WO2023152415A1

WO2023152415A1 - Compounds for the treatment and/or prevention of an infection or disease caused by helicobacter or campylobacter

Info

Publication number: WO2023152415A1
Application number: PCT/ES2023/070071
Authority: WO
Inventors: Andrés GONZÁLEZ RODRÍGUEZ; Ángel LANAS ARBELOA; Javier CASADO APASTEGUI; Eduardo CHUECA LAPUENTE; Elena PIAZUELO ORTEGA; Sandra SALILLAS BERGES; Javier Sancho Sanz
Original assignee: Fundación Instituto de Investigación Sanitaria Aragón; Consorcio Centro De Investigación Biomédica En Red; Instituto Aragonés De Ciencias De La Salud; Universidad De Zaragoza
Priority date: 2022-02-10
Filing date: 2023-02-10
Publication date: 2023-08-17
Also published as: ES2948122A1

Abstract

The present invention relates to a compound of formula I, wherein the groups R₁ to R₅ and Cy have the meaning described in the description, for use thereof in the treatment and/or prevention of an infection or disease caused by the infection of a bacterium of the genus Helicobacter or Campylobacter.

Description

DESCRIPTION

Compounds for the treatment and/or prevention of an infection or disease caused by Helicobacter or Campylobacter

The present invention relates to a compound of formula I for use in the treatment and/or prevention of an infection or disease caused by infection with a bacterium of the genus Helicobacter or Campylobacter.

BACKGROUND OF THE INVENTION

H. pylori is considered the most prevalent bacterial pathogen in humans. A recent study suggests that more than half of the world population is infected (Hooi J.K.Y., et al. Global prevalence of Helicobacter pylori infection: systematic review and metaanalysis. Gastroenterology 2017; 153: 420-429). The organism penetrates the mucosal layer that protects the stomach and adheres to and colonizes the gastric epithelial cells. Unless the infection is adequately treated, the bacterium can persist in the stomach throughout the patient's life, constituting the most common cause of chronic gastritis and contributing to the pathogenesis of more serious clinical manifestations such as peptic ulcer, gastric adenocarcinoma, and lymphoma of the stomach. mucosa-associated lymphoid tissue (MALT lymphoma) (Kusters J.G., et al. Pathogenesis of Helicobacter pylori infection. Clin Microbiol Rev 2006; 19: 449-490; and Moss S.F. The clinical evidence linking Helicobacter pylori to gastric cancer. Cell Mol Gastroenterol Hepatol 2017;3:183-191). In 1994, H. pylori was classified as a type I carcinogen (definitive human carcinogen) for gastric cancer by the International Agency for Research on Cancer. About 60% of intestinal-type gastric adenocarcinomas and 98% of MALT lymphomas of the stomach are associated with H. pylori infection. Likewise, chronic infection with this organism significantly increases the patient's risk of developing other non-gastric lymphomas, such as diffuse large B-cell lymphoma and ocular adnexal lymphoma. The bacterium has also been implicated in various extra-gastrointestinal disorders, such as ischemic heart and cerebrovascular diseases, atherosclerosis, among others.

H. pylori is a microaerophilic Gram-negative bacterium of the class

Epsilonproteobacteria, to which other clinically relevant pathogens belong as C. jejuni. Campylobacter is one of the four main causes of diarrheal disease and is considered the most frequent bacterial cause of gastroenteritis in the world (Kaakoush NO, et al. Epidemiology of Campylobacter infection. Clin Microbiol Rev 2015; 28: 687-720). Campylobacter infections are usually mild but can be fatal in very young children, the elderly, and immunosuppressed individuals. The bacterium colonizes the intestinal epithelium causing acute gastroenteritis, watery diarrhea, fever, nausea and vomiting. Occasionally, the infection can trigger serious extraintestinal events such as bacteremia, meningitis, and Guillain-Barré syndrome, or severe gastrointestinal pathologies such as pseudomembranous colitis, appendicitis, and massive gastrointestinal bleeding. Within the genus Campylobacter, the species C. jejuni is associated with most reported infections in humans (Igwaran A., et al. Human campylobacteriosis: A public health concern of global importance. Heliyon 2019; 5: e02814).

Given the alarming development and accumulation of antimicrobial resistance mechanisms by strains of H. pylori and C. jejuni throughout the world, with the consequent decrease in the efficacy of antimicrobial treatments considered first-line, in 2017 the QMS classified these two pathogens as “high priority” for the search and development of new antibiotics (Tacconelli E., et al. Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis. Lancet Infect Dis 2018; 18 : 318-327).

Thus, it would be desirable to have safe drugs that are highly effective as bactericides against pathogenic epsilonproteobacteria such as Helicobacter pylori and Campylobacter jejuni species and selective against relevant members of the human microbiota.

DESCRIPTION OF THE INVENTION

In a first aspect, the present invention refers to a compound of formula I for its use in the treatment and/or prevention of an infection caused by a bacterium of the genus Helicobacter or Campylobacter or for its use in the treatment and/or prevention of a disease caused by infection with a bacterium of the genus Helicobacter or Campylobacter, and preferably where the bacterium of the genus Helicobacter is of the species Helicobacter pylori and where the bacterium of the genus Campylobacter is of the species Campylobacter jejuni:

where:

R ₁ is a group H or halogen;

R ₂ is a group H or halogen;

R ₃ is a group H, halogen, C _1-4 alkyl, -COC _1-4 alkyl; -COOH, -COOC _1-4 alkyl, -OH, -NH ₂ , -SH or -NO ₂ , where C _1-4 alkyl is optionally substituted by one or more R ₉ groups;

R ₄ is an H or C _1-4 alkyl group;

R ₅ is a group H, -COOH or -COOC _1-4 alkyl;

Cy is a group of formula la, Ib, le or Id:

R ₆ is a group H, halogen, C _1-4 alkyl, -COOH or -COOC _1-4 alkyl;

R ₇ is a group H, halogen, C _1-4 alkyl, -COOH, -COC _1-4 alkyl or -NO ₂ , where C _1-4 alkyl is optionally substituted by one or more R ₁₀ groups; R ₈ is H or halogen;

R ₉ is a halogen group; and

R ₁₀ is a halogen group.

In another embodiment the invention relates to a compound of formula I for the use defined above where:

R ₁ is a group H or halogen;

R ₂ is a group H or halogen;

R ₄ is an H or C _1-4 alkyl group;

R ₅ is a group H, -COOH or -COOC _1-4 alkyl;

Cy is a group of formula la, Ib, le or Id:

R ₆ is a group H, halogen, C _1-4 alkyl, -COOH or -COOC _1-4 alkyl;

R ₇ is a group H, CH ₃ , Cl, Br, -COOH, -COC ₂ H ₅ , -COCH ₃ or -NO ₂ , and preferably H, CH ₃ , Cl, Br, -COOH, -COCH ₃ or - NOT ₂ ;

R ₈ is H or halogen;

R ₉ is a halogen group; and

R10 is a halogen group.

In another embodiment the invention relates to a compound of formula I for the use defined above where R ₁ is a group H, Cl or Br, preferably where R ₁ is H or Br, and more preferably where R ₁ is H.

In another embodiment the invention relates to a compound of formula I for the use defined above where R ₂ is a group H, Cl or Br, and preferably where R ₂ is H or Br.

In another embodiment the invention relates to a compound of formula I for the use defined above wherein R ₃ is a group H, Br, Cl, methyl, ethyl, -COC ₂ H ₅ , -COCH ₃ ; -COOH, -COOCH ₃ , -OH, -NH ₂ , -SH or -NO ₂ .

In another embodiment the invention relates to a compound of formula I for use defined above where R ₄ is a group H or CH ₃ .

In another embodiment the invention relates to a compound of formula I for the use defined above where R ₅ is a group H or -COOH.

In another embodiment the invention relates to a compound of formula I for the use defined above, where Cy is a group la.

In another embodiment the invention relates to a compound of formula I for the use defined above where R ₆ is a group H, Cl, Br, CH ₃ or -COOH, and preferably H, Br, CH ₃ or -COOH.

In another embodiment the invention relates to a compound of formula I for the use defined above where R ₇ is a group H, CH ₃ , Cl,

Br, -COOH, -COC ₂ H ₅ , -COCH ₃ or -NO ₂ , and preferably H, CH ₃ , Cl,

Br, -COOH, -COCH ₃ O -NO ₂ .

In another embodiment the invention relates to a compound of formula I for the use defined above where R ₈ is a group H, Br or Cl, and preferably H or Br.

In another embodiment the invention relates to a compound of formula I for the use defined above where R ₉ is a Cl or Br group.

In another embodiment the invention relates to a compound of formula I for the use defined above where R ₁₀ is a Cl or Br group.

R ₁ is a group H, Cl or Br, preferably where R ₁ is H or Br, and more preferably where R ₁ is H; and R ₂ is a group H, Cl or Br, and preferably where R ₂ is H or Br.

R ₁ is a group H, Cl or Br, preferably where R ₁ is H or Br, and more preferably where R ₁ is H; and

R ₃ is a group H, Br, Cl, methyl, ethyl, -COC ₂ H ₅ , -COCH ₃ ; -COOH, -COOCH ₃ , -OH, -NH ₂ , -SH or -NO ₂ .

R ₄ is an H or CH ₃ group.

R ₅ is a group H or -COOH.

R ₆ is a group H, Cl, Br, CH ₃ or -COOH, and preferably H, Br, CH ₃ or -COOH.

R ₇ is a group H, CH ₃ , Cl, Br, -COOH, -COC ₂ H ₅ , -COCH ₃ or -NO ₂ , and preferably H, CH ₃ , Cl, Br, -COOH, -COCH ₃ or - NOT ₂ .

R ₁ is a group H, Cl or Br, preferably where R ₁ is H or Br, and more preferably where R ₁ is H; and R ₈ is a group H, Br or Cl, and preferably H or Br. In another embodiment the invention relates to a compound of formula I for the use defined above where:

Cy is a group of formula la.

R ₁ is a group H, Cl or Br, preferably where R ₁ is H or Br, and more preferably where R ₁ is H; R ₂ is a group H, Cl or Br, and preferably where R ₂ is H or Br; and Cy is a group la.

R ₁ is a group H, Cl or Br, preferably where R ₁ is H or Br, and more preferably where R ₁ is H;

R ₃ is a group H, Br, Cl, methyl, ethyl, -COC ₂ H ₅ , -COCH ₃ ; -COOH, -COOCH ₃ , -OH, -NH ₂ , -SH or -NO ₂ ; and Cy is a group la.

R ₄ is a H or CH ₃ group; and

Cy is a group la.

R ₅ is a group H or -COOH; and

Cy is a group la. In another embodiment the invention relates to a compound of formula I for the use defined above where:

R ₆ is a group H, Cl, Br, CH ₃ or -COOH, and preferably H, Br, CH ₃ or -COOH; and Cy is a group la.

R ₇ is a group H, CH ₃ , Cl, Br, -COOH, -COC ₂ H ₅ , -COCH ₃ or -NO ₂ , and preferably H, CH ₃ , Cl, Br, -COOH, -COCH ₃ or - NOT ₂ ; and

Cy is a group la.

R ₁ is a group H, Cl or Br, preferably where R ₁ is H or Br, and more preferably where R ₁ is H; R ₈ is a group H, Br or Cl, and preferably H or Br; and

Cy is a group la.

R ₂ is a group H, Cl or Br, and preferably where R ₂ is H or Br; and

R ₄ is an H or CH ₃ group.

In another embodiment the invention relates to a compound of formula I for the use defined above where: R ₂ is a group H, Cl or Br, and preferably where R ₂ is H or Br; and R ₅ is a group H or -COOH.

In another embodiment the invention relates to a compound of formula I for the use defined above where: R ₂ is a group H, Cl or Br, and preferably where R ₂ is H or Br; and

In another embodiment the invention relates to a compound of formula I for the use defined above where: R ₂ is a group H, Cl or Br, and preferably where R ₂ is H or Br;

Cy is a group la; and R ₈ is a group H, Br or Cl, and preferably H or Br.

In another embodiment the invention relates to a compound of formula I for the use defined above where: R ₂ is a group H, Cl or Br, and preferably where R ₂ is H or Br; and R ₈ is a group H, Br or Cl, and preferably H or Br.

R ₃ is a group H, Br, Cl, methyl, ethyl, -COC ₂ H ₅ , -COCH ₃ ; -COOH, -COOCH ₃ , -OH, -NH ₂ , -SH or -NO ₂ ; and R ₄ is an H or CH ₃ group.

R ₃ is a group H, Br, Cl, methyl, ethyl, -COC ₂ H ₅ , -COCH ₃ ; -COOH, -COOCH ₃ , -OH, -NH ₂ , -SH or -NO ₂ ; and R ₅ is a group H or -COOH.

R ₃ is a group H, Br, Cl, methyl, ethyl, -COC ₂ H ₅ , -COCH ₃ ; -COOH, -COOCH ₃ , -OH, -NH ₂ , -SH or -NO ₂ ; and

R ₃ is a group H, Br, Cl, methyl, ethyl, -COC ₂ H ₅ , -COCH ₃ ; -COOH, -COOCH ₃ , -OH, -NH ₂ , -SH or -NO ₂ ; and Cy is a group of formula la.

R ₃ is a group H, Br, Cl, methyl, ethyl, -COC ₂ H ₅ , -COCH ₃ ; -COOH, -COOCH ₃ , -OH, -NH ₂ , -SH or -NO ₂ ; and R ₈ is a group H, Br or Cl, and preferably H or Br.

R ₄ is a H or CH ₃ group; and

R ₅ is a group H or -COOH.

R ₄ is a H or CH ₃ group; and R ₆ is a group H, Cl, Br, CH ₃ or -COOH, and preferably H, Br, CH ₃ or -COOH.

R ₄ is a H or CH ₃ group; and

R ₄ is a H or CH ₃ group; and R ₈ is a group H, Br or Cl, and preferably H or Br.

R ₅ is a group H or -COOH; and

R ₅ is a group H or -COOH; and R ₈ is a group H, Br or Cl, and preferably H or Br.

R ₆ is a group H, Cl, Br, CH ₃ or -COOH, and preferably H, Br, CH ₃ or -COOH; and R ₇ is a group H, CH ₃ , Cl, Br, -COOH, -COC ₂ H ₅ , -COCH ₃ or -NO ₂ , and preferably H, CH ₃ , Cl, Br, -COOH, -COCH ₃ or -NO ₂ . In another embodiment the invention relates to a compound of formula I for the use defined above where:

R ₆ is a group H, Cl, Br, CH ₃ or -COOH, and preferably H, Br, CH ₃ or -COOH; and R ₈ is a group H, Br or Cl, and preferably H or Br.

R ₇ is a group H, CH ₃ , Cl, Br, -COOH, -COC ₂ H ₅ , -COCH ₃ or -NO ₂ , and preferably H, CH ₃ , Cl, Br, -COOH, -COCH ₃ or - NOT ₂ ; and R ₈ is a group H, Br or Cl, and preferably H or Br.

R ₂ is a group H, Cl or Br, and preferably where R ₂ is H or Br;

R ₄ is a H or CH ₃ group; and

R ₅ is a group H or -COOH.

R ₄ is a H or CH ₃ group; and

In another embodiment the invention relates to a compound of formula I for the use defined above where: R ₂ is a group H, Cl or Br, and preferably where R ₂ is H or Br; R ₄ is a H or CH ₃ group; and R ₈ is a group H, Br or Cl, and preferably H or Br.

R ₅ is a group H or -COOH; and

R ₆ is a group H, Cl, Br, CH ₃ or -COOH, and preferably H, Br, CH ₃ or -COOH; and

R ₆ is a group H, Cl, Br, CH ₃ or -COOH, and preferably H, Br, CH ₃ or -COOH; and R ₈ is a group H, Br or Cl, and preferably H or Br. In another embodiment the invention relates to a compound of formula I for the use defined above where: R ₂ is a group H, Cl or Br, and preferably where R ₂ is H or Br;

R ₃ is a group H, Br, Cl, methyl, ethyl, -COC ₂ H ₅ , -COCH ₃ ; -COOH, -COOCH ₃ , -OH, -NH ₂ , -SH or -NO ₂ ;

R ₄ is a H or CH ₃ group; and

R ₅ is a group H or -COOH.

R1 is a group H, Cl or Br, preferably where R1 is H or Br, and more preferably where R1 is H; R ₂ is a group H, Cl or Br, and preferably where R ₂ is H or Br;

R ₄ is a H or CH ₃ group; and

R ₅ is a group H or -COOH.

R ₄ is a H or CH ₃ group;

R ₅ is a group H or -COOH; and

Cy is a group of formula la.

R ₄ is a H or CH ₃ group; R ₅ is a group H or -COOH; and Cy is a group of formula la.

R ₄ is a H or CH ₃ group; and

R ₆ is a group H, Cl, Br, CH ₃ or -COOH, and preferably H, Br, CH ₃ or -COOH; and Cy is a group of formula la.

R ₄ is a H or CH ₃ group; and

R ₂ is a group H, Cl or Br, and preferably where R ₂ is H or Br;

R ₄ is a H or CH ₃ group; and

R ₂ is a group H, Cl or Br, and preferably where R ₂ is H or Br;

R ₄ is a H or CH ₃ group;

Cy is a group of formula la.

R ₂ is a group H, Cl or Br, and preferably where R ₂ is H or Br;

R ₄ is a H or CH ₃ group; and

R ₇ is a group H, CH ₃ , Cl, Br, -COOH, -COC ₂ H ₅ , -COCH ₃ or -NO ₂ , and preferably H, CH ₃ , Cl, Br, -COOH, -COCH ₃ or -NO ₂ ; and Cy is a group of formula la.

R ₂ is a group H, Cl or Br, and preferably where R ₂ is H or Br;

R ₄ is a H or CH ₃ group; and R ₈ is a group H, Br or Cl, and preferably H or Br; and

Cy is a group of formula la.

R ₁ is a group H, Cl or Br, preferably where R ₁ is H or Br, and more preferably where R ₁ is H; R ₂ is a group H, Cl or Br, and preferably where R ₂ is H or Br;

R ₄ is a H or CH ₃ group; R ₈ is a group H, Br or Cl, and preferably H or Br; and Cy is a group of formula la.

R ₄ is a H or CH ₃ group;

R ₇ is a group H, CH ₃ , Cl, Br, -COOH, -COC ₂ H ₅ , -COCH ₃ or -NO ₂ , and preferably H, CH ₃ , Cl, Br, -COOH, -COCH ₃ or - NOT ₂ ; R ₈ is a group H, Br or Cl, and preferably H or Br; and Cy is a group of formula la.

R ₄ is a H or CH ₃ group;

R ₅ is a group H or -COOH;

In another embodiment the invention relates to a compound of formula I for use defined above where:

R ₄ is a H or CH ₃ group;

R ₅ is a group H or -COOH;

R ₆ is a group H, Cl, Br, CH ₃ or -COOH, and preferably H, Br, CH ₃ or -COOH; R ₇ is a group H, CH ₃ , Cl, Br, -COOH, -COC ₂ H ₅ , -COCH ₃ or -NO ₂ , and preferably H, CH ₃ , Cl, Br, -COOH, -COCH ₃ O - NOT ₂ ; R ₈ is a group H, Br or Cl, and preferably H or Br; and

Cy is a group of formula la.

R ₄ is a H or CH ₃ group;

R ₅ is a group H or -COOH; and

R ₄ is a H or CH ₃ group;

R ₅ is a group H or -COOH; and

In another embodiment the invention relates to a compound of formula I for use defined above where: R ₂ is a group H, Cl or Br, and preferably where R ₂ is H or Br;

R ₄ is a H or CH ₃ group;

R ₅ is a group H or -COOH; and

R ₄ is a H or CH ₃ group;

R ₅ is a group H or -COOH; and

R ₄ is a H or CH ₃ group;

R ₅ is a group H or -COOH; and

R ₄ is a H or CH ₃ group;

R ₅ is a group H or -COOH; and

R ₄ is a H or CH ₃ group;

R ₅ is a group H or -COOH;

R ₇ is a group H, CH ₃ , Cl, Br, -COOH, -COC ₂ H ₅ , -COCH ₃ or -NO ₂ , and preferably H, CH ₃ , Cl, Br, -COOH, -COCH ₃ or - NOT ₂ .; and

Cy is a group of formula la.

R ₄ is a H or CH ₃ group;

R ₅ is a group H or -COOH; and

R ₇ is a group H, CH ₃ , Cl, Br, -COOH, -COC ₂ H ₅ , -COCH ₃ or -NO ₂ , and preferably H,

CH ₃ , Cl, Br, -COOH, -COCH ₃ or -NO ₂ ; and

Cy is a group of formula la.

R ₄ is a H or CH ₃ group;

R ₂ is a group H, Cl or Br, and preferably where R ₂ is H or Br;

R ₄ is a H or CH ₃ group;

R ₂ is a group H, Cl or Br, and preferably where R ₂ is H or Br;

R ₄ is a H or CH ₃ group;

R ₅ is a group H or -COOH; R ₈ is a group H, Br or Cl, and preferably H or Br; and

Cy is a group of formula la.

R ₂ is a group H, Cl or Br, and preferably where R ₂ is H or Br;

R ₄ is a H or CH ₃ group;

R ₅ is a group H or -COOH; and R ₈ is a group H, Br or Cl, and preferably H or Br; and Cy is a group of formula la.

R ₄ is a H or CH ₃ group;

R ₅ is a group H or -COOH;

R ₄ is a H or CH ₃ group;

R ₅ is a group H or -COOH;

R ₄ is a H or CH ₃ group;

R ₅ is a group H or -COOH;

R ₆ is a group H, Cl, Br, CH ₃ or -COOH, and preferably H, Br, CH ₃ or -COOH; R ₇ is a group H, CH ₃ , Cl, Br, -COOH, -COC ₂ H ₅ , -COCH ₃ or -NO ₂ , and preferably H, CH ₃ , Cl, Br, -COOH, -COCH ₃ or - NOT ₂ ; and

Cy is a group of formula la.

R ₂ is a group H, Cl or Br, and preferably where R ₂ is H or Br;

R ₄ is a H or CH ₃ group;

R ₅ is a group H or -COOH;

R ₆ is a group H, Cl, Br, CH ₃ or -COOH, and preferably H, Br, CH ₃ or -COOH;

Cy is a group of formula la.

R ₂ is a group H, Cl or Br, and preferably where R ₂ is H or Br;

R ₄ is a H or CH ₃ group;

R ₅ is a group H or -COOH;

R ₂ is a group H, Cl or Br, and preferably where R ₂ is H or Br;

R ₃ is a group H, Br, Cl, methyl, ethyl, -COC ₂ H ₅ , -COCH ₃ ; -COOH, -COOCH ₃ , -OH, -NH ₂ , -SH or -NO ₂ ; R ₄ is a H or CH ₃ group;

R ₅ is a group H or -COOH;

R ₄ is a H or CH ₃ group;

R ₅ is a group H or -COOH;

R ₆ is a group H, Cl, Br, CH ₃ or -COOH, and preferably H, Br, CH ₃ or -COOH; R ₈ is a group H, Br or Cl, and preferably H or Br; AND

Cy is a group of formula la.

R ₄ is a H or CH ₃ group;

R ₅ is a group H or -COOH;

R ₇ is a group H, CH ₃ , Cl, Br, -COOH, -COC ₂ H ₅ , -COCH ₃ or -NO ₂ , and preferably H, R ₈ is a group H, Br or Cl, and preferably H or br; and

Cy is a group of formula la.

R1 is a group H, Cl or Br, preferably where R1 is H or Br, and more preferably where R1 is H; R ₂ is a group H, Cl or Br, and preferably where R ₂ is H or Br; R ₃ is a group H, Br, Cl, methyl, ethyl, -COC ₂ H ₅ , -COCH ₃ ; -COOH, -COOCH ₃ , -OH, -NH ₂ , -SH or -NO ₂ ;

R ₄ is a H or CH ₃ group;

R ₅ is a group H or -COOH;

R ₇ is a group H, CH ₃ , Cl, Br, -COOH, -COC ₂ H ₅ , -COCH ₃ or -NO ₂ , and preferably H, R ₈ is a group H, Br or Cl, and preferably H or br; and Cy is a group of formula Ib.

R ₂ is a group H, Cl or Br, and preferably where R ₂ is H or Br;

R ₄ is a H or CH ₃ group;

R ₅ is a group H or -COOH;

R ₇ is a group H, CH ₃ , Cl, Br, -COOH, -COC ₂ H ₅ , -COCH ₃ or -NO ₂ , and preferably H, R ₈ is a group H, Br or Cl, and preferably H or br; and Cy is a group of formula le.

R ₂ is a group H, Cl or Br, and preferably where R ₂ is H or Br;

R ₄ is a H or CH ₃ group;

R ₅ is a group H or -COOH;

R ₇ is a group H, CH ₃ , Cl, Br, -COOH, -COC ₂ H ₅ , -COCH ₃ or -NO ₂ , and preferably H; R ₈ is a group H, Br or Cl, and preferably H or Br; and Cy is a group of formula Id.

In another embodiment the invention relates to a compound of formula I for the use defined above selected from:

A10

5A15

In another embodiment the invention relates to a compound of formula I for the use defined above selected from A1 to A30.

In another embodiment the invention relates to a compound of formula I for the use defined above selected from A1, A2, A4 to A6, A8 to A10, A12 to A17, A19 to A22, A26 to A28, A31 and A32.

In another embodiment the invention refers to 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 refers to a pharmaceutical composition comprising at least one compound of formula I defined above, for its use in the treatment and/or prevention of an infection caused by a bacterium of the genus Helicobacter or Campylobacter or for their use in the treatment and/or prevention of a disease caused by an infection caused by a bacterium of the genus Helicobacter or Campylobacter, and preferably where the bacterium of the genus Helicobacter is of the species Helicobacter pylori and where the bacterium of the genus Campylobacter is of the species Campylobacter jejuni.

In another embodiment, the invention refers 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 cancer, gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma (MALT lymphoma).

Another aspect of the invention refers to the use of a compound of formula I or a pharmaceutical composition thereof for the manufacture of a medicament for the treatment and/or prevention of a disease caused by infection by a bacterium of the genus Helicobacter or Campylobacter. , and preferably where the bacterium of the genus Helicobacter is of the species Helicobacter pylori and where the bacterium of the genus Campylobacter is of the species Campylobacter jejuni.

Another aspect of the invention refers to a method of treatment and/or prevention of a disease caused by infection by a bacterium of the genus Helicobacter or Campylobacter, and preferably where the bacterium of the genus Helicobacter is of the species Helicobacter pylori and where the bacterium of the genus Campylobacter is of the species Campylobacter jejuni, for a subject in need thereof, especially a human being, which comprises administering to said subject an effective amount of a compound of formula I as defined above or a pharmaceutical composition thereof .

In the above definitions, the term C1-4 alkyl, as a group or part of a group, means a straight or branched chain alkyl group 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 expression "optionally substituted by one or more" means the possibility of a group being 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 capable of being substituted. If present, such substituents may be the same or different and may 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 purposes of this invention, treatment includes, but is not limited to, alleviating, lessening, or eliminating one or more symptoms of the disease; reduce the degree of disease, stabilize (ie, not worsen) the disease state, retard or slow the progression of the disease, alleviate or improve the disease state, and remit (either totally or partially). Examples include, among others, "eradicate the infection", "total eradication of the microorganism" and "decreased colonization of the microorganism".

As used herein, the term "prevention" refers to preventing the onset of disease from occurring in a patient who is predisposed to or has risk factors, but 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 may contain one or more acidic protons and thus may 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. By pharmaceutically acceptable salts is meant those salts which, in the 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. The pharmaceutically acceptable salts are widely known by 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 the 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 from which they are caused to precipitate or crystallize. These complexes are known as solvates. As used herein, the term "solvate" refers to a complex of variable stoichiometry formed by 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, ie in amorphous form and crystalline forms. Also, the compounds of the present invention may have the ability to crystallize in more than one way, a feature known as polymorphism. Polymorphs can be distinguished 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 their polymorphic forms ("polymorphs"), are included within the scope of the present invention.

Some compounds of the present invention may exist as diastereoisomeric vapors 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 synthesis 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 they are 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 features of the invention will emerge in part from the description and in part from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

Fig. 1 shows the in vitro inhibition of the biological activity of the transcriptional regulator HsrA. (A) Under optimized in vitro conditions, recombinant HsrA protein specifically recognizes and binds to DNA sequences from its 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. . The optimal protein concentration for the inhibition assays of the compound of interest is that minimum concentration of HsrA that manages to completely complex the target promoter, in this case 6 μM. (B) The compound of interest inhibits the binding of the HsrA protein to its target promoter, preventing the formation of HsrA-DNA complexes. The inhibition capacity of the compound of interest on the in vitro activity of HsrA is directly proportional to the concentration of the inhibitor, managing to completely inhibit the activity of the protein with some of the inhibitor concentrations tested in the assay.

Fig. 2 Shows the interaction model between the HsrA protein of Helicobacter pylori and the experimentally tested ligand. (A) The ligand (compound A0) binds to the C-terminal domain of the protein and directly interacts with amino acid residues involved in DNA recognition and binding that make up a helix-turn-helix (HTH) motif. The DNA-binding helix-turn-helix (HTH) motif in the HsrA regulator it comprises amino acids 165 to 199 within the C-terminal domain and is formed by two a-helices separated by a small turn without secondary structure. This motif has been highlighted in dark color in figures 2A and 2B and further indicated with an arrow. (B) Surface model of the HsrA protein showing the ligand binding site, located in a pocket or small cavity in the C-terminal domain. The surface corresponding to the HTH motif has been highlighted in dark color and indicated with an arrow (C) The binding of the A0 ligand to the protein is stabilized by non-covalent interactions with the residues Leu126, lleul 35, Leu152, Lys194, Metí 95, Pro198, Leu199 and Thr203, which are indicated in the figure. The images have been obtained using the PyMoL program

Fig. 3 shows the in vitro inhibition of the essential biological activity of the transcriptional regulator CosR. The compound of interest totally inhibits the binding of the CosR protein to its target promoter at some of the concentrations tested, preventing the formation of CosR-DNA complexes.

EXAMPLES

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

General procedures

1. Cloning of the hsrA and cosR gene genes

Genomic DNA of Helicobacter pylori strain 26695 (ATCC 700392) was purified by the phenol-chloroform method. The biomass obtained from 100 mL of culture of Helicobacter pylori 26695 in brain heart infusion (BHI) broth supplemented with 4% fetal bovine serum (FBS) for 48 h under microaerobic conditions was resuspended in 400 μL of 10 mM Tris-buffer. CI (pH 8) and 1 mM EDTA. To this cell suspension, 1% SDS and 10 μL of proteinase K (10 mg/mL) were added and incubated for 1 h at 55°C. After this time, the sample was washed with 1 volume (same volume as the sample) of phenol, then with 1 volume of phenol-chloroform (1:1) and finally two washes with 1 volume of chloroform. Genomic DNA was precipitated overnight at -20°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 10 mM Tris-CI buffer (pH 8) and 1 mM EDTA. The complete sequence of the hsrA (hp1043) gene was amplified by PCR from the purified genomic DNA of Helicobacter pylori strain 26695 (ATCC 700392) using the 5'-GGAATTCCATATGCGCGTTCTACTGATTG-3' (SEQ ID NO: 1) and 5' primers. -CCCAAGCTTTTACTCTTCACACGCCGG-3' (SEQ ID NO: 2) and the high fidelity Pfu DNA polymerase enzyme (Agilent). The resulting PCR product was digested with the restriction enzymes Ndel and Hindi II 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.

Likewise, the Campylobacter CosR protein gene was cloned, following the same methodology as above. The complete sequence of the cosR gene was amplified by PCR from the purified genomic DNA of Campylobacter jejuni strain NCTC 11351, using the primers 5'-GGAATTTCCATATGAGAATTTAGTTATAGAAG-3' (SEQ ID NO: 3) and 5'-CGGGATCCTTAAGATTTTTAGGGAAGCAGAAACGG-3'. (SEQ ID NO: 4) and the high fidelity Pfu DNA polymerase enzyme. The resulting PCR product was digested with Ndel and BamHI restriction enzymes and cloned between the same restriction sites of the pET-28a expression vector. The final vector was partially sequenced to check the integrity of the gene.

2. Recombinant Expression and Purification of the HsrA and CosR Proteins

Competent Escherichia coli BL21 (DE3) cells were transformed with the vector pET-28a containing the hsrA gene from Helicobacter pylori ATCC 700392 or the cosR gene from Campylobacter jejuni NCTC 11351. This vector allows the recombinant protein to be expressed as a fusion protein at a peptide of 6 histidine residues at its N-terminal end. Transformed Escherichia coli cells were cultured in Luria-Bertani (LB) medium supplemented with 50 pg/mL of kanamycin under conditions of vigorous shaking and 37ºC until reaching an optical density of 0.8 measured at a wavelength of 600 nm. The expression of the recombinant HsrA protein was induced by adding 1 mM IPTG to the culture medium, after which the cells were allowed to grow under the same conditions of temperature and shaking for 6 hours. After this time, the culture was centrifuged at 8,000 rpm for 10 min at 4ºC and the cell biomass obtained was washed with cold PBS pH 7.4.

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

3. In Vitro Biological Activity of HsrA and CosR v Inhibition Assays

The HsrA and CosR proteins are response regulators with DNA-binding activity, acting as transcriptional regulators. The biological activity of both proteins is determined by the electrophoretic mobility shift assay (EMSA). In this assay, the transcriptional regulator is brought into contact 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 with respect to free DNA, given the larger molecular size of the complex. As the HsrA target promoter region, a 288 bp sequence corresponding to the promoter of the porGDAB operon was amplified by PCR from the genomic DNA of Helicobacter pylori ATCC 700392 using 5'-CCCCACACTTGCCCCATACAGAC-3' (SEQ ID NO: 5) and 5' as primers. '-

GCATGCCATCTAATTTGAAACATGG-3' (SEQ ID NO: 6). As a target promoter region of CosR, a 420 bp sequence corresponding to the promoter of the sodB gene was amplified by PCR from the genomic DNA of Campylobacter jejuni NCTC. 11351 using as primers 5'-CTGCGAAAGCACCTAGTAATGC-3' (SEQ ID NO: 7) and 5'-GTAACATAAGTATTGTGATGTTTTCCATG-3' (SEQ ID NO: 8). The promoters thus synthesized were purified using the commercial ¡Illustra™ GFX POR DNA and Gel Band Purification kit (GE Healthcare) and their concentration was quantified using a NanoVue Plus spectrophotometer (GE Healthcare). For biological activity assays of freshly purified recombinant HsrA protein, increasing concentrations of the protein, from 2 to 7 pM, were mixed with 120 ng of DNA in 10 mM bis-Tris buffer (pH 7.5), 40 mM KOI. , 100 pg/mL BSA, 1 mM DTT, and 5% (v/v) glycerol, in a final reaction mix volume of 20 µL. As non-specific 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. PCC 7120. For biological activity assays of freshly purified recombinant CosR protein, increasing concentrations of the protein from 20 to 700 nM were mixed with 120 ng of DNA in 10 mM bis-Tris buffer (pH 7.5), 40 mM KOI, 100 pg/mL BSA, 1 mM DTT, and 5% (v/v) glycerol, in a final reaction mix volume of 20 µL. As non-specific 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. PCC 7120. The protein (HsrA or CosR) and DNA mixtures were incubated at 25°C for 30 minutes and subsequently separated by non-denaturing 6% polyacrylamide gel electrophoresis using 25 mM Tris and 190 mM glycine buffer for the electrophoretic chamber. Polyacrylamide gels were stained with SYBR® Safe DNA Gel Stain (Invitrogen) and processed with Gel Doc 2000 Image Analyzer (Bio-Rad).

For biological activity inhibition assays, 6 pM of the HsrA protein or 600 nM of the CosR protein were mixed with 2; 1 ; 0.5; 0.1 and 0.05 mM of the compound A0 described in the present invention. Since the compound of interest was 100% dissolved in DMSO, the protein in the presence of the same amount of DMSO was included as a negative control, instead of the inhibitor. 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 for HsrA and sodB for CosR) and 120 ng of competitor DNA (pkn22) were added to each mixture. The rest of the procedure was carried out under the same conditions as the previously described activity assay. Each assay was performed in triplicate. 4. Antimicrobial activity against Helicobacter pylori of the HsrA inhibitor

The antimicrobial activity of compound A0 was tested against two different strains of Helicobacter pylori, the strain ATCC 700392 and the strain resistant to clarithromycin ATCC 700684. Both strains were cultured on blood agar (base) No. 2 (OXOID) supplemented with 8% of defibrinated horse blood (OXOID) in a humid microaerobic atmosphere (85% N ₂ , 10% CO ₂ , 5% O ₂ ) at 37ºC. The minimum inhibitory concentration (MIC) of each compound was determined by the microdilution method using flat-bottomed 96-well polystyrene plates. For the assays, the bacteria were subcultured in Brain Heart Infusion (BHI) broth supplemented with 4% fetal bovine serum (FBS) for 48 h under microaerobic conditions. After this time, the cultures were diluted with BHI broth + 4% SFB to an optical density of 0.01 measured at a wavelength of 600 nm. For the plate microdilution assay, 100 μL of the bacterial cultures thus adjusted 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 μL of the inhibitory compound. A0, at a concentration of 10.24 mg/mL in 100% DMSO. Serial double dilutions of the compound were made, evaluating the antibacterial activity of a range of concentrations between 256 and 0.125 mg/L. In all the plates positive and negative growth controls were incorporated with BHI broth + 4% SFB without the presence of inhibitors, inoculated and not inoculated with the bacteria. Controls with DMSO and clarithromycin were also incorporated. All plates were incubated under microaerobic conditions at 37°C and visually examined after 48 h of incubation. The minimal inhibitory concentration (MIC) 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 compound A0 was also determined. Once the MIC assay was completed, 10 μL aliquots of two dilutions before and two after the MIC value were extracted and plated on blood agar (base) No. 2 (OXOID) supplemented with 8% defibrinated horse blood (OXOID). in a humid microaerobic atmosphere (85% N ₂ , 10% CO ₂ , 5% O ₂ ). The agar plates were incubated at 37°C for 48h. The CMB value was defined as the lowest concentration of compound that prevented the growth of >99.9% of Helicobacter pylori cells subcultured on this inhibitor-free medium. Each experiment was performed in triplicate, twice, to confirm the results.

5. Antimicrobial activity against Campylobacter jejuni, Escherichia coli and Staphylococcus epidermidis of the HsrA inhibitor

The antibacterial activity of compound A0 was tested against Campylobacter jejuni ATCC 33560. Both Helicobacter pylori and Campylobacter jejuni are pathogenic species of the Epsilonproteobacteria family, both microorganisms have very similar nutritional requirements and grow optimally under the same in vitro culture conditions. In this way, for the determination of the MIC and the CMB against Campylobacter jejuni, the same protocol, media and culture conditions described above for the Helicobacter pylori strains were used.

The antimicrobial activity of compound A0 was also determined against strains of two representative species of normal human microbiota: Escherichia coli and Staphylococcus epidermidis. The calculation of the MIC and CMB of both strains was performed using the broth microdilution technique, following the recommendations of the European Committee for Antimicrobial Susceptibility Testing (EUCAST).

For the calculation of the MIC, the strains Escherichia coli ATCC 25922 and Staphylococcus epidermidis ATCC 12228 were grown on Mueller-Hilton agar medium overnight at 37ºC. Starting from this culture, several representative colonies of each strain were resuspended in sterile saline solution until the bacterial suspension adjusted to a turbidity similar to the 0.5 standard on the McFarland scale (~1.5 x 10 ⁸ CFU/mL). From this standardized suspension, the inoculum was prepared in Mueller-Hinton broth at 1 x10 ⁶ CFU/mL. Sterile 96-well flat bottom microtiter plates were used in the assay. 75 μL of Mueller-Hinton liquid medium was added to each plate in all wells except for the first column, where 120 μL was dispensed. In the corresponding wells of the first column, 30 μL of the different compounds were added, previously prepared in working solutions at 0.64 mg/mL in DMSO. Two controls were also included in each plate, a viability control in which the antimicrobial was replaced by the same amount of its solvent (30 μL of DMSO) and a sensitivity control in which kanamycin was incorporated, from a stock at 0 0.64mg/mL. Subsequently, serial doubling dilutions were made from column 1 to the end (eliminating the corresponding 75 μL from the last column) and then 75 μL of freshly prepared inoculum were added to each well of the plate, reaching in all cases a final concentration of 5x10 ⁵ CFU/mL. The assay made it possible to evaluate the antimicrobial activity of the compounds of interest in a concentration range of 64 pg/mL up to 0.5 pg/mL. The plates were incubated at 37ºC overnight and the MIC value was read visually. due to the absence of turbidity. Each determination was performed at least twice, in duplicate.

To determine the CMB, aliquots of 10 μL of each one of the wells corresponding to at least one or two dilutions on each side of the CMI value of each compound were seeded in Mueller-Hilton agar in duplicate. The plates were incubated at 37°C overnight. The CMB value was considered to be the lowest concentration of the compound that prevented the growth of 99.9% of the initial bacterial population after subculture in an antimicrobial-free medium.

6. Study of cytotoxicity in HeLa cells

The cytotoxic capacity of compound A0 on human cells was studied using the resazurin reduction assay in HeLa cells. For this assay, the compound PrestoBlue™ from the ThermoFisher commercial house was used. Cells were seeded in sterile 96-well plates, at a rate of 10,000 cells/well, using Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 1% GlutaMAX™ (ThermoFisher) and 1% penicillin antibiotic solution. -streptomycin (Sigma-Aldrich). The plates so seeded were incubated for 24 h at 37ºC under a saturated humidity atmosphere with 5% CO ₂ . After this time, the cells were exposed in triplicate to double serial dilutions of the compound of interest, in order to evaluate its cytotoxic capacity in a concentration range of 0.125 to 128 mg/L. The amount of total DMSO (vehicle) incorporated in all cases was 1.2% v/v, including DMSO cytotoxicity controls in all assays. After 24 h of exposure to each treatment, PrestoBlue 10% v/v was added and incubated for 2 hours at 37°C. The fluorescence emitted by resofurin in viable HeLa cells was recorded with a Synergy HT reader (Biotek).

The active ingredient in PrestoBlue is resazurin, a blue, non-fluorescent compound that is non-toxic to cells. Resazurin crosses the membrane of living cells and is irreversibly reduced inside living cells to resorufin, a pink-colored compound that fluoresces. The amount of resorufin produced in each well of the plate will be proportional to the number of viable cells and can be quantified by an absorbance (absorption wavelength: 570 nm) or fluorescence (excites at 530 nm and emits at 590 nm) reader. Once the fluorescence emission of the HeLa cells has been collected after exposure to each concentration of the compound of interest, a percentage curve of cell viability versus compound concentration is constructed, taking the fluorescence emitted by the wells as 100% viability. of untreated cells. The mean cytotoxic concentration value (CC ₅₀ ) is determined from this curve, that is, the concentration of the compound that causes the death of 50% of the cells in the culture.

7. Calculation of the therapeutic index (TI)

Taking into account the CC ₅ o of the compound of interest calculated in the cytotoxicity studies in HeLa cells and the minimum inhibitory concentration (MIC) obtained from previous studies of antimicrobial activity, the therapeutic index (TI) of the compound of interest can be calculated. interest against each microorganism, as a dimensionless numerical value resulting from the CC ₅₀ /MIC ratio. This value provides an idea about the efficacy and safety of a drug since it constitutes the ratio between the concentration at which it causes the therapeutic effect versus that which causes toxicity and adverse effects in the patient. The higher the TI value, the higher the safety of the drug. A TI value > 10 implies a high safety window for the compound or drug.

8. Acute toxicity in mice

Acute toxicity by oral ingestion was evaluated in mice following the Organization for Economic Co-operation and Development (OECD) class method, with slight modifications. The assay was developed in the Nanotoxicology and Immunotoxicology Unit (UNATI) of the I IS Aragón with the prior approval of the Ethics Advisory Commission for Animal Experimentation of the University of Zaragoza (Ref. PI58/20). SWISS female mice weighing 25-30 g, specific pathogen free (SPF), were supplied by Janvier Labs (France). Animals were marked to allow individual identification and kept in their cages for 7 days prior to dosing to allow acclimation to laboratory conditions. The test substance was administered in a single dose via intragastric tube. The concentration of each dose was adjusted so as not to administer more than 1 mL/100 g of animal weight. Two groups of 3 animals are they were fasted for 4 hours (water ad libitum), weighed individually and received a dose of compound A0 dissolved at the moment in corn oil. One group of animals received a dose of 50 mg/kg of body weight and another a dose of 300 mg/kg of body weight. After the treatments, the animals were observed individually for the first 30 min and then every 1 h for the first 4 h. Two daily observations were then made for the first 5 days, and daily thereafter, for a total of 14 days. Animals were weighed daily and individual records were systematically made for each animal regarding the appearance of skin, fur, mucous membranes, respiratory rate, activity pattern, and somatomotor behavior. At the end of the trial, all animals were humanely euthanized and subjected to gross necropsy. Samples of spleen, liver, kidneys, heart and brain were extracted from all animals, which were fixed in formalin for subsequent pathological analysis by staining with hematoxylin-eosin. The histopathological studies were carried out in the Pathological Anatomy Laboratory of the Lozano Blesa Clinical University Hospital by specialized personnel.

9. Analysis of the protein-ligand interaction by molecular docking (Docking)

The binding affinity to the target protein of different low molecular weight ligands with a similar chemical structure was estimated by molecular docking. The three-dimensional structure of the Helicobacter pylori HsrA protein (2HQR, model 1, A chain) was extracted from the Protein Data Bank database. The 3D structures of all ligands were built using the Corina Classic program and minimized using AMMOS (https://mobyle.rpbs.univ-paris-diderot.fr/). The protein and ligands were prepared using AutoDockTools 1.5.6 (https://autodock.schipps.edu/). The molecular structure of the protein was considered rigid, free twisting of the rotatable bonds in the ligand molecules was allowed. Molecular docking analysis was performed using the AutoDock Vina 1.2 program. For each of the ligands, the interaction free energies (AG) of the 20 most thermodynamically favored protein-ligand conformations were analyzed after blind docking. The conformation with the lowest binding energy between the ligand and the C-terminal domain (DNA-binding domain) of the target protein was considered as a model of interaction. The molecular interaction between both molecules was visualized using the PyMol 2.5.2 program (https://pymol.org). Results

1. Inhibition of the activity of the response regulators HsrA and CosR

2. Compound A0 inhibits 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 in the cell as a transcriptional regulator, it binds specifically to the promoter region of target genes and regulates the transcription of these genes. Previous studies have shown that HsrA constitutes an effective therapeutic target against Helicobacter pylori, since the inhibition of its biological activity leads to the death of the microorganism. Through gel retardation assays, it is shown that the compound of interest in the present invention inhibits the binding of HsrA to its target promoter by GDAB, as observed in Figure 1. Compound A0 also inhibits the biological activity of the essential CosR protein of Campylobacter jejuni. Through gel retardation assays, it is shown that the compound of interest in the present invention inhibits the binding of CosR to its target sodB promoter, as observed in Figure 3. Antimicrobial activity against Helicobacter pylori

Compound A0 shows potent bactericidal activity against Helicobacter pylori strains. The minimum inhibitory concentration range observed (MIC = 0.031 - 0.063 mg/L, Table 1 ) shows an antimicrobial activity 16 to 32 times higher than metronidazole (MIC = 1 mg/L) against the Helicobacter pylori strains used in the study.

3. Antimicrobial activity against Campylobacter jejuni

Compound A0 also demonstrates strong bactericidal activity against another epsilonproteobacteria of clinical interest, Campylobacter jejuni, with a MIC value for the ATCC 33560 control strain of 0.25 mg/L (Table 1). This antimicrobial activity against Campylobacter is similar to that evidenced by ciprofloxacin (MIC = 0.5 mg/L) and higher than that obtained with doxycycline (MIC = 1 mg/L), erythromycin (MIC = 2 mg/L) and gentamicin. (MIC = 1 mg/L), using the same study strain.

Table 1. Antimicrobial activity of compound A0 against strains of Helicobacter pylori, Campylobacter jejuni and representatives of normal human microbiota.

MIC: Minimum inhibitory concentration, CMB: Minimum bactericidal concentration, both expressed in mg/L.

4. Antimicrobial activity representatives of normal human microbiota

The antimicrobial activity of compound A0 against representative bacterial species of the normal human microbiota such as Escherichia coli and Staphylococcus epidermidis is very low, with MIC values > 64 mg/L (Table 1). This result suggests a selective antimicrobial activity of the compound of interest against epsilonproteobacteria and a low rate of adverse side effects on the normal microbiota, if administered to humans as a new antimicrobial agent.

5. Toxicity and therapeutic index

Compound A0 shows low toxicity both in vitro and in vivo and a high therapeutic index against Helicobacter pylori and C. jejuni (Table 2). Thus, the mean dose of the compound needed to cause cytotoxicity in a human (HeLa) cell line is >2000 times the mean dose needed to inhibit the in vitro growth of Helicobacter pylori and >500 times the required dose. to inhibit the in vitro growth of Campylobacter jejuni, which guarantees a wide therapeutic margin in the treatment of infections caused by both microorganisms.

Acute toxicity in mice after oral ingestion is low, with a LD ₅₀ value > 300 mg/kg (Table 2). This value implies lower toxicity than widely prescribed drugs such as aspirin (LD ₅₀ = 250 mg/kg), voltaren (LD ₅₀ = 95 mg/kg) or diazepam (LD ₅₀ = 48 mg/kg) and antibiotics such as isoniazid (LD ₅₀ = 133mg/kg). All animals exposed to doses of 50 mg/kg and 300 mg/kg of body weight survived. There were no macroscopic signs of toxicity such as weight loss, changes in the skin, fur, mucous membranes, respiratory rate, activity pattern. and somatomotor behavior during the 14 days of study. No histological changes specific to toxicity were observed in any of the organs studied (spleen, liver, kidneys, heart and brain). In two of the three mice exposed to the 300 mg/kg dose, mild inflammatory infiltrates with the presence of eosinophils were observed in lobular and sinusoidal areas of the liver. Although these nonspecific histological findings could indicate a hypersensitivity reaction to the compound of interest, they were not accompanied by other typical alterations (eosinophils in portal spaces, centrilobular cholestasis, apoptosis) that could confirm a definitive diagnosis of liver toxicity.

Table 2. Cytotoxicity in HeLa cells, median lethal dose in mice, and therapeutic indices of compound A0 against Helicobacter pylori and Campylobacter jejuni.

CC ₅₀ : Median cytotoxic concentration, LD50 : Median lethal dose.

The therapeutic index (TI) is the ratio between the CC ₅₀ and the MIC. An IT value > 10 implies a high safety profile of the compound or drug.

6. Analysis of protein-ligand interaction by molecular docking. Study of the binding affinity to the target protein of different low molecular weight ligands with similar chemical structure

A bioinformatics prediction of the best interaction model between several ligands of similar chemical structure and the HsrA target protein was performed. The ligand whose antimicrobial activity was experimentally demonstrated (A0) was initially used to define the most probable binding site with the protein by blind docking. By means of this procedure, all the possible conformations of protein-ligand interaction are analyzed and finally they are organized or classified according to the value of the free energy of interaction (AG), which is a measure of the affinity of the complex. The lower the free energy of interaction, the greater the binding affinity between both molecules. Given the functional independence of the two HsrA domains, only the ligands that interacted with the C-terminal DNA-binding domain were considered as possible inhibitors of the biological activity of the target protein, being defined as the interaction model the conformation with the lowest binding energy for each ligand. The results of the analysis are shown in Figure 2 and Table 3.

As can be seen in Figure 2C, at the protein binding site, within the C-terminal domain, the ligand establishes the following non-covalent interactions with the closest amino acids: (1) the phenyl group of the ligand establishes hydrophobic interactions with aliphatic side chain of residue Leu 126, (2) Cl forms a hydrogen bond with the OH group of the side chain of Thr203, (3) the thiophene ring establishes hydrophobic interactions with the aliphatic side chain of amino acids Lys194 and Leu152, (4) the NO ₂ group establishes electrostatic interactions with the NH ₃ ⁺ group of Lys 194, while (5) the carbonyl group of the ligand establishes a hydrogen bond with the OH group of the Thr203 side chain. The residues Lys194, Meti 95, Pro198, Leu199 and Thr203 are part of the HTH motif. According to this interaction model, the inhibition of the biological activity of HsrA as a transcriptional regulator after ligand binding is caused by spherical impediment and blocking of the conformational mobility of the HTH motif, which prevents recognition of specific DNA sequences and binding to HsrA. the promoter regions of their target genes. Ligand binding can also cause destabilization of the C-terminal domain structure.

Once the most probable interaction site of the experimentally evaluated ligand (A0) was defined, small rational modifications to the chemical structure of this ligand were designed in silico, mainly through changes or additions of new radicals, both polar and nonpolar, to its rings. phenyl and thiophene, and modifications in the position and chemical nature of the thiophene aromatic ring (Table 3). Next, the free energy of interaction between each of the designed chemical structures and the most probable binding site of the experimental ligand in the HsrA protein, previously defined and visualized in Figure 2, was analyzed by molecular docking.

As shown in Table 3, small modifications incorporated in silico to the chemical structure of the experimental ligand (A0) allowed obtaining a series of low molecular weight analogues, which maintained the same interaction model of the experimental ligand in the coupling assays. molecular. Notably, some of the chemical modifications made in the bioinformatics simulation increased the affinity of the ligand for the protein, which is reflected in a lower interaction energy (ΔG, kcal/mol). However, other modifications appeared to slightly decrease the binding affinity relative to the experimental ligand.

Table 3. Free energy of interaction between the Helicobacter pylori transcriptional regulator HsrA and different low molecular weight ligands that share a similar chemical structure, according to docking analysis.

The molecular docking and the calculation of the interaction energies were carried out using the AutoDock Vina software.

The groups Cy and R ₁ to R ₈ correspond to the groups of the formula I of the compounds of the invention.

Claims

CLAIMS 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 Campylobacter or for use in the treatment and/or prevention of a disease caused by infection by a bacterium of the genus Helicobacter or Campylobacter. genus Helicobacter or Campylobacter:

I where:

R ₁ is a group H or halogen;

R ₂ is a group H or halogen;

R ₃ _{is a group H, halogen, C 1-4} _alkyl , -COC1-4 alkyl; -COOH, -COOC _1-4 _alkyl , -OH, -NH ₂ , -SH or -NO ₂ , _where C _1-4 alkyl is optionally substituted by one or more R ₉ groups;

R ₄ is a H or C _1-4 alkyl group _;

_R ₅ is a group H, -COOH or -COOC _1-4 alkyl;

Cy is a group of formula la, Ib, le or Id:

R ₆ is _a group H, halogen _, C _1-4 alkyl, -COOH or -COOC _1-4 alkyl;

R ₇ is a group H, halogen, C _1-4 alkyl _, -COOH, -COC _1-4 _alkyl or -NO ₂ , where C _1-4 _alkyl is optionally substituted by one or more R ₁₀ groups;

R ₈ is H or halogen;

R ₉ is a halogen group; and

R ₁₀ is a halogen group. The compound of formula I for the use according to claim 1, wherein R ₁ is a group H, Cl or Br. The compound of formula I for the use according to any of claims 1 or

2, wherein R ₂ is a group H, Cl or Br. The compound of formula I for use according to any of claims 1 to

3, where R ₃ is a group H, Br, Cl, methyl, ethyl, -COC ₂ H ₅ , -COCH ₃ ; -COOH, -COOCH ₃ , -OH, -NH ₂ , -SH or -NO ₂ . The compound of formula I for use according to any of claims 1 to

4, where R ₄ is an H or CH ₃ group.

The compound of formula I for use according to any of claims 1 to

5, where R ₅ is a H or -COOH group.

The compound of formula I for use according to any of claims 1 to

6, where Cy is a group la.

8. The compound of formula I for use according to any of claims 1 to

7, where R ₆ is a H, Cl, Br, CH ₃ or -COOH group.

The compound of formula I for use according to any of claims 1 to

8, where R ₇ is a group H, CH ₃ , Cl, Br, -COOH, -COC ₂ H ₅ , -COCH ₃ or -NO ₂ .

The compound of formula I for use according to any of claims 1 to

9, where R ₈ is a group H, Br or Cl, and preferably H or Br.

eleven . The compound of formula I for use according to any of claims 1 to

10, where R ₉ is a Cl or Br group.

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

A1

5

13. The compound of formula I for the use according to claim 12, selected from A1 to A30.

The compound of formula I for use according to any one of claims 1 to 13, wherein the bacterium of the genus Helicobacter is of the species Helicobacter pylori and wherein the bacterium of the genus Campylobacter is of the species Campylobacter jejuni.

15. A pharmaceutical composition comprising at least one compound of formula I according to any of claims 1 to 14, for use in the treatment and/or prevention of an infection caused by a bacterium of the genus Helicobacter or Campylobacter or for use in the treatment and/or prevention of a disease caused by an infection caused by a bacterium of the genus Helicobacter or Campylobacter.

The compound of formula I or a pharmaceutical composition thereof for the use according to any of claims 1 to 14, wherein the disease caused by an infection is a gastrointestinal pathology. The compound of formula I or a pharmaceutical composition thereof for the use according to claim 16, wherein 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).