ES2754900A1 - 4-Phenyldihydropyridine derivatives for the treatment and/or prevention of an infection or disease caused by Helicobacter (Machine-translation by Google Translate, not legally binding) - Google Patents
4-Phenyldihydropyridine derivatives for the treatment and/or prevention of an infection or disease caused by Helicobacter (Machine-translation by Google Translate, not legally binding) Download PDFInfo
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- ES2754900A1 ES2754900A1 ES201831019A ES201831019A ES2754900A1 ES 2754900 A1 ES2754900 A1 ES 2754900A1 ES 201831019 A ES201831019 A ES 201831019A ES 201831019 A ES201831019 A ES 201831019A ES 2754900 A1 ES2754900 A1 ES 2754900A1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
- C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D211/80—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members
- C07D211/84—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen directly attached to ring carbon atoms
- C07D211/90—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/4422—1,4-Dihydropyridines, e.g. nifedipine, nicardipine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Description
D E S C R I P C I Ó ND E S C R I P C I Ó N
Derivados de 4-fenildihidropiridina para el tratamiento y/o prevención de una infección o enfermedad causada por Helicobacter 4-Phenyldihydropyridine derivatives for the treatment and / or prevention of an infection or disease caused by Helicobacter
La presente invención se refiere a derivados de 4-fenildihidropiridina de fórmula I o a composiciones farmacéuticas de los mismos para su uso en el tratamiento y/o prevención de una infección o una enfermedad causada por dicha infección, donde la infección está causada por bacterias del género Helicobacter, preferiblemente por la especie Helicobacter pylori. 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.
ANTECEDENTES DE LA INVENCIÓNBACKGROUND OF THE INVENTION
Helicobacter pylori está considerado como el patógeno de mayor prevalencia en humanos. Un estudio reciente sugiere que más de la mitad de la población mundial se encuentra infectada. La prevalencia de la infección varía usualmente según las condiciones socio-económicas, oscilando desde casi un 20 % en países de elevados ingresos como Suiza o Suecia, hasta casi el 90% de la población infectada en países como Nigeria. La prevalencia de la infección por Helicobacter pylori en España se estima en la actualidad en el 54,9% de la población (Hooi J.K.Y., et al. Global prevalence of Helicobacter pylori infection: systematic review and meta-analysis. Gastroenterology 2017; 153 (2): 420-429). 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 es una bacteria Gram-negativa microaerofílica de la clase Epsilonproteobacteria, a la cual pertenecen otros patógenos de relevancia clínica como Campylobacter jejuni. El mecanismo de transmisión de la infección por Helicobacter pylori no está perfectamente dilucidado. Es posible que la bacteria se transmita de persona a persona a través de la saliva o por vía fecal-oral, tras la ingesta de agua o alimentos contaminados. El microorganismo atraviesa la capa mucosa que protege al estómago con la ayuda de flagelos y se adhiere a las células epiteliales gástricas mediante adhesinas. La bacteria produce una variedad de enzimas como la ureasa, que le permiten neutralizar la acidez gástrica y crear un microambiente neutro a su alrededor. Helicobacter pylori sintetiza grandes cantidades de enzima ureasa que acumula en el citoplasma, espacio periplasmático y superficie celular. La enzima cataliza la hidrólisis de la urea presente en el estómago hasta amoníaco y dióxido de carbono. Los iones amonio son capaces de neutralizar la acidez estomacal, pero además generan daño en las células epiteliales estomacales produciendo necrosis y favoreciendo patologías gástricas diversas. Otras enzimas y citotoxinas producidas por Helicobacter pylori como VacA y CagA están igualmente asociadas al daño de la mucosa gástrica y al potencial oncogénico de este microorganismo (Kusters J.G., et al. Pathogenesis of Helicobacter pylori Infection. Clin Microbiol Rev 2006; 19 (3): 449 490). 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 like 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. carbon. 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 the oncogenic potential of this microorganism (Kusters JG, et al. Pathogenesis of Helicobacter pylori Infection. Clin Microbiol Rev 2006; 19 (3) : 449 490).
El tratamiento erradicador actual de la infección por Helicobacter pylori consiste en la combinación de al menos dos antibióticos, asociados a un inhibidor de la bomba de protones (IBP). La pauta más utilizada actualmente y la aceptada como de "primera línea” por el Colegio Americano de Gastroenterología y el último consenso de Maastricht consiste en la administración de Amoxicilina (1 g cada 12 horas) junto a Claritromicina (500 mg cada 12 horas) y un IBP a dosis estándar cada 12 horas, o sustituyendo Amoxicilina por Metronidazol (400 mg cada 12 horas). Este tratamiento se conoce como la "triple terapia” y requiere habitualmente de 10 a 14 días, alcanzando tasas de erradicación de un 70-85%. Sin embargo, en los últimos años esta terapia de primera línea ha disminuido significativamente sus tasas de erradicación, hasta niveles tan bajos como el 50% de los casos tratados. La causa fundamental de esta pérdida de eficacia radica en el creciente desarrollo de resistencia antimicrobiana por parte del microorganismo (Yang J.C., et al. Treatment of Helicobacter pylori infection: current status and future concepts. World J Gastroenterol 2014; 20 (18): 5283-5293; y Safavi M., et al. Treatment of Helicobacter pylori infection: current and future insights. World J Clin Cases 2016; 4 (1): 5-19).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 the cases treated. 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).
En Helicobacter pylori, la proteína HsrA constituye un regulador de respuesta que actúa como activador de la transcripción. HsrA regula la expresión de un gran número de genes y operones implicados en una variedad de procesos fisiológicos esenciales del microorganismo, sincronizando las funciones metabólicas y la virulencia con la disponibilidad de nutrientes, la división celular y la homeostasis redox. HsrA es una proteína esencial para la viabilidad de Helicobacter pylori, la deleción génica o la inhibición de la actividad biológica de la proteína implican la muerte celular. HsrA es una proteína de ~25 kDa, con dos dominios estructurales y funcionales definidos: un dominio N-terminal con función regulatoria y un dominio C-terminal de unión al DNA, ambos dominios están conectados por una corta secuencia de aminoácidos flexible sin estructura secundaria definida. La proteína dimeriza a través de su dominio N-terminal formando un dímero estable tanto in vitro como in vivo. El regulador reconoce y se une a los promotores de sus genes diana cubriendo una región de unos 30 pb cercana pero no solapante al elemento -10 del promotor. La unión del regulador a sus promotores diana pudiera favorecer el contacto de la RNA polimerasa con el DNA e influir positivamente en el reclutamiento de la enzima, ejerciendo un papel activador de la transcripción. La proteína HsrA ha sido clonada y caracterizada extensivamente en el laboratorio, se conoce su estructura cristalográfica y se cuenta con ensayos relativamente sencillos para evaluar su actividad biológica y la inhibición de esta actividad por potenciales inhibidores. HsrA es una proteína típica de las epsilonbacterias y no existe contraparte o proteína de secuencia similar en humanos, lo cual minimiza el riesgo de reactividad cruzada y efectos colaterales indeseables de potenciales inhibidores de HsrA sobre la microbiota normal o sobre biomoléculas humanas (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; Schar 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 I.N., 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; y Olekhnovich I.N., 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).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 defined secondary structure. 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 by covering a region of about 30 bp close but not overlapping to the -10 element 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; Schar 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 Olekhnovi ch 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).
Por tanto, sería deseable disponer de nuevos fármacos inhibitorios frente al regulador transcripcional HsrA de Helicobacter pylori. Therefore, it would be desirable to have new inhibitory drugs against the transcriptional regulator HsrA of Helicobacter pylori.
DESCRIPCIÓ N DE LA INVENCIÓNDESCRIPTION OF THE INVENTION
En un primer aspecto, la presente invención se refiere a un compuesto de fórmula I para su uso en el tratamiento y/o prevención de una infección causada por una bacteria del género Helicobacter o para su uso en el tratamiento y/o prevención de una enfermedad causada por una infección causada por una bacteria del género Helicobacter: 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 :
donde:where:
Ri es C1-4 alquilo, -NH2 o -CN, donde C1-4 alquilo está opcionalmente sustituido por un grupo -OCONH2, -OR5 o -SR5;Ri is C1-4alkyl, -NH2 or -CN, where C1-4alkyl is optionally substituted by a group -OCONH2, -OR5 or -SR5;
R2 es C1-4 alquilo opcionalmente sustituido por un grupo -OR5 o -SR5;R2 is C1-4 alkyl optionally substituted by a group -OR5 or -SR5;
R3 es C1-4 alquilo o Cy1, donde C1-4 alquilo está opcionalmente sustituido por un grupo R7;R3 is C1-4alkyl or Cy1, where C1-4alkyl is optionally substituted by a group R7;
A es un grupo de fórmula II, III, IV, V o VI:A is a group of formula II, III, IV, V or VI:
V VIV VI
R4 es -NO2, halógeno o -OCF3;R4 is -NO2, halogen, or -OCF3;
R5 es C1-4 alquilo opcionalmente sustituido por un grupo -NH2 o -OR6;R5 is C1-4alkyl optionally substituted by a group -NH2 or -OR6;
R6 es C1-4 alquilo opcionalmente sustituido por un grupo -NH2;R6 is C1-4alkyl optionally substituted by a group -NH2;
R7 es -OC1-4 alquilo, -NRsRg, Cy2, -COC1-4 alquilo o C2-4 alquenilo, donde C2-4 alquenilo está opcionalmente sustituido por un grupo fenilo; R7 is -OC1-4 alkyl, -NRsRg, Cy2, -COC1-4 alkyl or C2-4 alkenyl, where C2-4 alkenyl is optionally substituted by a phenyl group;
Cyi es un heterociclo saturado de 4 a 6 miembros unido al resto de la mólecula a través de un átomo de C o N disponible, que contiene 1 o 2 heteroátomos de N, donde Cy1 está opcionalmente sustituido por un grupo C1-4 alquilo, y donde el grupo C1-4 alquilo está opcionalmente sustituido por uno o dos grupos fenilo;Cyi is a 4-6 membered saturated heterocycle attached to the remainder of the molecule through an available C or N atom, containing 1 or 2 N heteroatoms, where Cy1 is optionally substituted by a C1-4alkyl group, and where the C1-4 alkyl group is optionally substituted by one or two phenyl groups;
Cy2 es un fenilo o un heterociclo saturado o aromático de 5 o 6 miembros que contiene de 1 a 3 heteroátomos seleccionados de N y O, donde Cy2 se une al resto de la molécula a través de un átomo de N o C, cuando Cy2 es un heterociclo saturado puede estar opcionalmente fusionado a un anillo de piperidina formando un anillo espiránico, y donde Cy2 está opcionalmente sustituido por uno o más grupos R10; R8 es C1-4 alquilo;Cy2 is a phenyl or saturated or aromatic 5- or 6-membered heterocycle containing 1 to 3 heteroatoms selected from N and O, where Cy2 binds to the rest of the molecule through an N or C atom, when Cy2 is a saturated heterocycle may be optionally fused to a piperidine ring forming a spiranic ring, and where Cy2 is optionally substituted by one or more R10 groups; R8 is C1-4 alkyl;
R9 es C1-4 alquilo opcionalmente sustituido por uno o más grupos fenilo, y donde cada grupo fenilo está opcionalmente sustituido por un grupo halógeno;R9 is C1-4alkyl optionally substituted by one or more phenyl groups, and where each phenyl group is optionally substituted by a halogen group;
cada R10 independientemente es vinilo, fenilo o Cy3, donde el grupo fenilo está sustituido por un grupo R11;each R10 independently is vinyl, phenyl or Cy3, where the phenyl group is substituted by an R11 group;
R11 es halógeno; yR11 is halogen; and
Cy3 es un heterociclo saturado de 5 o 6 miembros, que contiene 1 o 2 heterátomos de N, que se une al resto de la molécula a través de un átomo de N o C disponible y que está opcionalmente sustituido por un grupo C1-4 alquilo, donde el grupo C1-4 alquilo está opcionalmente sustituido por uno o dos grupos fenilo.Cy3 is a 5- or 6-membered saturated heterocycle, containing 1 or 2 N heteroatoms, that binds to the rest of the molecule through an available N or C atom and is optionally substituted by a C1-4alkyl group , where the C1-4 alkyl group is optionally substituted by one or two phenyl groups.
En otra realización la invención se refiere al compuesto de fórmula I según el uso definido anteriormente, donde la bacteria del género Helicobacter es de la especie Helicobacter pylori. 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 species Helicobacter pylori.
En otra realización la invención ser refiere al compuesto de fórmula I según el uso definido anteriormente, donde R1 es metilo, isopropilo, -CH2OR5, -CH2SR5, -CH2OCONH2, -NH2 o -CN.In another embodiment the invention refers to the compound of formula I according to the use defined above, where R1 is methyl, isopropyl, -CH2OR5, -CH2SR5, -CH2OCONH2, -NH2 or -CN.
En otra realización la invención ser refiere al compuesto de fórmula I según el uso definido anteriormente, donde R1 es metilo, isopropilo, -CH2OCH2CH2NH2, -CH2OCH2CH2OR6, -CH2SCH2CH2NH2, -CH2SCH2CH2O R6, -CH2OCONH2, -NH2 o -CN.In another embodiment the invention relates to the compound of formula I according to the use defined above, where R1 is methyl, isopropyl, -CH2OCH2CH2NH2, -CH2OCH2CH2OR6, -CH2SCH2CH2NH2, -CH2SCH2CH2O R6, -CH2OCONH2, -NH2 or -CN.
En otra realización la invención ser refiere al compuesto de fórmula I según el uso definido anteriormente, donde R1 es metilo, isopropilo, -CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2NH2, -CH2SCH2CH2NH2, -CH2S CH2CH2O CH2CH2NH2, -CH2OCONH2, -NH2 o -CN.In another embodiment the invention refers to the compound of formula I according to the use defined above, where R1 is methyl, isopropyl, -CH2OCH2CH2NH2, -CH2OCH2CH2OCH2CH2NH2, -CH2SCH2CH2NH2, -CH2S CH2CH2O CH2CH2NH2, -CH2OCONH2, -NH2 or -CN.
En otra realización la invención ser refiere al compuesto de fórmula I según el uso definido anteriormente, donde R1 es C1-4 alquilo, y preferiblemente donde R1 es metilo.In another embodiment the invention refers to the compound of formula I according to the use defined above, where R1 is C1-4alkyl, and preferably where R1 is methyl.
En otra realización la invención ser refiere al compuesto de fórmula I según el uso definido anteriormente, donde R2 es C1-4 alquilo.In another embodiment the invention refers to the compound of formula I according to the use defined above, where R2 is C1-4 alkyl.
En otra realización la invención ser refiere al compuesto de fórmula I según el uso definido anteriormente, donde R2 es metilo, etilo, propilo, isopropilo, butilo, isobutilo, sec-butilo o tert-butilo, preferiblemente donde R2 es metilo, etilo o isopropilo, y más preferiblemente donde R2 es metilo.In another embodiment the invention refers to the compound of formula I according to the use defined above, where R2 is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl or tert-butyl, preferably where R2 is methyl, ethyl or isopropyl , and more preferably where R2 is methyl.
En otra realización la invención ser refiere al compuesto de fórmula I según el uso definido anteriormente, donde R3 es C1-4 alquilo opcionalmente sustituido por un grupo R7.In another embodiment the invention refers to the compound of formula I according to the use defined above, where R3 is C1-4 alkyl optionally substituted by a group R7.
En otra realización la invención ser refiere al compuesto de fórmula I según el uso definido anteriormente, donde R4 es -NO2, -Cl o -OCF3, y preferiblemente -NO2 o -Cl.In another embodiment the invention refers to the compound of formula I according to the use defined above, where R4 is -NO2, -Cl or -OCF3, and preferably -NO2 or -Cl.
En otra realización la invención ser refiere al compuesto de fórmula I según el uso definido anteriormente, donde A es un grupo de fórmula II o III.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.
En otra realización la invención ser refiere al compuesto de fórmula I según el uso definido anteriormente, donde R7 es -OC1-4 alquilo, -NR8R9, Cy2 o -COC1-4 alquilo.In another embodiment the invention refers to the compound of formula I according to the use defined above, where R7 is -OC1-4 alkyl, -NR8R9, Cy2 or -COC1-4 alkyl.
En otra realización la invención ser refiere al compuesto de fórmula I según el uso definido anteriormente, donde R8 es metilo.In another embodiment the invention refers to the compound of formula I according to the use defined above, where R8 is methyl.
En otra realización la invención ser refiere al compuesto de fórmula I según el uso definido anteriormente, donde R9 es C1-4 alquilo sustituido por uno o dos grupos fenilo, y donde cada grupo fenilo está opcionalmente sustituido por un grupo halógeno.In another embodiment the invention relates to the compound of formula I according to the use defined above, where R9 is C1-4alkyl substituted by one or two phenyl groups, and where each phenyl group is optionally substituted by a halogen group.
En otra realización la invención ser refiere al compuesto de fórmula I según el uso definido anteriormente, donde Rg es -CH2Ph o -CH2CH2C(Ph)(4-F-Ph).In another embodiment the invention refers to the compound of formula I according to the use defined above, where Rg is -CH2Ph or -CH2CH2C (Ph) (4-F-Ph).
En otra realización la invención ser refiere al compuesto de fórmula I según el uso definido anteriormente, donde Cy1 es un heterociclo saturado de 4 a 6 miembros, preferiblemente de 4 o 5 miembros, unido al resto de la molécula a través de un átomo de C disponible, que contiene 1 heteroátomo de N, donde Cy1 está opcionalmente sustituido por un grupo C1-4 alquilo, y donde el grupo C1-4 alquilo está opcionalmente sustituido por uno o dos grupos fenilo.In another embodiment the invention refers to the compound of formula I according to the use defined above, where Cy1 is a saturated 4- to 6-membered heterocycle, preferably 4- or 5-membered, attached to the rest of the molecule via a C atom available, containing 1 heteroatom of N, where Cy1 is optionally substituted by a C1-4 alkyl group, and where the C1-4 alkyl group is optionally substituted by one or two phenyl groups.
En otra realización la invención ser refiere al compuesto de fórmula I según el uso definido anteriormente, donde Cy1 es un grupo de fórmula VII o VIII:In another embodiment the invention refers to the compound of formula I according to the use defined above, where Cy1 is a group of formula VII or VIII:
V il VIIIV il VIII
En otra realización la invención ser refiere al compuesto de fórmula I según el uso definido anteriormente, donde Cy2 es un fenilo opcionalmente sustituido por uno o más grupos R10.In another embodiment the invention relates to the compound of formula I according to the use defined above, where Cy2 is a phenyl optionally substituted by one or more R10 groups.
En otra realización la invención ser refiere al compuesto de fórmula I según el uso definido anteriormente, donde R11 es F o Cl, y preferiblemente Cl.In another embodiment the invention refers to the compound of formula I according to the use defined above, where R11 is F or Cl, and preferably Cl.
En otra realización la invención ser refiere al compuesto de fórmula I según el uso definido anteriormente, donde Cy3 es 1-vinilpiperazina o pirrolidina.In another embodiment the invention refers to the compound of formula I according to the use defined above, where Cy3 is 1-vinylpiperazine or pyrrolidine.
En otra realización la invención ser refiere al compuesto de fórmula I según el uso definido anteriormente, donde el compuesto de fórmula I se selecciona de: 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:
Lemildipino, Lercanidipino, Levamlodipino,Lemildipine, Lercanidipine, Levamlodipine,
Levniguldipino, Manidipino, Nicardipino,Levniguldipine, Manidipine, Nicardipine,
Nifedipino, Niguldipino, Niludipino,Nifedipine, Niguldipine, Niludipine,
Nilvadipino, Nimodipino, Nisoldipino,Nilvadipine, Nimodipine, Nisoldipine,
Nitrendipino, Olradipino, Palonidipino, Nitrendipine, Olradipine, Palonidipine,
En otra realización la invención ser refiere al compuesto de fórmula I según el uso definido anteriormente, donde el compuesto de fórmula I se selecciona de: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:
Nicardipino, Nisoldipino, Nimodipino,Nicardipine, Nisoldipine, Nimodipine,
Felodipino, Nitrendipino, Lercanidipino,Felodipine, Nitrendipine, Lercanidipine,
En otra realización la invención ser refiere al compuesto de fórmula I según el uso definido anteriormente, donde el compuesto de fórmula I se selecciona de: 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:
Nicardipino, Nisoldipino, Nimodipino, Nicardipine, Nisoldipine, Nimodipine,
Nitrendipino, Lercanidipino, y Nifedipino.Nitrendipine, Lercanidipine, and Nifedipine.
En otra realización la invención ser refiere al compuesto de fórmula I según el uso definido anteriormente, donde la enfermedad causada por una infección es una patología gastrointestinal, y preferiblemente donde la patología gastrointestinal se selecciona de gastritis aguda, gastritis crónica, duodenitis, dispepsia funcional, úlcera gástrica, úlcera duodenal, adenocarcinoma gástrico y linfoma de tejido linfoide asociado a mucosa (linfoma MALT).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).
La presente invención también se refiere a una composición farmacéutica que comprende al menos un compuesto de la invención (o una sal o solvato farmacéuticamente aceptable del mismo) y uno o más excipientes farmacéuticamente aceptables. Los excipientes deben ser “aceptables” en el sentido de ser compatibles con los demás ingredientes de la composición y de no ser perjudiciales para quién tome dicha composición.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 in the composition and not being harmful to whoever takes the composition.
Así, otro aspecto de la presente invención se refiere a una composición farmacéutica que comprende al menos un compuesto de fórmula I definido anteriormente, para su uso en el tratamiento y/o prevención de una infección causada por una bacteria del género Helicobacter o para su uso en el tratamiento y/o prevención de una enfermedad causada por una infección causada por una bacteria del género Helicobacter, y preferiblemente donde la bacteria del género Helicobacter es de la especie Helicobacter pylori. 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 its use in the treatment and / or prevention of a disease caused by an infection caused by a bacterium of the Helicobacter genus, and preferably where the bacterium of the Helicobacter genus is of the Helicobacter pylori species .
En otra realización la invención se refiere a la composición farmacéutica definida anteriormente, donde la enfermedad causada por una infección es una patología gastrointestinal, y preferiblemente donde la patología gastrointestinal se selecciona de gastritis aguda, gastritis crónica, duodenitis, dispepsia funcional, úlcera gástrica, úlcera duodenal, adenocarcinoma gástrico y linfoma de tejido linfoide asociado a mucosa (linfoma MALT).In another embodiment the invention relates to the defined pharmaceutical composition 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 lymphoid tissue lymphoma associated with mucosa (MALT lymphoma).
En las definiciones anteriores, el término C1-4 alquilo, como grupo o parte de un grupo, significa un grupo alquilo de cadena lineal o ramificada que contiene de 1 a 4 átomos de C e incluye los grupos metilo, etilo, propilo, isopropilo, butilo, isobutilo, sec-butilo y tert-butilo. In the above definitions, the term C1-4alkyl, as a group or part of a group, means a straight or branched chain alkyl group containing from 1 to 4 C atoms and includes methyl, ethyl, propyl, isopropyl groups, butyl, isobutyl, sec-butyl and tert-butyl.
Un radical halógeno o su abreviatura halo significa fluoro, cloro, bromo o yodo.A halogen radical or its abbreviation halo means fluoro, chloro, bromo or iodo.
La expresión "opcionalmente sustituido por uno o más" significa la posibilidad de un grupo de estar sustituido por uno o más, preferiblemente por 1, 2, 3 ó 4 sustituyentes, más preferiblemente por 1, 2 ó 3 sustituyentes y aún más preferiblemente por 1 ó 2 sustituyentes, siempre que dicho grupo disponga de suficientes posiciones disponibles susceptibles de ser sustituidas. Si están presentes, dichos sustituyentes pueden ser iguales o diferentes y pueden estar situados sobre cualquier posición disponible.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.
A lo largo de la presente descripción, el término “tratamiento” se refiere a eliminar, erradicar, erradicar totalmente, reducir o disminuir la causa o efectos de una enfermedad. Para los propósitos de esta invención, tratamiento incluye, aunque sin quedar limitados a los mismos, aliviar, disminuir o eliminar uno o más síntomas de la enfermedad; reducir del grado de enfermedad, estabilizar (es decir, no empeorar) el estado de la enfermedad, retrasar o ralentizar la progresión de la enfermedad, aliviar o mejorar el estado de la enfermedad y remitir (ya sea total o parcial). Ejemplos incluyen entre otros, “erradicar la infección”, “erradicación total del microorganismo” y “disminución de la colonización del microorganismo”.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."
Tal como se utiliza en la presente invención, el término “prevención” se refiere a prevenir la aparición de la enfermedad que se presente en un paciente que está predispuesto o tiene factores de riesgo, pero que todavía no presenta síntomas de la enfermedad. Prevención también incluye prevenir la reaparición de una enfermedad en un sujeto que previamente ha padecido dicha enfermedad. 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.
Los compuestos de la presente invención contienen uno o más nitrógenos básicos y podrían por tanto formar sales con ácidos, tanto orgánicos como inorgánicos. Algunos compuestos de la presente invención podrían contener uno o más protones ácidos y por tanto podrían formar también sales con bases.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.
No hay limitación en el tipo de sal que se puede utilizar, con la condición de que cuando se usen con fines terapéuticos sean farmacéuticamente aceptables. Se entiende por sales farmacéuticamente aceptables aquellas sales que, a criterio médico, son adecuadas para el uso en contacto con los tejidos de seres humanos u otros mamíferos sin provocar una toxicidad indebida, irritación, respuesta alérgica o similar. Las sales farmacéuticamente aceptables son ampliamente conocidas por cualquier experto en la materia.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.
Los compuestos de fórmula I y sus sales pueden diferir en ciertas propiedades físicas, pero son equivalentes a efectos de la invención. Todas las sales de los compuestos de fórmula I quedan incluidas dentro del ámbito de la invención.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.
Los compuestos de la presente invención pueden formar complejos con disolventes en los que se hacen reaccionar o desde los que se hacen precipitar o cristalizar. Estos complejos se conocen como solvatos. Tal como se utiliza aquí, el término solvato se refiere a un complejo de estequiometría variable formado por un soluto (un compuesto de fórmula I o una sal del mismo) y un disolvente. Ejemplos de disolventes incluyen los disolventes farmacéuticamente aceptables como agua, etanol y similares. Un complejo con agua se conoce como hidrato. Los solvatos de los compuestos de la invención (o sus sales), incluyendo hidratos, quedan incluidos dentro del ámbito de la invención. Los compuestos de fórmula I pueden existir en diferentes formas físicas, es decir en forma amorfa y formas cristalinas. Asimismo, los compuestos de la presente invención pueden tener la capacidad de cristalizar de más de una forma, una característica que se conoce como polimorfismo. Los polimorfos se pueden diferenciar por varias propiedades físicas bien conocidas por los entendidos en la materia como por ejemplo sus difractogramas de rayos X, puntos de fusión o solubilidad. Todas las formas físicas de los compuestos de fórmula I, incluyendo todas sus formas polimórficas (“polimorfos”), quedan incluidas dentro del ámbito de la presente invención. 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 of 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 feature known as polymorphism. Polymorphs can be differentiated by several 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.
Algunos compuestos de la presente invención podrían existir en forma de varios diastereoisómeros y/o varios isómeros ópticos. Los diastereoisómeros pueden separarse mediante técnicas convencionales como la cromatografía o la cristalización fraccionada. Los isómeros ópticos pueden ser resueltos mediante el uso de técnicas convencionales de resolución óptica, para dar los isómeros ópticamente puros. Esta resolución puede realizarse sobre los intermedios de síntesis que sean quirales o bien sobre los productos de fórmula I. Los isómeros ópticamente puros también pueden ser obtenidos individualmente empleando síntesis enantioespecíficas. La presente invención cubre tanto los isómeros individuales como sus mezclas (por ejemplo mezclas racémicas o mezclas de diastereoisómeros), tanto si se obtienen por síntesis como mezclándolos físicamente.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.
Los compuestos de la presente invención pueden ser administrados en forma de cualquier formulación farmacéutica, la naturaleza de la cual, como es bien sabido, dependerá de la naturaleza del principio activo y de su vía de administración. En principio se puede utilizar cualquier vía de administración, por ejemplo oral, parenteral, nasal, ocular, rectal, y tópica.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.
A lo largo de la descripción y las reivindicaciones la palabra "comprende" y sus variantes no pretenden excluir otras características técnicas, aditivos, componentes o pasos. Para los expertos en la materia, otros objetos, ventajas y características de la invención se desprenderán en parte de la descripción y en parte de la práctica de la invención. Las siguientes figuras y ejemplos se proporcionan a modo de ilustración, y no se pretende que sean limitativos de la presente invención.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 figures and examples are provided by way of illustration, and are not intended to be limiting of the present invention.
DESCRIPCIÓ N DE LA S FIGURASDESCRIPTION OF THE FIGURES
Fig. 1. Inhibición de la actividad de unión al ADN de HsrA por compuestos de fórmula I. (A) Bajo condiciones in vitro apropiadas, según se ha descrito en el procedimiento, la proteína HsrA recombinante reconoce y se une específicamente a las secuencias de ADN de sus promotores diana. La unión de la proteína al ADN diana determina la formación de un complejo ADN-proteína estable, con un tamaño molecular superior al del ADN libre, experimentando un retardo en la movilidad electroforética. La unión específica del HsrA a su promotor diana PporGDAB y la formación de un complejo HsrA-ADN detectable por la técnica de EMSA es directamente proporcional a la concentración de la proteína recombinante y no logra ser inhibido por la presencia de un fragmento de ADN competidor inespecífico. El ADN diana libre va desapareciendo en presencia de concentraciones crecientes de la proteína recombinante para formar un complejo estable ADN-proteína de mayor peso molecular. Sin embargo, la concentración de ADN competidor se mantiene inalterable, dado que la proteína no reconoce ni se une a esta secuencia inespecífica. (B) Compuestos de fórmula I inhiben la unión de la proteína HsrA recombinante a sus promotores diana, evitando la formación de complejos HsrA-ADN. La capacidad de inhibición de los compuestos de fórmula I sobre la actividad de HsrA es directamente proporcional a la concentración del inhibidor. Según se aprecia en la figura 1B, compuestos de fórmula I logran inhibir totalmente la actividad de la proteína HsrA recombinante.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 the sequences of DNA 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 an 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 inhibitory 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.
EJEM PLO SEXAMPLE PLO S
A continuación se ilustrará la invención mediante unos ensayos realizados por los inventores, que pone de manifiesto la efectividad del producto de la invención.The invention will now be illustrated by tests carried out by the inventors, which shows the effectiveness of the product of the invention.
ProcedimientoProcess
Clonaje del gen del regulador de respuesta HsrACloning of the HsrA response regulator gene
El ADN genómico de Helicobacter pylori cepa 26695 (ATCC 700392) se purificó mediante el método de fenol-cloroformo. La biomasa obtenida a partir de 100 mL de cultivo de Helicobacter pylori 26695 en caldo infusión cerebro corazón (BHI) suplementado con 4% de suero fetal bovino (SFB) durante 48 h en condiciones microaeróbicas fue resuspendida en 400 pL de tampón 10 mM Tris-Cl (pH 8) y 1 mM EDTA. A esta suspensión celular se adicionó 1% SDS y 10 pL de proteinasa K (10 mg/mL) y se incubó durante 1 h a 55°C. Al cabo de este tiempo, la muestra se lavó con 1 volumen (mismo volumen de la muestra) de fenol, luego con 1 volumen de fenolcloroformo (1:1) y finalmente se realizaron dos lavados con 1 volumen de cloroformo. El DNA genómico se precipitó toda la noche a -20°C con 2 volúmenes de etanol absoluto frio y 0,1 volumen de 3M acetato de sodio (pH 5,2), el precipitado se lavó con etanol 70% y finalmente se resuspendió en tampón 10 mM Tris-Cl (pH 8) y 1 mM EDTA. Helicobacter pylori strain 26695 genomic DNA (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 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. Cl (pH 8) and 1 mM EDTA. To this cell suspension, 1% SDS and 10 pL proteinase K (10 mg / mL) were added and incubated for 1 h at 55 ° C. At the end of this time, the sample was washed with 1 volume (same volume of the sample) of phenol, then with 1 volume of phenolchloroform (1: 1) and finally, two washes with 1 volume of chloroform were carried out. 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 10mM Tris-Cl buffer (pH 8) and 1mM EDTA.
La secuencia completa del gen hsrA (hp1043) se amplificó por PCR a partir del ADN genómico purificado de la cepa 26695 (ATCC 700392) de Helicobacter pylori empleando los cebadores 5'-GGAATTCCATATGCGCGTTCTACTGATTG-3' (SEQ ID NO: 1) y 5'-CCCAAGCTTTTACTCTTCACACGCCGG-3' (SEQ ID NO: 2) y la enzima Pfu DNA polimerasa de alta fidelidad (Agilent). El producto de PCR resultante fue digerido con las enzimas de restricción Ndel y HindIII y clonado entre los mismos sitios de restricción del vector de expresión pET-28a (Novagen). El vector final fue parcialmente secuenciado para comprobar la integridad del gen.The entire sequence of the hsrA gene ( hp1043 ) was amplified by PCR from genomic DNA purified from Helicobacter pylori strain 26695 (ATCC 700392) using primers 5'-GGAATTCCATATGCGCGTTCTACTGATTG-3 '(SEQ ID NO: 1) and 5' -CCCAAGCTTTTACTCTTCACACGCCGG-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 HindIII 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.
Expresión y purificación de HsrA recombinanteRecombinant HsrA expression and purification
Se transformaron células competentes de E. coli BL21 (DE3) con el vector pET-28a conteniendo el gen hsrA de Helicobacter pylori ATCC 700392. Este vector permite expresar la proteína recombinante como una proteína de fusión a un péptido de 6 residuos de histidinas en su extremo N-terminal. Las células de E. coli transformadas fueron cultivadas en medio Luria-Bertani (LB) suplementado con 50 pg/mL de kanamicina bajo condiciones de agitación vigorosa y 37°C hasta alcanzar una densidad óptica de 0,8 medida a una longitud de onda de 600 nm. La expresión de la proteína HsrA recombinante se indujo mediante la adición al medio de cultivo de 1 mM de IPTG, tras lo cual las células se dejaron crecer bajo las mismas condiciones de temperatura y agitación durante 6 horas. Transcurrido este tiempo, el cultivo se centrifugó a 8,000 rpm durante 10 min a 4°C y la biomasa celular obtenida fue lavada con PBS pH 7,4 frio.Competent E. coli cells BL21 (DE3) were transformed with the vector pET-28A containing the gene HSRA Helicobacter pylori ATCC 700392. This vector allows express recombinant protein as a fusion protein to a peptide of six histidines residues 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 vigorous shaking conditions and 37 ° 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. At the end of this time, the culture was centrifuged at 8,000 rpm for 10 min at 4 ° C and the obtained cell biomass was washed with cold PBS pH 7.4.
La proteína HsrA recombinante es expresada en forma soluble en el citoplasma de E. coli BL21 (DE3). Para la purificación proteica, las células fueron resuspendidas en tampón 50 mM Tris-Cl (pH 8), con 500 mM NaCl, 10 mM imidazol y 1 mM PMSF. La ruptura celular se realizó mediante 10 ciclos ultrasónicos de 45 segundos en baño de hielo, con descansos de 30 segundos entre cada ciclo. Los detritos celulares se eliminaron mediante centrifugación a 18,000 rpm durante 20 min a 4°C. La proteína recombinante con cola de histidinas contenida en el sobrenadante fue purificada mediante cromatografía de afinidad a metales inmovilizados (IMAC) empleando la matriz Chelating Sepharose Fast Flow (GE Healthcare) cargada con iones Ni2+, previamente equilibrada con el tampón empleado en la lisis celular. La proteína recombinante unida a la matriz fue eluida empleando un gradiente de imidazol (0,01 a 1 M) y dializada frente al tampón 50 mM Tris-HCl (pH 8), 300 mM NaCl y 10% glicerol. Recombinant HsrA protein is expressed in soluble form in the E. coli BL21 cytoplasm (DE3). For protein purification, cells were resuspended in 50mM Tris-Cl buffer (pH 8), with 500mM NaCl, 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 Healthcare) matrix loaded with Ni2 + ions, previously equilibrated 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 Tris-HCl buffer (pH 8), 300mM NaCl and 10% glycerol.
La concentración de proteína fue determinada mediante el kit comercial BCA™ Protein Assay (Thermo Fisher Scientific). La cola de poli-histidinas fue eliminada del extremo N-terminal de HsrA recombinante mediante tratamiento con trombina (GE Healthcare), empleando 10 U de enzima por cada miligramo de proteína recombinante. Tras la restricción enzimática, la proteína HsrA recombinante fue obtenida en el volumen muerto de un segundo paso de purificación mediante IMAC-Ni2+. La proteína así purificada fue conservada a -20°C.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 thrombin treatment (GE Healthcare), 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-Ni2 +. The thus purified protein was stored at -20 ° C.
Actividad biológica y ensayos de inhibiciónBiological activity and inhibition assays
La proteína HsrA es un regulador de respuesta con actividad de unión al ADN, que actúa como regulador transcripcional. La actividad biológica de esta proteína se determina mediante el ensayo del cambio de movilidad electroforética (EMSA). En este ensayo, el regulador transcripcional se pone en contacto con un fragmento de ADN correspondiente a la región promotora de sus genes diana. Si la proteína es activa, se unirá de forma específica a sus promotores diana y formará un complejo proteína-ADN que experimentará un retardo en la movilidad electroforética respecto al DNA libre, dado el mayor tamaño molecular del complejo. Como región promotora diana de HsrA se amplificó por PCR una secuencia de 288 pb correspondiente al promotor del operón porGDAB a partir del ADN genómico de Helicobacter pylori ATCC 700392 empleando como cebadores 5'- CCCCACACTTGCCCCATACAGAC-3' (SEQ ID NO: 3) y 5'-GCATGCCATCTAATTTGAAACATGG-3' (SEQ ID NO: 4). El promotor sintetizado fue purificado mediante el kit comercial illustra™ GFX PCR DNA and Gel Band Purification kit (GE Healthcare) y su concentración fue cuantificada mediante espectrofotómetro NanoVue Plus (GE Healthcare). Para los ensayos de actividad biológica de la proteína HsrA recombinante recién purificada, se mezclaron concentraciones crecientes de la proteína, desde 2 hasta 7 pM, con 120 ng de ADN en tampón 10 mM bis-Tris (pH 7,5), 40 mM KCl, 100 pg/mL BSA, 1 mM DTT y 5% (v/v) de glicerol, en un volumen final de mezcla de reacción de 20 pL. Como ADN competidor inespecífico se adicionó a todas las mezclas 120 ng de un fragmento de 121 pb correspondiente a una porción de la secuencia codificadora del gen pkn22 (alr2502) de la cianobacteria Anabaena sp. PCC 7120. La mezcla de proteína y ADN se incuba a 25°C durante 30 minutos y posteriormente se separa mediante electroforesis no desnaturalizante en gel de poliacrilamida 6% empleando el tampón 25 mM Tris y 190 mM glicina para la cámara electroforética. Los geles de poliacrilamida fueron teñidos con el colorante SYBR® Safe DNA gel stain (Invitrogen) y procesados con Gel Doc 2000 Image Analyzer (Bio-Rad).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 promoter region target HSRA PCR amplified sequence of 288 bp corresponding to the promoter of the operon porGDAB from genomic DNA of Helicobacter pylori ATCC 700392 using as primers 5'CCCCACACTTGCCCCATACAGAC-3 '(SEQ ID NO: 3) and 5 '-GCATGCCATCTAATTTGAAACATGG-3' (SEQ ID NO: 4). The synthesized promoter was purified using the commercial illustra ™ 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 pM, were mixed with 120 ng of DNA in 10mM bis-Tris buffer (pH 7.5), 40mM KCl , 100 pg / mL BSA, 1 mM DTT and 5% (v / v) glycerol, in a final volume of reaction mixture of 20 pL. 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 protein and DNA mixture is incubated at 25 ° C for 30 minutes and subsequently separated by non-denaturing 6% polyacrylamide gel electrophoresis using the 25mM Tris buffer and 190mM glycine 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).
Para los ensayos de inhibición de la actividad biológica, 6 ^M de la proteína HsrA recombinante se mezclaron con 2; 1; 0,5 y 0,1 mM del compuesto de fórmula I descrito en la presente invención. Dado que los compuestos analizados se encontraban disueltos al 100% en DMSO, se incluyó como control negativo de inhibición la proteína en presencia de la misma cantidad de DMSO, en sustitución de los compuestos. La mezcla de proteína e inhibidor en tampón 10 mM bis-Tris (pH 7,5), 40 mM KCl, 100 ^g/mL BSA, 1 mM DTT y 5% (v/v) de glicerol se incubó durante 10 min a 25°C. Transcurrido este tiempo se adicionó a cada mezcla 120 ng de promotor diana (porGDAB) y 120 ng de ADN competidor (pkn22). El resto del procedimiento se llevó a cabo en las mismas condiciones del ensayo de actividad anteriormente descrito. Cada ensayo se realizó por triplicado y se analizó la actividad inhibitoria de al menos 6 compuestos diferentes con fórmula I descrita en la presente invención.For assays of inhibition of biological activity, 6 ^ M 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 10mM bis-Tris buffer (pH 7.5), 40mM KCl, 100 ^ g / mL BSA, 1mM 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.
Actividad antibacteriana frente a Helicobacter pylori de inhibidores de HsrA La actividad antibacteriana de 6 compuestos inhibitorios de fórmula I de la presente invención fue testada frente a dos cepas diferentes de Helicobacter pylori, la cepa ATCC 700392 y la cepa resistente a claritromicina ATCC 700684. Ambas cepas fueron cultivadas en agar sangre (base) N° 2 (OXOID) suplementado con 8% de sangre de caballo desfribinada (OXOID) en atmósfera microaeróbica húmeda (85% N2, 10% CO2, 5% O2) a 37°C. Se determinó la concentración mínima inhibitoria (MIC) de cada compuesto mediante el método de microdilución empleando placas de poliestireno de 96 pocillos con fondo plano. Para los ensayos, las bacterias fueron subcultivadas en caldo infusión cerebro corazón (BHI) suplementado con 4% de suero fetal bovino (SFB) durante 48 h en condiciones microaeróbicas. Al cabo de este tiempo, los cultivos fueron diluidos con caldo BHI 4% SFB hasta una densidad óptica de 0,01 medida a una longitud de onda de 600 nm. Para el ensayo de microdilución en placas, 100 ^L de los cultivos bacterianos así ajustados fueron dispensados en cada uno de los pocillos de la placa, excepto la primera columna de la placa, que recibió 200 ^L de suspensión bacteriana con 5 ^L del compuesto inhibitorio de fórmula I, a una concentración de 10,24 mg/mL en 100% DMSO. Cada uno de los compuestos de fórmula I ensayados fue adicionado al primer pocillo de cada fila de la placa. Se realizaron diluciones dobles seriadas de cada compuesto, evaluando la actividad antibacteriana de un rango de concentraciones entre 256 y 0,125 ^g/mL de cada uno de los compuestos. En todas las placas se incorporaron controles de crecimiento positivo y negativo con caldo BHI 4% SFB sin la presencia de inhibidores, inoculado y no inoculado con las bacterias. Se incorporaron además controles con DMSO y claritromicina. Todas las placas fueron incubadas bajo condiciones microaeróbicas a 37°C y examinadas visualmente tras 48 h de incubación. El valor MIC de cada compuesto se definió como la mínima concentración del compuesto que inhibió completamente el crecimiento visible de la bacteria tras 48 h.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 ATCC 700392 strain and the clarithromycin resistant strain ATCC 700684. Both strains were grown on blood agar (base) No. 2 (OXOID) supplemented with 8% of defibrillated horse blood (OXOID) in a humid microaerobic atmosphere (85% N2, 10% CO2, 5% O2) 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 4% SFB BHI broth to an optical density of 0.01 measured at a wavelength of 600 nm. For the microdilution assay in plates, 100 ^ L 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 ^ L of bacterial suspension with 5 ^ L 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 ^ g / mL of each of the compounds. Positive and negative growth controls were incorporated in all the plates with 4% SFB BHI broth 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 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.
Se determinó además la concentración mínima bactericida (MBC) de cada uno de los 6 compuestos de fórmula I de la presente invención. Una vez culminado en el ensayo de MIC, se extrajeron alícuotas de 10 pL de dos diluciones anteriores y dos posteriores al valor MIC y se sembraron en agar sangre (base) N° 2 (OXOID) suplementado con 8% de sangre de caballo desfribinada (OXOID) en atmósfera microaeróbica húmeda (85% N2, 10% CO2, 5% O2). Las placas de agar se incubaron a 37°C durante 48 h. El valor MBC de cada compuesto se definió como la mínima concentración del compuesto que impidió el crecimiento de >99,9% de las células de Helicobacter pylori subcultivadas sobre este medio libre de inhibidores. Cada experimento se realizó por triplicado, dos veces, para confirmar los resultados.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 removed and plated on No. 2 (OXOID) blood (base) agar supplemented with 8% defibrillated horse blood ( OXOID) in a humid microaerobic atmosphere (85% N2, 10% CO2, 5% O2). 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> 99.9% of the Helicobacter pylori cells subcultured on this inhibitor-free medium. Each experiment was performed in triplicate, twice, to confirm the results.
Energética de unión de los inhibidores a HsrAEnergetic binding of inhibitors to HsrA
La unión específica de 6 compuestos inhibitorios de fórmula I de la presente invención al regulador de respuesta esencial HsrA de Helicobacter pylori fue confirmada mediante calorimetría de titulación isoterma (ITC). Esta técnica no sólo mide la afinidad de cada compuesto por la proteína diana, sino que determina la contribución de las componentes entálpica y entrópica, así como la estequiometría de la unión.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.
Los experimentos de unión se llevaron a cabo en el microcalorímetro de alta sensibilidad MicroCal Auto-iTC200 (Malvern Instruments). Una solución de 20 mM de HsrA en tampón 50 mM Tris-Cl (pH 8), 150 mM NaCl, 10% glicerol y 1% DMSO ubicada en la celda de medida, fue titulada con una solución de 200 pM del correspondiente compuesto inhibitorio disuelto en el mismo tampón anterior, ubicado en la jeringa de inyección.The binding experiments were carried out on the MicroCal Auto-iTC200 high sensitivity microcalorimeter (Malvern Instruments). A solution of 20 mM HsrA in 50 mM Tris-Cl buffer (pH 8), 150 mM NaCl, 10% glycerol and 1% DMSO located in the measurement cell, was titrated with a 200 pM solution of the corresponding dissolved inhibitory compound in the same buffer as above, located in the injection syringe.
Resultados Results
a) Inhibición de la actividad de HsrAa) Inhibition of HsrA activity
Los compuestos de fórmula I inhiben la función biológica de la proteína esencia1HsrA de Helicobacterpylori. La proteína HsrA es un regulador de respuesta con actividad de unión al ADN. Esta proteína actúa en la célula como regulador transcripcional, se une específicamente a la región promotora de genes diana y regula la transcripción de estos genes. Se demuestra que los compuestos de fórmula I inhiben la unión de HsrA a sus promotores diana, según se demuestra en la figura 1.The compounds of formula I inhibit the biological function of the Helicobacterpylori essence1HsrA protein . 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) Actividad antimicrobiana de inhibidores de HsrA sobre Helicobacter pylori b) Antimicrobial activity of HsrA inhibitors on Helicobacter pylori
El empleo de HsrA como diana terapéutica puede resultar efectivo para el tratamiento de enfermedades infecciosas causadas por Helicobacter pylori y patologías asociadas, dado que inhibidores de la actividad biológica de esta proteína demuestran una elevada actividad bactericida sobre cepas de este patógeno. La proteína HsrA es esencial para la viabilidad del microorganismo, por lo que la inhibición de su actividad biológica conlleva a la muerte del patógeno y potencialmente a la erradicación de la infección. En la tabla 1 se describen los valores de concentración mínima bactericida (MBC) de varios compuestos de fórmula I frente a dos cepas de 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, since 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.
Tabla 1. Actividad bactericida de compuestos de fórmula I sobre Helicobacter pylori Table 1. Bactericidal activity of compounds of formula I on Helicobacter pylori
c) Afinidad y estequiometría de unión entre compuestos de fórmula I y HsrA La unión específica de los compuestos de fórmula I a la proteína HsrA fue confirmada mediante Calorimetría de Titulación Isotérmica (ITC). Como se aprecia en la tabla 2, todos los compuestos ensayados se unen a la proteína con una afinidad en el orden micromolar. En todos los casos, la estequiometría de unión fue de 1:1; es decir, una molécula de inhibidor por cada molécula de proteína monomérica.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 order micromolar. In all cases, the binding stoichiometry was 1: 1; that is, one inhibitor molecule for each monomeric protein molecule.
Tabla 2. Afinidad de unión HsrA/inhibidoresTable 2. HsrA / Inhibitor Binding Affinity
Claims (15)
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Non-Patent Citations (3)
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| K MAZUMDAR ET AL. Potential role of the cardiovascular non-antibiotic amlodipine in the treatment of microbial infections.. International Journal of Antimicrobial Agents, 2010, Vol. 36, Páginas 295-302 resumen * |
| KA KUMAR ET AL. Amlodipine: a cardiovascular drug with powerful antimicrobial property.. Acta Microbio. Polonia, 2003, Vol. 52, Páginas 285-292 [en línea][recuperado el 06/05/2019]. Recuperado de Internet (URL:https://www.ncbi.nlm.nih.gov/pubmed/14743981), resumen * |
| P B YAZBEK ET AL. Challenges to the treatment and new perspectives for the eradication of Helicobacter pylori. Digestive Diseases and Sciences, 2015, Vol. 60, Páginas 2901-2912 [en línea][recuperado el 06/05/2019]. 1573-2568, resumen en la base de datos EMBASE (ELSEVIER), nº de acceso EMB-2015067291 * |
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