 1, wherein the compound of formula I is selected from:

Nicardipine, Nisoldipine, Nimodipine,

Nitrendipine, Lercanidipine, and Nifedipine.

13. The compound of formula I according to the use of claim 12, wherein the compound of formula I is selected from:

Nimodipine, and Nitrendipine.

14. The compound of formula I according to the use of any of claims 1 to 13, wherein the disease caused by an infection is a gastrointestinal pathology.

15. The compound of formula I according to the use of claim 14, wherein the gastrointestinal pathology is selected from acute gastritis, chronic gastritis, duodenitis, functional dyspepsia, gastric ulcer, duodenal ulcer, gastric adenocarcinoma and lymphoid tissue lymphoma associated with mucosa (MALT lymphoma).

16.- Pharmaceutical composition comprising at least one compound of formula I according to any of claims 1 to 13, for use in the treatment and / or prevention of an infection caused by a bacterium of the genus Helicobacter or for use in treatment and / or prevention of a disease caused by an infection caused by a bacterium of the Helicobacter genus.