WO2020234424A1 - Benzo[c][1,2,5]oxadiazole for the treatment of diseases caused by helicobacter - Google Patents

Abstract

The present invention relates to new compounds which are benzo[c][1,2,5]oxadiazole derivatives, and the use thereof in the treatment of infectious diseases caused by Helicobacter pylori. Also, the present invention relates to a pharmaceutical composition and to a combined preparation both comprising said compounds

Description

BENZO[C][1 ,2,5]OXADIAZOLE FOR THE TREATMENT OF DISEASES

CAUSED BY HELICOBACTER

The invention relates to benzo[c][1 ,2,5]oxadiazole derivatives, and the use thereof as antibiotics and in particular for the treatment of diseases caused by bacteria, in particular of diseases caused by Helicobacter.

STATE OF ART

Helicobacter pylori ( Hp ) is a Gram-negative bacterium that establishes life-long infections in humans by colonising their gastric mucosa, usually during childhood. Hp infection is the most common bacterial infection all over the world with a prevalence that varies from 10 to 94 % in different countries. Hp transmission follows person-to- person, oral-oral, faecal-oral, waterborne or iatrogenic routes. In most cases, Hp infection is asymptomatic, but it can progressively damage the gastric mucosa by inducing chronic gastritis and then diseases from peptic ulcer to gastric mucosa- associated lymphoid tissue (MALT) lymphoma and gastric adenocarcinoma. Hp is the main cause of gastric cancer all over the world. Up to now, it is the only bacterium classified as a group I carcinogen. Eradication of Hp infection is the gold standard in peptic ulcer disease treatment and might prevent gastric cancer, the third most common cause of cancer death. Effective and affordable treatments are needed to reduce the impact of Hp infection in those diseases. Moreover, Hp infection has been suggested to be involved in other extra-gastrointestinal disorders and may modify the bioavailability and absorption of essential nutrients and the plasma levels of metabolic hormones.

Conventional therapy against Hp consists of empirical triple or, more often now, quadruple treatments based on a proton pump inhibitor plus a combination of two or three broad spectrum antimicrobials such as clarithromycin (Cla), amoxicillin, metronidazole (Mnz), tetracycline and bismuth salts. Increasing Hp resistance to these common antimicrobial drugs, especially to Cla and Mnz, is growing worldwide, reducing the effectiveness of available therapies. Therefore, new specific treatments are required, and eradication strategies need to be adapted geographically. Ongoing research on vaccines against Hp has not been successful until now, probably due to Hp genetic variability and to the complex host immune response against the bacterium. Current alternatives proposed include the application of therapeutic regimes based on local patterns of antimicrobial susceptibility and the use of personalized treatments consisting of the pre-identification of Hp susceptibility/resistance to conventional antimicrobials by molecular or by cultured-guided methods. The use of antimicrobial peptides and the development of novel compounds acting on specific Hp targets have also been proposed. Following the latter approach, specific Hp targets, essential for the bacterium and absent in humans, have been identified. One such target is Hp flavodoxin (Hp-FId), a small acidic redox protein that contains one molecule of tightly bound flavin mononucleotide cofactor.

Flavodoxins are bacterial proteins that take part in a variety of electron transfer reactions. In particular, Hp-FId accepts electrons from the pyruvate ferredoxin oxidoreductase complex (PFOR), which catalyses the oxidative decarboxylation of pyruvate, and transfers them to flavodoxin quinone reductase. Both Hp-FId and Hp- PFOR are essential for the bacterium survival and compounds interfering with this pathway might be suited for Hp eradication therapies.

Unlike most other flavodoxins, Hp-FId contains a pocket near the active site where small molecules could bind interfering with binding or electron transfer to Hp-FId partner proteins. Because flavodoxins as such are not present in humans, and the Fld- like domain found in human cytochrome P450 reductase lacks an equivalent pocket near its active site, no side effects are anticipated for a therapy targeting Hp-FId.

The biophysical properties of Hp-FId have been characterized and several small interacting molecules that bind to it have been identified through experimental screening. Some of them inhibit Hp-Fld-mediated pyruvate decarboxylation and act as specific bactericidal agents for Hp.

Also, document WO2015104433A1 discloses benzo[c][1 ,2,5]oxadiazole derivatives and the use thereof as antibiotics and in particular for the treatment and/or prevention of diseases caused by bacteria, in particular of diseases caused by Helicobacter. Similarly, the document ES2304221A1 relates to compound for use in a pharmaceutical composition for treating infectious diseases caused by Helicobacter pylori.

The present invention represents an improvement over the prior art, since it relates to new benzo[c][1 ,2,5]oxadiazole derivatives showing better bactericidal properties than the known ones. Besides, their toxicity for mice after oral administration is greatly reduced compared to benzo[c][1 ,2,5]oxadiazole derivatives known in the state of the art. These novel derivatives are effective against metronidazole-, clarithromycin- and rifampicin-resistant clinical isolates of Hp.

DESCRIPTION OF THE INVENTION

The present invention discloses new compounds for their use in the treatment of infectious diseases, and more specifically, diseases caused by Helicobacter pylori ( also referred to in the present invention as Hp or H. pylori).

It has been demonstrated (see examples) that the compounds of the invention completely inhibit Hp bacteria in the micro molar concentration range by the inhibition of pyruvate oxidation in which Hp flavodoxin participates.

Then, a first aspect of the present invention relates to a compound of formula (I):

a pharmaceutically acceptable salt, an isomer or a solvate thereof, wherein

Ri is a NO2 or NH₂ group,

R₂ is H or halogen, preferably halogen is selected from F, Cl, Br or I,

X is -S-, -SO- or -S0₂-

R₃ is H or (C₁-C₃) alkyl group optionally substituted by at least one group selected from: -OH, -NH_2, halogen (F, Cl, Br or I) or combinations thereof,

n is an integer of 1 to 3,

with the proviso that R₂ is halogen when X is -S- and R3 is -CH₃.

In an embodiment, the invention relates to a compound of formula (I) wherein X is -S- or -SO-.

In another embodiment, the invention relates to a compound of formula (I) wherein R₂ is H or Cl. In another embodiment, the invention relates to a compound of formula (I) wherein R3 is CH₃.

In another embodiment, the invention relates to a compound of formula (I) wherein R3 is H or a (C₁-C₃) alkyl substituted by at least one -OH group, more preferably R₃ is propyl substituted by two OH groups and even more preferably is -CH₂-CHOH- CH₂OH.

In another embodiment, the invention relates to a compound of formula (I) wherein n is 1 .

In another embodiment, the invention relates to a compound of formula (I) wherein X is -SO- and R2 is H.

In another embodiment, the invention relates to a compound of formula (I) wherein Ri is a NO2 or NH₂ group, R2 is H or Cl, X is -S-, -SO- or -SO2- , R3 is CH₃ and n is 1 .

In an embodiment, the invention relates to a compound of formula (I) wherein Ri is a NO2 or IMH2 group, R2 is H, X is -SO- or -SO2-, R3 is CH₃ and n is 1.

In an embodiment, the invention relates to a compound of formula (I) selected from: 5-chloro-7-((4-methoxybenzyl)thio)benzo[c][1 ,2,5]oxadiazol-4-amine

7-((4-methoxybenzyl)sulfinyl)benzo[c][1 ,2,5]oxadiazol-4-amine

4-((4-methoxybenzyl)sulfinyl)-7-nitrobenzo[c][1 ,2,5]oxadiazole

7-((4-methoxybenzyl)sulfonyl)benzo[c][1 ,2,5]oxadiazol-4-amine

3-(4-(((7-nitrobenzo[c][1 ,2,5]oxadiazol-4-yl)thio)methyl)phenoxy)propane-1 ,2-diol 3-(4-(((7-aminobenzo[c][1 ,2,5]oxadiazol-4-yl)thio)methyl)phenoxy)propane-1 ,2-diol, or 3-(4-(((7-aminobenzo[c][1 ,2,5]oxadiazol-4-yl)sulfinyl)methyl)phenoxy)propane-1 ,2- diol.

The compounds of the present invention contain one or more basic nitrogens and they could therefore form salts with acids, both organic and inorganic. Examples of such salts include: salts with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, perchloric acid, sulfuric acid or phosphoric acid; and salts with organic acids such as methanesulfonic acid, trifluoromethane sulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, fumaric acid, oxalic acid, acetic acid, maleic acid, ascorbic acid, citric acid, lactic acid, tartaric acid, malonic acid, glycolic acid, succinic acid and propionic acid, among others. Some compounds of the present invention may contain one or more acid protons and thus may also form salts with bases. Examples of such salts include: salts with inorganic cations such as sodium, potassium, calcium, magnesium, lithium, aluminum, zinc, etc; and salts formed with pharmaceutically acceptable amines such as ammonia, alkylamines, hydroxyalkylamines, lysine, arginine, /V-methylglucamine, procaine and the like.

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

The salts of a compound of formula (I) can be obtained during the final isolation and purification of the compounds of the invention or can be prepared by treatment of a compound of formula (I) with a sufficient amount of the desired acid or base to give the salt in a conventional manner. The salts of the compounds of formula (I) can in turn be transformed into other salts of compounds of formula (I) by ion exchange by an ion exchange resin.

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

The compounds of the present invention can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as solvates. As used herein, the term solvate refers to a variable stoichiometry complex formed by a compound of formula (I) or a salt thereof(solute) and a solvent. Examples of solvents include the pharmaceutically acceptable solvents such as water, ethanol and the like. A complex with water is known as hydrate. Solvates of the compounds of the invention (or salts thereof), including hydrates, are included within the scope of the invention. It must be understood that the present invention encompasses all the isomers of the compounds of formula (I), i.e. all the geometric, tautomeric and optical forms, and their mixtures (for example, racemic mixtures). When there are more chiral centres in the compounds of formula (I), the present invention includes, within its scope, all the possible diastereomers, including their mixtures. The different isomeric forms may be separated or resolved therebetween using conventional methods or any given isomer can be obtained using conventional synthetic methods or by means of stereospecific, stereoselective or asymmetric synthesis.

The compounds of formula (I) may exist in different physical forms, i.e. in amorphous form and crystalline forms. Also, the compounds of the present invention can have the ability to crystallize from more than one form, a characteristic which is known as a polymorphism. The polymorphs can be differentiated by various physical properties well known to those skilled in the art such as 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.

Unless stated otherwise, the compounds of the invention also include compounds that only differ in the presence of one or more isotopically enriched atoms. For example, compounds having said structure, except for the substitution of a hydrogen by a deuterium or tritium, or the substitution of a carbon by a carbon enriched in ¹³C or ¹⁴C or a nitrogen enriched in ¹⁵N, are within the scope of this invention.

The term“C₁-C₃ alkyl”, in the present invention, refers to radicals of hydrocarbon, linear or branched chains having 1 to 3 carbon atoms, preferably 1 , and which bind to the rest of the molecule by a single bond, for example, methyl, ethyl, n-propyl, i-propyl. The Ci- C₃ alkyl groups may be optionally substituted by one or more hydroxyl, amine or halogen substituents or combinations of these substituents.

A second aspect of the invention relates to the compound of formula (I), a pharmaceutically acceptable salt, an isomer or a solvate thereof, for use as a medicament.

Another aspect of the invention relates to compound of formula (I), a pharmaceutically acceptable salt, an isomer or a solvate thereof for use in the treatment of infectious diseases caused by Helicobacter, more preferably by Helicobacter pylori. In an embodiment of the present invention relates, the compound of formula (I), a pharmaceutically acceptable salt, an isomer or a solvate thereof, is used for the treatment of infectious diseases caused by NTCT 1 1 637, 26695 or SS1 strain of Helicobacter pylori.

In a preferred embodiment, the present invention relates to compound 4-((4- methoxybenzyl)sulfinyl)-7-nitrobenzo[c][1 ,2,5]oxadiazole for use in the treatment of infectious diseases caused by NTCT 1 1 637, 26695 or SS1 strain of Helicobacter pylori.

In a preferred embodiment, the present invention relates to compound 5-chloro-7-((4- methoxybenzyl)thio)benzo[c][1 ,2,5]oxadiazol-4-amine for use in the treatment of infectious diseases caused by NTCT 1 1 637 or 26695 strain of Helicobacter pylori.

In a preferred embodiment, the present invention relates to compound 7-((4- methoxybenzyl)sulfinyl)benzo[c][1 ,2,5]oxadiazol-4-amine for use in the treatment of infectious diseases caused by SS1 strain of Helicobacter pylori.

In a preferred embodiment, the compound of formula (I), a pharmaceutically acceptable salt, an isomer or a solvate thereof, is administered orally.

The dosage and frequency of the doses will vary depending on the nature and severity of the disease to be treated, the age, the general condition and weight of the patient, as well as the particular compound administered and the route of administration, among other factors.

Nevertheless, in a preferred embodiment, the compound of formula (I) , a pharmaceutically acceptable salt, an isomer or a solvate thereof, is administered to a subject in a dose between 0.1 and 20 mg/1 OOgr bw (body weight) /day, more preferably in a dose of 20 mg/1 OOgr bw/day.

In another embodiment, the compound of formula (I), a pharmaceutically acceptable salt, an isomer or a solvate thereof, is administered to a subject once daily, twice daily or three times daily; more preferably, once daily or twice daily; even more preferably, once daily. Infectious diseases caused by Helicobacter pylori are related to gastrointestinal pathologies such as chronic gastritis, gastric or duodenal ulcers, lymphoma, MALT lymphoma (mucosal lymphoma associated with lymphoid tissue) and gastric cancer. Therefore, a more preferred embodiment comprises the use of the compounds of the invention for the treatment of infectious diseases such as chronic gastritis, gastric and/or duodenal ulcers, lymphoma and gastric cancer.

Both gastric cancer and gastric lymphoma, in particular, MALT lymphoma, are diseases which may be caused by H. pylori and this bacterium has been classified into the Group I of carcinogens by the International Research Agency.

Throughout the present description, the term "treatment" is intended to be eliminated, reducing or decreasing the cause or effects of a disease. For the purposes of this invention, treatment includes, but is not limited to, the same, alleviating, slowing or eliminating one or more symptoms of the disease; to reduce the degree of disease, stabilize (ie, do not worsen) the state of the disease, delaying or slowing the progression of the disease, alleviating or improving the condition of the disease and recessing (either total or partial).

For its therapeutic application, the compounds of the formula (I), the isomers, salts or solvates thereof, will preferably be in a pharmaceutically acceptable or substantially pure form, that is, having a pharmaceutically acceptable grade of purity, excluding the usual pharmaceutical additives such as diluents and carriers and not including material considered toxic at normal dosage levels. The levels of purity for the active ingredient are preferably greater than 50%, more preferably, greater than 70%, more preferably, greater than 90%. In a preferred embodiment, they are greater than 95% of the compound of formula (I) or of the salts, solvates or isomers thereof.

The compounds of the present invention able to inhibit flavodoxin constitute a very good alternative to the treatment of infectious diseases, particularly those caused by Helicobacter, more specifically by H. pylori.

The compounds of the invention, when administered individually at single daily doses for 8 days in a mice model of Hp infection (see examples), reduced significantly the Hp gastric colonization rates and were even able to eradicate the infection in up to 60% of the mice. These flavodoxin inhibitors constitute a novel family of antimicrobials that may help fight the constant increase of Hp antimicrobial-resistant strains.

Another aspect of the present invention relates to a combined preparation that comprises, at least, a compound as defined in the first aspect of the invention (compound of formula (I), a pharmaceutically acceptable salt, an isomer or a solvate thereof) and other active ingredient, wherein said active ingredient may be an antiacid; another antibiotic, such as but not limited to amoxicillin, metronidazole, clarithromycin or levofloxacin; a proton pump inhibitor, such as but not limited to omeprazole, pantoprazole or rabeprazole; or any combination thereof.

Another aspect of the present invention relates to the combined preparation for use in the treatment of infectious diseases caused by Helicobacter, more preferably, by Helicobacter pylori. Therefore, the other active ingredient provides a combined therapy and can be administered separately, simultaneously or sequentially with respect to compound of formula (I) defined in the first aspect of the invention.

Another aspect of the present invention relates to a pharmaceutical composition comprising at least the compound defined in the first aspect of the invention (compound of formula (I), a pharmaceutically acceptable salt, an isomer or a solvate thereof) or at least the combined preparation as previously defined. The pharmaceutical composition, in addition to these compounds, may comprise pharmaceutically acceptable excipients.

The excipients must be "acceptable" in the sense of being compatible with the other ingredients of the composition and should not be detrimental to taking said composition.

Another aspect of the present invention relates to the pharmaceutical composition defined above as a medicament, preferably for use in the treatment of infectious diseases caused by Helicobacter, more preferably by Helicobacter pylori and even more preferably by NTCT 1 1637, 26695 or SS1 strain of Helicobacter pylori.

The composition may be administered by any suitable route of administration, for which reason said preparation is formulated in the dosage form suitable for the chosen route of administration. In a particular embodiment, the administration of the compound of formula (I), which is provided by this invention, is carried out by oral, topical, rectal or parenteral (including subcutaneous, intraperitoneal, intradermal, intramuscular, intravenous, etc.) route. Different pharmaceutical forms of administration of drugs and excipients are known for the skilled person in the art.

In a preferred embodiment the invention relates to the pharmaceutical composition as defined above, wherein the composition is suitable for oral administration .

Solid compositions for oral administration include tablets, tablets, granulates, capsules and prolonged release capsules. In any case, the method of manufacture is based on a simple mixture, dry granulation or wet granulation of the active ingredient with excipients. These excipients may be, for example, diluents such as lactose, microcrystalline cellulose, mannitol or calcium phosphate; binding agents such as starch, gelatin or polyvinylpyrrolidone; disintegrants such as sodium carboxymethyl starch or croscarmellose sodium; and lubricating agents such as magnesium stearate, stearic acid or talc.

Powders and granulates for the preparation of oral suspensions can be obtained by the addition of water, mixing the active ingredient with dispersing or wetting agents; suspending agents and preservatives. Other excipients, for example sweeteners, flavorants and colorants may also be added.

As liquid forms for oral administration, emulsions, solutions, suspensions, syrups and elixirs containing commonly used inert diluents, such as distilled water, ethanol, sorbitol, glycerol, polyethylene glycols (macrogols) and propylene glycol. Said compositions may also contain adjuvants as wetting agents, suspending agents, sweeteners, flavors, preservatives and buffers.

In a particular embodiment, the invention relates to a composition wherein the compound of formula (I) is dissolved in oil, preferably in olive oil, to improve the solubility of said compound and, therefore, to improve its effectiveness. In such a way that a non-aqueous solvent and more preferably the olive oil functions as a coadjuvant, as a solvent and/or as a carrier of the compound of formula (I).

Thus, in another embodiment the invention relates to the pharmaceutical composition as defined above, wherein the solvent is a non-aqueous solvent, more preferably the solvent is an oil and even more preferably the solvent is olive oil. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skilled in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. Throughout the description and claims the word "comprise" and its variations are not intended to exclude other technical features, additives, components, or steps. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples and drawings are provided by way of illustration and are not intended to be limiting of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Mice weight variation during H. pylori infection and treatment over in vivo efficacy experiments. Four trials (A, B, C and D) have been carried out in order to determine the in vivo efficacy of the compounds of the invention at five different doses (0.1 , 1 , 5, 10 and 20 mg/100 g mouse). Mice were weighted at the start of the experiment, at mid-experiment and the day of the sacrifice. Results are represented as weight per mouse for each group. Each line represents a group of animals which received the same treatment.

FIG. 2. Viability of HeLa cells treated with the different tested compounds.

FIG. 3. Histological study of in vivo toxicity of inhibitors 120 and IV described in example 1 and belonging to the state-of-the-art inhibitors.

FIG. 4. Data of gastric colonization for compounds IV, 120 and SS16 and doses tested are presented along with those of the corresponding control groups in Figure 4A, while the percentages of Hp-eradicated mice after treatment are shown in Figure 4B.

EXAMPLES

Example 1 : Synthesis of the compounds of the invention Several compounds within the formula (I) defined in the present invention have been synthesized as described below.

General synthetic and analytic procedures

All reagents were of analytical grade and were used as obtained from commercial sources. Reactions were carried out using anhydrous solvents. ¹H NMR, ¹³C NMR and ¹⁹F NMR spectra were acquired at room temperature at 400, 100 and 376 MHz, respectively, using a 5 mm probe. Chemical shifts (d) are reported in parts per million from tetramethylsilane with the solvent resonance as the internal standard. Coupling constants (J) are quoted in hertz. The splitting patterns are reported as s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), m (multiplet), bs (broad singlet). Proton test spectra were acquired to determine the types of carbon signals, and Heteronuclear Multiple Bond Correlation spectra were acquired to confirm the position of heteroatoms. Special precautions such as degassing of the sample were not taken. Purity of compounds was determined to be > 95 % by HPLC chromatography, using a Waters HPLC system equipped with a 600-E pump, a 2996 Photodiode Array detector, and a 2707 autosampler. High-resolution mass measurements were made using a microTOF (time-of-flight) analyzer, and spectra were recorded from methanolic solutions using the positive electrospray ionization mode (ESI+). Reactions were magnetically stirred and whenever possible monitored by TLC. TLC was performed on precoated silica gel polyester plates and products were visualized using ultraviolet light (254 nm), ninhydrin, potassium permanganate and phosphomolybdic acid solution followed by heating. Column chromatography was performed using silica gel (Kiesegel 60, 230-400 mesh).

Synthetic reactions

Compounds of the present invention SS15, SS16, SS17, SS21 have been readily synthesized using the routes described in Schemes 1 -4 from compound IV, that is a compound already disclosed in the prior art (ES2304221 A1 ):

Reduction of nitro-compound IV with iron and hydrochloric acid, by using dichloromethane and methanol as solvents, afforded the desired product SS15 and also some of the previously synthesized molecule 120 (Scheme 4) from which it was separated by column chromatography. Scheme 1. Reduction of nitro-compound IV to a mixture of amino-compound 120 and amino-chlorinated compound SS15

Amino-sulfoxide SS16 and amino-sulfone SS21 could be obtained by thio-ether oxidation of molecules 120 (already disclosed in WO2015104433A1 ) and IV, respectively, with hydrogen peroxide in the presence of glacial acetic acid (Schemes 2 and 3).

Scheme 2. Oxidation of sulphur compound 120 to sulfoxide SS16

Scheme 3. Oxidation of sulphur compound IV to sulfoxide SS21

The amino-sulfone SS17 was synthesized by oxidation of compound 120 using meta- chloroperbenzoic acid in ethyl acetate as the oxidant.

Compound SS17 was obtained as a mixture with compound SS16, from which it was separated by flash chromatography (Scheme 4).

Scheme 4. Oxidation of sulphur 120 to a mixture of sulfoxide SS16 and sulfone SS17

Synthetic procedures

4-((4-methoxybenzyl)thio)-7-nitrobenzo[c][1 ,2,5]oxadiazole (IV). 4-chloro-7- nitrobenzo[c][1 ,2,5]oxadiazole (Alfa Aesar), (4-methoxyphenyl)methanethiol (Alfa Aesar) and pyridine were dissolved in ethanol (Scheme 5) and the mixture was stirred under argon atmosphere at 79°C for 2 hours. Purification of the residue by flash chromatography gave the desired product IV (64 % yield).

5-chloro-7-((4-methoxybenzyl)thio)benzo[c][1 ,2,5]oxadiazol-4-amine (SS15). A mixture of dichloromethane (2.83 ml_), cone hydrochloric acid (170 mI_) and methanol (1 .29 ml.) was set in a flask containing compound IV (25 mg, 0.08 mmol). After adding iron powder (30.42 mg, 0.54 mmol), the reaction was stirred at room temperature until disappearance of the nitro-compound, as judged by TLC. The mixture was poured into water (3 ml.) and extracted with dichloromethane (4x3 ml_). The organic layers were combined, dried over anhydrous magnesium sulphate, filtered and then evaporated in vacuo. TLC monitoring revealed that a mixture of two products had been obtained, one of them being a compound previously synthesised, namely 120, and the other one being SS15. The crude was purified by column chromatography over silica gel (eluent: Hex/EtOAc: 6/4). Removal of solvents under reduced pressure yielded 16 mg (61 % yield) of pure SS15. ¹H-NMR (400 MHz, DMSO-cfe): d 7.23 (s, 1 H), 7.14-7.12 (m, 2H), 6.94 (s, 2H), 6.83-6.81 (m, 2H), 4.19 (s, 2H), 3.70 (s, 3H) ppm. ¹³C-NMR (100 MHz, DMSO-cfe): d 158.3, 149.6, 144.6, 137.8, 132.2, 130.0, 129.1 , 113.8, 108.1 , 106.4, 55.0, 37.1 ppm. HRMS (ESI⁺) m/z [M+Na]⁺calc for Ci₄Hi₂CIN₃Na0₂S 344.0231 , found 344.0232.

7-((4-methoxybenzyl)sulfinyl)benzo[c][1 ,2,5]oxadiazol-4-amine (SS16). Compound 120 (495 mg, 1 .72 mmol) was dissolved in a mixture of glacial acetic acid (20.70 mL) and 33 % aqueous hydrogen peroxide (1 .38 mL), and kept at 25 ^eC for 2.5 h. The yellowish-brown mixture obtained was quenched with saturated NaHC0₃ solution (50 mL) and extracted with dichlorometane (5x30 mL). The organic layers were dried over anhydrous magnesium sulphate, filtered and, once the solvent was evaporated under reduced pressure, the crude was purified by column chromatography over silica gel (eluent: Hex/AcOEt: 2/8) to give 378 mg (73 % yield) of the desired product. ¹ H-NMR (400 MHz, DMSO-cfe) : d 7.38 (d, J=8 Hz, 1 H), 7.31 (s, 2H), 6.94-6.92 (m, 2H), 6.80- 6.78 (m, 2H), 6.33 (d, J=8 Hz, 1 H), 4.41 (d, J=12.8 Hz, 1 H), 4.21 (d, J=12.8 Hz, 1 H), 3.70 (s, 3H) ppm. ¹³C-NMR (100 MHz, DMSO-cfe): d 158.9, 145.7, 144.5, 140.3, 135.7, 131 .3, 121 .9, 1 13.6, 1 1 1 .5, 103.1 , 58.2, 55.0 ppm. HRMS (ESI⁺) m/z [M+Na]⁺calc for CuHisNsNaOsS 326.0570, found 326.0575.

7-((4-methoxybenzyl)sulfonyl)benzo[c][1 ,2,5]oxadiazol-4-amine (SS17). To a stirred solution of compound 120 (29 mg, 0.1 mmol) in ethyl acetate (1 .43 ml.) at room temperature, 3-chloroperbenzoic acid (34.5 mg, 0.2 mmol)) was added. TLC monitoring was done till the completion of the reaction (1 .5 h). Traces of 3-chloroperbenzoic acid and 3-chlorobenzoic acid were neutralized with saturated aqueous NaHCC>3 solution (2x2 ml_). After extracting with ethyl acetate (3 x 3 ml_), the combined organic layers were dried over anhydrous magnesium sulphate, filtered and the solvent was evaporated under reduced pressure. TLC monitoring revealed that a mixture of SS16 and SS17 had been obtained. The crude was purified by flash chromatography (eluent: Hex/EtOAc: 3/7) to provide 27 mg (85 % yield) of pure SS17.¹ H-NMR (400 MHz, DMSO-cfe) : d 7.95 (s, 2H), 7.57 (d, J=8 Hz, 1 H), 7.01 -6.98(m, 2H), 6.82-6.80 (m, 2H), 6.29 (d, J=8 Hz, 1 H), 4.55 (s, 2H), 3.70 (s, 3H) ppm. ¹³C-NMR (100 MHz, DMSO-cfe) : d

159.2, 146.0, 144.0, 143.5, 140.9, 131 .9, 120.6, 1 13.7, 106.6, 102.0, 59.7, 55.0 ppm. HRMS (ESI⁺) m/z [M+Na]⁺calc for Ci₄Hi₃N₃Na0₄S 342.0519, found 342.0523.

4-((4-methoxybenzyl)sulfinyl)-7-nitrobenzo[c][1 ,2,5]oxadiazole (SS21 ). Compound IV (96 mg, 0.30 mmol) was dissolved in a mixture of glacial acetic acid (5.66 mL) and 33 % hydrogen peroxide (242 pL) and the reaction was stirred at 25 ^eC for 24 h. The obtained orange-brown oily mixture was extracted with dichloromethane (3 x 10 mL). The combined organic layers were dried over anhydrous magnesium sulphate, filtered and the solvent was evaporated under reduced pressure. Flash chromatography of the mixture was done in order to purify the desired product. In this way 84.3 mg (88 % yield) of pure SS21 , which was stored in a sealed flask under an argon atmosphere at 4 °C until testing to avoid spontaneous decomposition because of its sensitivity to oxygen, were obtained. ¹ H-NMR (400 MHz, CDCI₃): d 8.47 (d, J=7.2 Hz, 1 H), 7.75 (d, J=7.2 Hz, 1 H), 6.89-6.87 (m, 2H), 6.77-6.74 (m, 2H), 4.61 (d, J=13.6 Hz, 1 H), 4.32 (d, J= 13.6 Hz, 1 H), 3.77 (s, 3H) ppm. ¹³C-NMR (100 MHz, CDCI₃): d 160.2, 146.1 , 142.6,

142.3, 131 .3, 129.1 , 128.7, 1 19.6, 1 14.1 , 59.5, 55.3 ppm. HRMS (ESI⁺) m/z [M+Na]⁺ calc for Ci₄HnN₃Na0₅S 356.0312, found 356.0308. Compounds of the present invention AM_P085, AM_P167 and AM_P202 have been synthesized using the routes described in in Schemes 6-8 from compound AM_P096:

Scheme 6: Synthesis of compound AM_P085

Scheme 7: Synthesis of compound AM_P167

Scheme 8: Synthesis of compound AM_P202

Compound AM_P096 was synthetized according to Scheme 9:

Synthetic procedures

2,3-di(A¹-oxidanyl)propyl 4-methylbenzenesulfonate (AM P102)

Solketal (Alfa Aesar), pyridine and 4-toluenesulfonyl chloride (Alfa Aesar) were dissolved in anhydrous dichloromethane. The mixture was stirred at room temperature for 16 hours. Purification of the residue by flash chromatography gave the desired product AM_P102 (85% yield). ¹ H NMR (300 MHz, CDCI3): 7.82-7.74 (m, 2H), 7.38- 7.31 (m, 2H), 4.31 -4.22 (m, 1 H), 4.06-3.92 (m, 3H), 3.75 (dd, J=9.0 Hz, J=5.1 Hz, 1 H), 2.44 (s, 3H), 1 .33 (s, 3H), 1 .30 (s, 3H) ppm.

(4-(2,3-di(A¹-oxidanyl)propoxy)phenyl)methanol compound with neopentane (1 :1 ) (AM P104)

Dry potassium carbonate was added to a solution of compound AM J³102 and 4- (hydroxymethyl)phenol (Alfa Aesar) in anhydrous DMF. The mixture was kept at 90°C for 16 hours and the residue was poured into deionized water and extracted with dichloromethane. The crude product was then purified by flash chromatography over silica gel to give AM_P104 (51 % yield). ¹ H NMR (300 MHz, CDCh): 7.32-7.25 (m, 2H), 6.93-6.87 (m, 2H), 4.61 (s, 2H), 4.53-4.43 (m, 1 H), 4.17 (dd, J=8.4 Hz, J=6.3 Hz, 1 H), 4.06 (dd, J=9.6 Hz, J=5.4 Hz, 1 H), 3.94 (dd, J=9.6 Hz, J=6.0 Hz, 1 H), 3.90 (dd, J=8.4 Hz, J=5.7 Hz, 1 H), 1 .83 (s_a, 1 H), 1 .47 (s, 3H), 1 .41 (s, 3H) ppm.

S-(4-(2.3-di(A¹-oxidanyl)propoxy)benzyl) ethanethioate compound with neopentane (1 :1 ) (AM P100)

Triphenylphosphine and DIAD were dissolved in anhydrous THF and stirred at 0°C under argon atmosphere for 20 minutes. Thioacetic acid (Alfa Aesar) and AM P104 were then added to the crude and it was stirred at 0°C for 1 hour. After that, it was kept at room temperature for an additional hour. The solvent was evaporated and the crude was purified by flash chromatography to obtain pure AM__.P100 (61 % yield). ¹ H NMR (300 MHz, CDCh): 7.23-7.16 (m, 2H), 6.87-6.80 (m, 2H), 4.51 -4.40 (m, 1 H), 4.15 (dd, J=8.4 Hz, J=6.6 Hz, 1 H), 4.07 (s, 2H), 4.03 (dd, J=9.6 Hz, J=5.4 Hz, 1 H), 3.96-3.83 (m, 2H), 2.33 (s, 3H), 1 .45 (s, 3H), 1 .40 (s. 3H) ppm.

(4-(2.3-di(l1 -oxidanyl)propoxy)phenyl)methanethiol compound with neopentane (1 :1 ) (AM P1 1 1 ) A solution of compound AM__P100 and dry potassium carbonate in dry methanol was stirred under argon atmosphere at room temperature for 15 minutes. The mixture was acidificated with HCI and extracted with dichloromethane. Flash chromatography was done in order to purify the desired product AM_P111 (67% yield). ¹ H NMR (400 MHz, CDCIs): 7.26-7.20 (m, 2H), 6.89-6.82 (m, 2H), 4.51 -4.43 (m, 1 H), 4.16 (dd, J=8.4 Hz, J=6.4 Hz, 1 H), 4.04 (dd, J=9.6 Hz, J=5.6 Hz, 1 H), 3.92 (dd, J=9.6 Hz, J=6.0 Hz, 1 H), 3.89 (dd, J=8.4 Hz, J=5.8 Hz, 1 H), 3.70 (d, J=7.6 Hz, 2H), 1 .73 (t, J=7.6 Hz, 1 H), 1 .46 (s, 3H), 1 .40 (s, 3H) ppm.

4-((4-((2,2-dimethyl-1 ,3-dioxolan-4-yl)methoxy)benzyl)thio)-7- nitrobenzofc1f1 ,2,51oxadiazole (AM P096)

Compound AM_P1 1 1 , 4-chloro-7-nitrobenzo[c][1 ,2,5]oxadiazole (Alfa Aesar) and anhydrous pyridine were dissolved in anhydrous DMF under argon atmosphere. The mixture was stirred under argon atmosphere at 80 °C for 4 hours. After the completion of the reaction, the DMF was removed under reduced pressure and the crude was purified by flash chromatography to give the desired product AMJP096 (45% yield). ¹ H NMR (400 MHz, CDCI₃): d 8.34 (d, J=8.0 Hz, 1 H), 7.38-7.31 (m, 2H), 7.18 (d, J=8.0 Hz, 1 H), 6.93-6.86 (m, 2H), 4.51 -4.43 (m, 1 H), 4.47 (s, 2H), 4.16 (dd, J=8.4 Hz, J=6.4 Hz, 1 H), 4.04 (dd, J=9.6 Hz, J=5.6 Hz, 1 H), 3.93 (dd, J=9.6 Hz, J=5.6 Hz, 1 H), 3.89 (dd, J=8.4 Hz, J=5.6 Hz, 1 H), 1 .45 (s, 3H), 1 .39 (s, 3H) ppm.

3-(4-(((7-nitrobenzo[c][1 ,2,5]oxadiazol-4-yl)thio)methyl)phenoxy)propane-1 ,2-diol (AM_P085)

To a vigorously stirred solution of AM P096 in acetonitrile, bismuth chloride and deionized water were added. The mixture was kept at room temperature for 5 hours and then concentrated in vacuo. Purification of the residue by flash chromatography afforded AM_P085 (79% yield). ¹ H NMR (400 MHz, Acetone-d₆): 8.56 (d, J=8.0 Hz, 1 H), 7.63 (d, J=8.0 Hz, 1 H), 7.52-7.40 (m, 2H), 7.00-6.93 (m, 2H), 4.66 (s, 2H), 4.14- 4.05 (m, 2H), 4.02-3.93 (m, 2H), 3.83-3.77 (m, 1 H), 3.71 -3.60 (m, 2H) ppm.

3-(4-(((7-aminobenzo[c][1 ,2,5]oxadiazol-4-yl)thio)methyl)phenoxy)propane-1 ,2- diol (AM_P167)

Compound AM P096 was dissolved in a mixture of ethanol, acetic acid and deionized water. After the addition of iron powder, the mixture was sonicated (JP Selecta ultrasonic bath, 40 kHz) at 30°C for 30 minutes. The reaction mixture was filtered to remove excess iron, quenched with saturated potassium carbonate solution and extracted with dichloromethane. The crude was dissolved in acetonitrile and then, bismuth chloride and deionized water were added. The mixture reaction was kept at room temperature for 16 hours. The purification of the crude by flash chromatography afforded AM_P167 (58% yield). ¹H NMR (400 MHz, MeOD): 7.08 (d, J=7.6 Hz, 1 H), 7.05-6.98 (m, 2H), 6.81 -6.73 (m, 2H), 6.19 (d, J=7.6 Hz, 1 H), 4.07 (s, 2H), 4.01 -3.96 (m, 1 H), 3.96-3.86 (m, 2H), 3.66 (dd, J=1 1 .2 Hz, J=4.8 Hz, 1 H), 3.61 (dd, J=1 1 .2 Hz, J=5.2 Hz, 1 H) ppm.

3-(4-(((7-aminobenzo[c][1 ,2,5]oxadiazol-4-yl)sulfinyl)methyl)phenoxy)propane- 1 ,2-diol (AM_P202)

Compound AM P167 was dissolved in a mixture of glacial acetic acid and 35 % aqueous hydrogen peroxide, and kept at room temperature for 1 .5 hours. After the completion of the reaction, the crude was purified by flash chromatography to give product AM_P202 (82% yield). ¹H NMR (400 MHz, MeOD): 7.39 (d, J=7.6 Hz, 1 H), 6.99-6.92 (m, 2H), 6.85-6.78 (m, 2H), 6.31 (d, J=7.6 Hz, 1 H), 4.54 (d, J=13.0 Hz, 1 H), 4.39 (d, J=13.0 Hz, 1 H), 4.03-3.96 (m, 1 H), 3.96-3.87 (m, 2H), 3.70-3.58 (m, 2H) ppm.

Example 2: Minimal Inhibitory Concentrations (MIC) on reference Ho strains (NCTC 11637, 26695, SS1 and UA 1182 )

For microdilution MIC testing, Hp reference strains NCTC 1 1637 (ATCC 43504, Mnz- resistant strain), 26695 (ATCC 700392) (Tomb, J. F. et al. The Complete Genome Sequence of the Gastric Pathogen Helicobacter pylori. Nature 1997, 389 (6649), 412) SS1 (Sydney Strain 1 ) (Lee, A. et al. Standardized Mouse Model of Helicobacter pylori Infection: Introducing the Sydney Strain. Gastroenterology 1997 , 112 ( 4), 1386-1397) and UA 1 182 (ATCC 700684) (Biosafety Level 2 pathogen) were grown in brain-heart infusion broth supplemented with 4 % fetal bovine serum at 37 °C under microaerophilic conditions and then diluted to an optical density at 600 nm of 0.01. Samples of 100 pL of the diluted bacterial suspension were dispensed in a 96-well plate, except for the first well of each row in which 200 pL of the bacterial suspension along with 2 pL of the compound (from a stock solution in dimethyl sulfoxide (DMSO) at 6.4 pg/pL) were added. Two-fold serial dilutions were made, which allowed to test the compounds in a concentration range from 64 to 0.031 pg/mL. Positive and negative controls consisted of brain-heart infusion broth supplemented with 4 % fetal bovine serum and inoculated or not with the diluted Hp bacterial suspension, respectively. We ensured that DMSO concentration was kept at 1 % v/v or below, so that no toxic effect was found for Hp cells. Plates were incubated at 37 ^eC for 48 hours under a microaerophilic atmosphere. MICs correspond to the lowest concentrations of compounds leading to a complete inhibition of Hp growth. The MIC values were determined by recording the colour change observed after addition of 30 mI_ of resazurin (0.1 mg/ml; Sigma-Aldrich) to each well in the 96-well dish. The MICs were additionally confirmed by spotting 10 pL of two consecutive wells (one with the highest concentration of compound that allowed bacterial growth, and the adjacent well with the lowest concentration of compound that prevented bacterial growth) onto Columbia Agar with Sheep Blood plates (Oxoid) followed by incubation at 37 °C for 48 h under microaerophilic conditions. Growth or absence of Hp growth allowed to determine the Minimal Bactericidal Concentration of the compounds. Each experiment was performed twice in triplicate. Both assays confirmed the MIC values obtained by the microdilution method.

MIC on drug-resistant Hp strains

Six Hp drug-resistant strains obtained at the University Hospital Lozano Blesa (Spain) from patients with gastric pathologies (dyspepsia and/or peptic ulcer disease) who had failed to at least two conventional therapies recommended in national guidelines were selected. These strains were isolated from gastric biopsies obtained during routine upper gastrointestinal endoscopic procedures and cultured. According to The European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints (EUCAST. European Committee on Antimicrobial Susceptibility Testing Breakpoint Tables for Interpretation of MICs and Zone Diameters European Committee on Antimicrobial Susceptibility Testing Breakpoint Tables for Interpretation of MICs and Zone Diameters. 2018), three of these Hp strains were metronidazole-resistant, two were metronidazole/clarithromycin resistant, and one was rifampicin -resistant. Compounds 120, SS15, SS16 and SS17 were tested on all of these strains. The determination of their MIC was performed by microdilution and followed by the colorimetric method using resazurin, as explained above.

Minimal Cytotoxic Concentrations (MCC₅o)

HeLa cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 pg/mL streptomycin at 37 °C in a 5% CO2 atmosphere. The toxicity of the compounds towards HeLa cells was determined by the XTT (2,3-Bis-(2-methoxy-4-nitro-5-sulphenyl)-2/-/-tetrazolium-5- carboxanilide sodium salt) method using the Cell Proliferation Kit II (Roche) as described (Cremades, N. et al. J. Discovery of Specific Flavodoxin Inhibitors as Potential Therapeutic Agents against Helicobacter pylori Infection. ACS Chem. Biol. 2009, 4 ( 1 1 ), 928-938. Galano, J. J. et al. Improved Flavodoxin Inhibitors with Potential Therapeutic Effects against Helicobacter pylori Infection. J. Med. Chem. 2013, 56 (15), 6248-6258). Briefly, 0.1 -pL volumes of compound dissolved in DMSO were added to give final compound concentrations of 25, 50, 75, 100, 250, 500, 750, and 1000 mM, in 96-well plates (with 30,000 cells in 100 mI_ in each well). The untreated control consisted in 30,000 cells per well in 100 mI_ of complete medium (with 0.1 % DMSO). Cell viability was calculated from the wells' absorbance at 450 and 650 nm. Cytotoxicity curves (i.e percentage of cell viability versus compound concentration) were used to calculate the compounds concentration producing 50 % cell lysis (MCC₅o). All experiments were performed twice in triplicate. Therapeutic Indexes (Tl) were calculated as MCC50/MIC.

Toxicity assays in mice

The in vivo toxicity of compounds was tested in 48 twenty-week old specific-pathogen- free female C57BL/6J mice obtained from Charles River Laboratories Espaha (Barcelona, Spain), fed with a standard commercial rodent diet and water ad libitum. Females were selected because of their less aggressiveness. Toxicity of compounds IV and 120 was tested at three different doses: 10, 20 and 40 mg/100 g body weight (bw). Assuming the administered compounds are uniformly distributed throughout the animal body, these doses range from 12 to 1600 times the MICs of the compounds. Ten groups of 4-5 animals were used. To nine of them, and for each compound, the corresponding daily dose was administered in volumes of 100 mI by oral gavage for 7 days. To the control group, vehicle alone (olive oil, 5 % DMSO) was administered. After treatment, mice were sacrificed by CO2 asphyxiation and blood was collected by cardiac puncture. An automatic analyser (VetScan, Abaxis) was used to determine biochemical alterations. Flistopathology evaluation was done on heart, stomach, liver, lung and kidney samples by haematoxylin-eosin staining of tissue sections.

Antibacterial assays in mice

In vivo analysis of the anti-Hp activity of the compounds was carried out in 6-week old (SPF) NMRI (Naval Medical Research Institute) mice (Charles River laboratories, France). The level of Hp gastric colonization in the mice model is similar in male and female, so assays were carried out in females because they are less aggressive than males under the hosting conditions. Four different experiments were performed to test compounds IV, 120, SS15 and SS16 at some of the following doses of 0.1 , 1 , 5, 10 and 20 mg/100 g bw. In all experiments, 7 mice were analysed for each studied condition. Mice were orogastrically infected at days 1 and 3 during the first week of the experiment with 100 mI_ of a suspension of Hp strain SS1 (5-10^s CFU/ml) known to colonize efficiently the mouse gastric mucosa. According to a previously established model of antimicrobial treatment of Hp infection (Correia, M. et al. Docosahexaenoic Acid Inhibits Helicobacter py/or/Growth In Vitro and Mice Gastric Mucosa Colonization. PLoS One 2012, 7, e35072), administration of the compounds started at day 8. One- hundred mI_ of each compound solubilised in 5-10 % DMSO (Euromedex)/95-90 % olive oil (Sigma-Aldrich) as vehicle at the defined doses were daily administered by oral gavage for 8 consecutive days. Non-infected control groups received orally 100 mI_ of peptone trypsin broth alone. In addition, infected and non-treated control groups were administered 100 mI_ of vehicle. No toxicity signs associated to the vehicle or the compounds treatment were noticed: i.e. no weight variations (Figure 1 ) and no appetite alterations. At 8 days post-treatment (day 24), mice were sacrificed by CO2 inhalation, stomachs were isolated and Hp gastric colonization determined as previously described (Ferrero, R. L. et al. Immune Responses of Specific-Pathogen-Free Mice to Chronic Helicobacter pylori ( Strain SS1 ) Infection. Infect. Immun. 1998, 66 (4), 1349- 1355). Plates were incubated for 5 days at 37 °C under a microaerophilic atmosphere created by the Anoxomat AN2CTS system (MART Microbiology B.V.). The antibacterial activity of the compounds was evaluated by counting the number of viable bacterial colonies expressed as CFU per gram of gastric tissue.

Statistical analysis

Statistical analyses of efficacy assays in mice were performed with GraphPad Prism 7 Software (GraphPad Software, Inc. CA, USA). Mann-Whitney U and Unpaired t tests were used to assess differences in mouse gastric colonization between Hp-infected vehicle-treated and compound-treated groups. Differences were considered statistically significant for p£0.05.

Intrinsic clearance determination of compounds in liver microsomes in male CD- 1 mice

The intrinsic clearance of compounds was studied in liver microsomes, which are vesicles of the hepatocyte endoplasmic reticulum that contain membrane phase I enzymes namely CYPs, flavine-containing monooxygenases, esterases, amidases and epoxide hydrolases, and also the phase II enzymes such as UDP- glucuronyltransferases. They are prepared by differential centrifugation. Liver microsomes are the model of choice (together with human hepatocytes) for drug metabolism studies.

The standard operating procedure is described below:

Assay compound stocks, NADPH and phosphate buffer (pH 7.4) are added to incubation tubes and pre-warmed at 37 °C for 3 minutes. Separately, microsomes are also pre-warmed at 37 °C for 3 minutes. Microsomes are then added to the incubation tubes and mixed well. Samples are taken at pre-determined time points (2, 5, 15, 30, 45 and 60 minutes). At each time point, a sample is removed from the incubation and quenched 1 :4 with ice-cold methanol containing internal standard. Incubation tubes are orbitally shaken at 37 °C throughout the experiment.

Standard final incubation conditions are 1 mM compound in buffer containing 0.5 mg/mL microsomal protein, 0.9% (v/v) acetonitrile and 0.1 % (v/v) DMSO.

Quenched samples are mixed thoroughly and protein precipitated at -20 °C for a minimum of 12 hours. Samples are then centrifuged at 4 °C. Supernatants are transferred to a fresh 96 well plate for analysis.

Positive control markers are included and they consist in: Diclofenac (metabolised by CYP2C9 and CYP3A4) and Diltiazem (Mainly metabolised by CYP3A4).

Analysis was carried out using a Thermo Scientific Q-Exactive Focus hybrid quadrupole-orbitrap mass spectrometer with Vanquish Flex UHPLC system.

RESULTS

Improving therapeutic indexes by modifying the oxidation state of chemical groups present in the flavodoxin inhibitor molecules

Several compound within the formula (I) of the present invention been synthesized and tested. All of them are less cytotoxic toward HeLa cells than their corresponding parent compounds (Table 1 and Table T). In fact, most derivatives are not cytotoxic at concentrations as high as 1 mM (Figure 2). The in vitro anti -Hp activities of some compounds have been evaluated using three or four Hp reference strains (Table 1 and Table T). The specific impact on Tls brought about by modifying the oxidation state of the nitro, sulphur groups of the inhibitors has been evaluated from pair wise comparisons of the compounds analysed.

Table 1. In vitro activity of flavodoxin inhibitors against H. pylori reference strains³

^aCompound already disclosed in the prior art

bThe Minimal Cytotoxic Concentration (MCC₅o) tested in HeLa cells

cMinimal Inhibitory Concentration (MIC) tested in three different H. pylori reference strains (NCTC 1 1637 (Mnz resistant), and 26695 and Sydney Strain 1 (Mnz susceptible))

dTherapeutic Indexes (Tl) have been calculated as MCC₅₀/MIC

Table 1’. In vitro activity of flavodoxin inhibitors against H. pylori reference strains³

The compound IV contains a nitro group, which, in this case, is attached to a benzoxadiazole ring. In this lead, reduction of the nitro group to amine lowers toxicity so much (>100-fold) that the concomitant moderate decrease in activity observed (around 10-fold) combines to a 10-fold higher Tl in compound 120, also disclosed in the prior art. The toxicity of compound IV is also greatly reduced (25-fold) by oxidation of the sulphur atom to sulfoxide, which causes a less pronounced decrease in activity and therefore also raises the Tl. Oxidation of the sulphur atom to sulfoxide in 120, compound SS16, similarly causes a smaller decrease in activity, while a possible further decrease in toxicity cannot be determined because 120 is already non-toxic at the higher concentration tested (1 mM). Further oxidation of the sulphur atom in 120 to sulfone, compound SS17, lowers the activity. However, chlorination of 120 increases the antimicrobial activity 2-4 fold and does not introduce toxicity, leading to the high Tls of compound SS15 (from >85 to >170, depending on the Hp reference strain tested).

Efficacy of compounds against antimicrobial-resistant Hp-strains

In addition, several nitro-reduced derivatives of compound IV (i.e 120, SS15, SS16) display, for the two Mnz(metronidazole)-sensitive strains tested (strains SS1 and 26695), Tls close to that of Mnz, or even higher. To explore the potential usefulness of these flavodoxin inhibitors towards other Hp drug-resistant strains, we have determined the activity of 6 representative commonly used antimicrobials on six Hp strains obtained from patients who have relapsed or are refractory to, at least, two conventional therapies (Gisbert, J. P. et al. IV Spanish Consensus Conference on Helicobacter pylori Infection Treatment. Gastroenterol Hepatol 2016, 39 (10), 697- 721 ). Three of these strains (isolates 1 -3) are Mnz-resistant, two are both Mnz- and Cla-resistant (isolates 4 and 6) and one is rifampicin -resistant (isolate 5) (Table 2).

Table 2. Antimicrobial resistance profiles of H. pylori drug-resistant clinical isolates following the EUCAST criteria. (S = sensitive; R = resistant)

Compound Isolate 1 Isolate 2 Isolate 3 Isolate 4 Isolate 5 Isolate 6

Amoxicillin S S S S S S

Clarithromycin S S S R S R

Tetracycline S S S S S S

Levofloxacin S s s s S S

Metronidazole R R R R S R Rifampicin S S S S R S

The analysis of the effect of some flavodoxin inhibitors on these clinical isolates (Table 3 and Table 4) confirms the activity trends previously observed when evaluated against reference strains (Table 1 ). According to EUCAST criteria, SS15 is effective against two Mnz-resistant strains (isolates 1 and 2) and against the two Cla- and Mnz-resistant strains (isolates 4 and 6). In addition, SS15 is also effective against the rifampicin- resistant strain (isolate 5). Only one Mnz-resistant strain (isolate 3) displayed low Tls for SS15 (Table 3).

Table 3. Tl values (MCC50/MIC) of some developed compounds against H. pylori drug-resistant clinical isolates ³

Compound Isolate 1 Isolate 2 Isolate 3 Isolate 4 Isolate 5 Isolate 6 120 144 72 9.0 144 9.0 72

SS15 161 322 10.0 80 40 322

³HeLa cells have been used to determine the Minimal Cytotoxic Concentration (MCC50), as reported in Table 1 . In bold, Tl values indicative of effectivity, according to EUCAST criteria.

Table 4. Doses tested for each compound in the mice efficacy³ experiments

Compounds

Dose (mg/100g bw)

0.1 1 5 10 20

^a Measured as the percentage of mice where Hp has been completely eradicated. NS: Non significant when comparing the Hp gastric colonisation in Hp infected mice treated vs non-treated.

Efficacy of compounds against Hp gastric colonization in mice

The anti-Hp activity of these novel compounds has also been investigated in the mouse model of Hp infection. First, the toxicity of the known inhibitors (IV and 120) at doses of 10, 20 and 40 mg/100 g bw has been evaluated. Daily administration of compound 120 for 8 days had no deleterious effects in stomach, liver, heart, lung and kidney at any dose, as determined by histopathological examination (Figure 3) and analysis of their biochemical parameters (data not shown). In Figure 3, hematoxylin-eosin staining of stomach slices of untreated mice (A; 40x) and treated mice (B-G) are exhibited. Images B-D correspond to mice treated with compound IV-a at 10 mg/100 g (B; 100x), 20 mg/100 g (C; 100x) and 40 mg/100 g (D; 100x), images E-G to mice treated with compound IV at 10 mg/100 g (E; 40x), 20 mg/100 g (F; 40x) and 40 mg/100 g (G; 10Ox). Compound IV did not induce significant alterations at any dose in liver, heart, lung or kidney either, but they did provoke histological alterations in stomach when administered at the highest dose of 40 mg/100 g (compound IV) (Figure 3). The histological alterations found in stomach included hyperplasia, inflammation and necrosis of the epithelium and the gastric mucosa. None of these compounds were associated with increased levels of tested biochemical parameters when compared to animals which did not receive the compounds. The lack of biochemical or histological alterations associated to the administration of high doses of 120 indicates that reduction of the nitro group in compound IV to amine removes the toxicity exerted in the stomach by compound IV when orally administered to mice at high doses. In the subsequent in vivo analysis on anti-Hp activity, the doses of compound IV have been kept below those that produce histological alterations in mice stomachs.

Several experiments have been conducted in mice infected with Hp SS1 to test the anti-Hp activity of the leads (compound IV) and of several derivatives: 120 and SS16. The doses ranged from 0.1 to 20 mg/100 g bw. As expected, in the experiments, all control mice have provided a clean background for interpretation: all mice in the infected non-treated groups have shown efficient stomach Hp colonization (10⁵ to 10⁶ CFU/g gastric tissue) and no Hp colonies have been found in any group corresponding to non-infected mice. The efficacy of the different compounds on Hp gastric colonization has been determined by comparing the CFU/g gastric tissue of each infected, treated-group with the corresponding infected, non-treated control group. Olive oil (90-95%)/DMSO (10-5%), used as vehicle to solubilise compounds, has not influenced the Hp gastric colonization (not shown). All the data gathered in the experiments for the testing of compound IV and 120 and compound of the present invention SS16 are presented in Figure 4. Data of gastric colonization for all compounds and doses tested are presented along with those of the corresponding control groups in Figure 4A, while the percentages of Hp-eradicated mice after treatment are shown in Figure 4B.

The parent compound IV and the amine-derivatives 120 and SS16, significantly lowered the colonization rate at most of the doses tested (Figure 4A) and eradicated the infection in some mice (Figure 4B). Compound IV lowered colonization at 0.1 , 1 , 5 and 10 mg/100 g bw doses (p=0.044; 0.041 ; 0.048 and 0.0002, respectively) and eradicated infection in 43 % (3/7) and 17 % (3/18) of mice treated with 0.1 and 10 mg/100 g bw, respectively. Compound 120 also inhibited the gastric colonization at 1 , 5 and 10 mg/100 g bw doses (p=0.0001 ; 0.0018 and 0.0004, respectively) and eradicated the infection in 13 % (4/31 ) of treated mice at 1 mg/100 g bw. Finally, compound SS16, the sulfoxide version of compound 120, decreased colonization at 1 , 10 and 20 mg/100g bw doses (p=0.005, 0.0002 and 0.0122, respectively) and eradicate the infection in 60 % (3/5) of animals at the higher dose and in 5 % (1/20) of mice at 10 mg/100 g.

Intrinsic clearance of compounds

Some compounds of the invention, reference compounds known in the state of the art IV and 120 and classical the antibiotics Diclofenac and Dilitazem have been tested.

The results are showed in Table 5:

Table 5. Half-life and intrisic clearance of compounds in

liver microsomes

Half-life Intrinsic clearance

Compound (ti ₂ ) (CL)

(min) (mI/min/mg)

IV < 3.0 > 460.0

120 < 3.0 > 460.0 93 < 3.0 > 460.0

5515 < 3.0 > 460.0

5516 4.5 307.2

5517 < 3.0 > 460.0 AMP85 < 3.0 > 460.0 AMP167 14.8 93.9

AMP202 1 15.5 12.0

Diclofenac

22.1 62.8

(reference)

Dilitazem

< 3.0 > 460.0

(reference)

Compounds IV, 120, SS15 and SS17 undergo a fast metabolism by liver microsomes, so their half-life is very short. The metabolism of compound AM P085 is peculiar due to it is carried out in two phases. First, it is almost completely metabolised and then it follows a more gradually tendency. Anyway, the half-life of this compound is also very brief. On the other hand, AM P202, AM P167 and SS16 are the most stable compounds (in order of appearance) developed. Accordingly, these three compounds are the most soluble ones in water, and this is not a coincidence; CYP metabolism is positively correlated with log D and, across a series of compounds, the most lipophilic analogue will tend to be more rapidly metabolised than its analogues with lower log D. As oxidative enzymes, CYPs will tend to oxidise lipophilic compounds at sites of significant electron density.

DISCUSSION

To try to further improve their therapeutic indexes, the inventors have synthesized new variants exhibiting different redox forms of the nitro or sulphur groups present in the known inhibitors. Their potential for Hp eradication has been tested in the mice model of infection, and their activity against drug -resistant clinical Hp isolates have been determined. The compounds of the invention exhibit much larger Tls for Hp reference strains than the known compound (Table 1 and T). In fact, some of these novel flavodoxin inhibitors display Tls comparable to those of Mnz and are effective against a variety of Hp clinical isolates resistant to common antimicrobials such as clarithromycin, metronidazole or rifampicin. Importantly, these inhibitors, used as sole agents against Hp infection in the mice model, can significantly lower the gastric Hp load (Figure 4A) and, in some mice, they have eradicated the infection (Figure 4B). Thus, compounds of the present invention constitute a promising new family of antimicrobial chemicals with potential against the increase of Hp drug-resistant strains.

In addition, the solubility of some inhibitors of the present invention has increased, therefore their bioavailability has increased and it is ensured a systemic effect thereof.

Claims

1 . Compound of formula (I):

a pharmaceutically acceptable salt, an isomer or a solvate thereof, wherein

Ri is a NO2 or NH₂ group,

R₂ is H or halogen,

X is -S-, -SO- or -S0₂-

R3 is H or (C1 -C3) alkyl group optionally substituted by at least one group selected from: -OH, -NH_2, halogen or combinations thereof,

n is an integer of 1 to 3,

with the proviso that R₂ is halogen when X is -S- and R₃ is CH₃.

2. Compound according to claim 1 , wherein X is -S- or -SO-.

3. Compound according to any of claims 1 or 2, wherein R₂ is H or Cl.

4. Compound according to claim 1 to 3, wherein R₃ is -CH₃ or -CH₂-CHOH-CH₂OH.

5. Compound according to any of claims 1 to 4, wherein n is 1.

6. Compound according to any of claims 1 to 5, wherein X is -SO- and R₂ is H.

7. Compound according to claim 1 , wherein the compound is selected from:

5-chloro-7-((4-methoxybenzyl)thio)benzo[c][1 ,2,5]oxadiazol-4-amine

7-((4-methoxybenzyl)sulfinyl)benzo[c][1 ,2,5]oxadiazol-4-amine

4-((4-methoxybenzyl)sulfinyl)-7-nitrobenzo[c][1 ,2,5]oxadiazole

7-((4-methoxybenzyl)sulfonyl)benzo[c][1 ,2,5]oxadiazol-4-amine

3-(4-(((7-nitrobenzo[c][1 ,2,5]oxadiazol-4-yl)thio)methyl)phenoxy)propane-1 ,2-diol 3-(4-(((7-aminobenzo[c][1 ,2,5]oxadiazol-4-yl)thio)methyl)phenoxy)propane-1 ,2-diol, and

3-(4-(((7-aminobenzo[c][1 ,2,5]oxadiazol-4-yl)sulfinyl)methyl)phenoxy)propane-1 ,2- diol.

8. Compound according to any of claims 1 to 7 for use as a medicament.

9. Compound according to any of claims 1 to 7 for use in the treatment of infectious diseases caused by Helicobacter.

10. Compound for use according to claim 9 wherein the infectious diseases are caused by Helicobacter pylori.

1 1 . Compound for use according to claim 10 wherein the infectious diseases are caused by NTCT 1 1637, 26695 or SS1 strain of Helicobacter pylori.

12. Compound for use according to any of claims 8-1 1 wherein the compound is administered orally.

13. Compound for use according to any of claims 8-12 wherein the compound is administered to a subject in a dose between 0.1 and 20 mg/100gr bw/day.

14. Compound for use according to any of claims 9-13 wherein the infectious disease is gastritis, gastroduodenal ulcer, lymphoma or gastric cancer.

15. Combined preparation that comprises, at least, a compound according to any of the claims 1 to 7 and other active ingredient, wherein the active ingredient is an antiacid, an antibiotic, a proton pump inhibitor or any combination thereof.

16. Combined preparation according to claim 15 for use in the treatment of infectious diseases caused by Helicobacter.

17. Combined preparation for use according to claim 16 wherein Helicobacter is Helicobacter pylori.

18. Pharmaceutical composition comprising at least a compound according to any of the claims 1 to 7 or at least the combined preparation according to the claim 15.

19. Pharmaceutical composition according to claim 18 for use in the treatment of infectious diseases caused by Helicobacter.

20. Pharmaceutical composition for use according to claim 19 wherein Helicobacter is Helicobacter pylori.