WO2021198284A1 - Use of a thienopyridone derivative in the treatment of cardiovascular diseases - Google Patents

Abstract

The invention relates to the use of a thienopyridone derivative of Formula (I) or its pharmaceutically acceptable salts and/or solvates, or a pharmaceutical composition comprising the same, in the treatment of cardiovascular diseases selected from the group consisting of: heart failure with preserved ejection fraction, heart failure with reduced ejection fraction, chronic heart failure, diabetic cardiomyopathy, AL-amyloid cardiomyopathy, cardiac ischemia, myocardial ischemia, acute heart failure, acute myocardial infarction, angina, and doxorubicin-induced cardiotoxicity.

Description

Use of a thienopyridone derivative in the treatment of cardiovascular diseases

TECHNICAL FIELD

The invention relates to the use of a thienopyridone derivative in the treatment of cardiovascular diseases.

TECHNICAL BACKGROUND

AMP-activated protein kinase (AMPK) is known as a key regulator to control cellular energy homeostasis. Over the last two decades, it has become apparent that AMPK regulates a number of other cellular functions, such as cardiac metabolism and cardiac contractile function as well as promoting anti-contractile, anti-inflammatory and anti-atherogenic actions in blood vessels. AMPK is thus thought to play a role, inter alia:

• in myocardial ischemia-reperfusion injury (inflammatory and oxidative damage to the cardiac muscle caused by reperfusion following a relief of ischemia), an important cardiopathic mechanism,

• in the development of cardiac hypertrophy,

• in anti-atherosclerotic processes in the vasculature,

• in the modulation of cardiac autophagy or apoptosis, which ultimately protects hearts from cellular damage and cell death in the context of diabetic cardiomyopathy, or other forms of heart failure and cardiomyopathy,

• in the reduction in arterial pressure, an important risk factor for cardiovascular diseases,

• in improving the energy supply in the failing heart by promoting ATP production and regulating several important physiological processes, delaying myocardial fibrosis, and reducing heart damage, to restore heart function.

A number of AMPK activators have thus been studied fortheir potential cardiovascular effects, including indirect activators such as metformin⁽¹⁾⁽⁵⁾, and resveratrol⁽², a plant-derived compound¹. In recent years, direct AMPK activators have been identified and tested in preclinical models. Among them, mention can be made of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) ^(1,(3,(4). More recently, studies have been conducted with A-799662⁽¹⁾ from Abbott, which is a thienopyridone with the following structure: However, A-799662 can only be administered by injection due to poor oral bioavailability, which represents a strong limitation that may have precluded its development. Besides, not all direct AMPK activators appear to be beneficial in the treatment of cardiovascular diseases. For instance, MK-8722 from Merck was shown to lead to cardiac hypertrophy*⁶¹.

Therefore, there remains the need for alternative compounds that could be administered orally in the treatment of cardiovascular diseases at clinically relevant doses and/or with reduced side effects.

The inventors have now shown that specific thienopyridone derivatives could satisfy this need. These compounds are direct AMPK activators that are encompassed within the generic formula disclosed in WO 2014/001554 but it has never been suggested so far to use them in the treatment of cardiovascular diseases. In addition, they have proven to be display a higher potency on AMPK activation and a highest oral bioavailability than other compounds disclosed in WO 2014/001554.

SUMMARY OF THE INVENTION

This invention relates to a thienopyridone derivative of Formula (I):

(I) or its pharmaceutically acceptable salts and/or solvates, or a pharmaceutical composition comprising the same, for use in the treatment of cardiovascular diseases selected from the group consisting of: heart failure with preserved ejection fraction, heart failure with reduced ejection fraction, chronic heart failure, diabetic cardiomyopathy, AL-amyloid cardiomyopathy, cardiac ischemia, myocardial ischemia, acute heart failure, acute myocardial infarction, angina, and doxorubicin-induced cardiotoxicity.

The present invention also relates to a method for the treatment of cardiovascular diseases selected from the group consisting of: heart failure with preserved ejection fraction, heart failure with reduced ejection fraction, chronic heart failure, diabetic cardiomyopathy, AL- amyloid cardiomyopathy, cardiac ischemia, myocardial ischemia, acute heart failure, acute myocardial infarction, angina, and doxorubicin-induced cardiotoxicity, comprising administering to a subject in need thereof an effective amount of a thienopyridone derivative as described above, or a pharmaceutical composition comprising an effective amount of a thienopyridone derivative as described above and a pharmaceutically acceptable support.

The present invention also relates to the use of a thienopyridone derivative as described above, or a pharmaceutical composition comprising the same, for the manufacture of a medicament for the treatment of cardiovascular diseases selected from the group consisting of: heart failure with preserved ejection fraction, heart failure with reduced ejection fraction, chronic heart failure, diabetic cardiomyopathy, AL-amyloid cardiomyopathy, cardiac ischemia, myocardial ischemia, acute heart failure, acute myocardial infarction, angina, and doxorubicin-induced cardiotoxicity.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows left ventricle (LV) end-diastolic and LV end-systolic diameters (EDD and ESD respectively), LV fractional shortening (FS), stroke volume (SV) and cardiac output (CO) determined by echocardiography, in ZSF-1 rats treated with PXL770 at 150 mg/kg twice a day for 8 days or 90 days, compared to untreated ZSF-1 rats and Lean rats. Figure 2 shows LV end-systolic pressure (ESP), LV end-systolic pressure-volume relation (ESPVR), LV end-diastolic pressure (EDP), LV Tau (index of LV relaxation) and LV end-diastolic pressure-volume relation (EDPVR) in ZSF-1 rats treated with PXL770 at 150 mg/kg twice a day for 90 days, compared to untreated ZSF-1 rats and Lean rats. Figure 3 shows the increase in myocardial tissue perfusion for ZSF-1 rats treated with PXL770 at 150 mg/kg twice a day for 90 days, compared to untreated ZSF-1 rats and Lean rats.

Figure 4 shows the exercise capacity of ZSF-1 rats treated with PXL770 at 150 mg/kg twice a day for 8 days or 90 days, compared to untreated ZSF-1 rats and Lean rats.

Figure 5 shows stroke volume, cardiac output and heart rate, measured by echocardiography in chronic heart failure rats treated with PXL770 at the dose of 150 mg/kg bid by oral gavage for 10 days.

Figure 6 shows myocardial tissue perfusion, measured by MRI, in chronic heart failure rats treated with PXL770 at the dose of 150 mg/kg bid by oral gavage for 5 days. DETAILED DESCRIPTION OF THE INVENTION

This invention pertains to specific uses of thienopyridone derivatives of Formula (I):

and their pharmaceutically acceptable salts and/or solvates.

The compound of formula (I) and a preparation process thereof have been disclosed in patent application WO 2014/001554.

Alternatively, said compound of formula (I) may be obtained by an improved process comprising the steps of: (a) reacting 6-acetyl-5-hydroxytetralin with an electrophilic benzyl source, preferably benzyl bromide, in the presence of a base; (b) reacting the compound obtained in step (a) with ethyl cyanoacetate in the presence of hexamethyldisilazane and acetic acid;

(c) reacting the compound obtained in step (b) with sulfur in the presence of a base;

(d) optionally forming a salt of the compound obtained in step (c), preferably a hydrochloride salt;

(e) reacting the compound obtained in step (c) or (d) with an electrophilic chlorine source, preferably N-chlorosuccinimide;

(f) reacting the compound obtained in step (e) with phenylacetyl chloride;

(g) reacting the compound obtained in step (f) with a base;

(h) reacting the compound obtained in step (g) with boron tribromide or trichloride, preferably boron trichloride; and

(i) optionally recovering the compound obtained in step (h).

Typically, step (B) can comprise a substep (bl) of heating the mixture obtained in step (A), preferably at a temperature close to reflux of the mixture, followed by a substep (b2) of cooling the resulting mixture, for instance at a temperature comprised between -15 °C and 35 °C. The expression "close to reflux of the mixture" refers typically to a temperature comprised between 90% and 100 % of the boiling point of the solvent system in step (A) (for instance, water/isopropanol or water/n-butyl acetate).

A distillation step, preferably under reduced pressure, can be carried out between the heating substep and substep (b2).

Step (B) allows a crystalline precipitate to form, which formation may be favored or triggered by adding seeds to steps (b2).

In a preferred embodiment, said precipitate is recovered by filtration in step (C). It may then be washed successively with one or more solvents, preferably water, n-butyl acetate and/or tert-butyl methyl ether.

Examples of pharmaceutically acceptable salts of the compound of formula (I) can be obtained by reacting the compound of formula (I) with various organic and inorganic bases by procedures usually known in the art to give the corresponding base-addition salt. Such bases are, for example, alkali metal hydroxides, including potassium hydroxide, sodium hydroxide and lithium hydroxide; alkali metal carbonates, including potassium carbonate and sodium carbonate; alkaline earth metal hydroxides, such as barium hydroxide and calcium hydroxide; alkaline earth metal carbonates; alkali metal alkoxides, for example potassium ethoxide and sodium propoxide; and various organic bases, such as piperidine, diethanolamine and N-methylglutamine. The aluminium salts of the compounds of formula (1) are likewise included.

The salts of the compound of formula (I) thus include aluminium, ammonium, calcium, copper, iron(lll), iron(ll), lithium, magnesium, manganese(lll), manganese(ll), potassium, sodium and zinc salts, but this is not intended to represent a restriction. Of the above-mentioned salts, preference is given to the mono-, di- and tri- sodium or potassium salts and most preferably to the potassium salts.

Any of the pharmaceutically acceptable salts of the compound of formula (I), or this compound itself, may be used in this invention in the form of one of its solvates. "Solvates" of the compounds are taken in the present invention to mean adductions of inert solvent molecules onto the compounds which form owing to their mutual attractive force. The nature of the solvate thus depends on the solvent used during the reaction of the base with the compound of formula (I). Examples of solvates include alcohol solvates, for instance methanol or ethanol solvates, and hydrates, including mono-, di-, tri- or tetra hydrates, but this is not intended to represent a restriction.

In a most preferred embodiment of this invention, the compound used in this invention is the monohydrate potassium salt of the compound of formula (I), corresponding to the following structure of formula (la):

(la)

This compound may be prepared according to a process comprising the steps of:

(A) reacting the compound of formula (I) with potassium carbonate in a solution comprising water and a solvent selected from n-butyl acetate and isopropanol:

(B) forming a precipitate; and

(C) recovering the precipitate obtained in step (B), preferably by filtration.

This process allows obtaining the monohydrate potassium salt of 2-chloro-4-hydroxy-3-(5- hydroxytetralin-6-yl)-5-phenyl-7H-thieno[2,3-b]pyridin-6-one, also known as PXL770, which is obtained in the form of a solid, such as a powder, having the following XRPD (X-Ray Powder Diffraction) peaks, as measured by means of a diffractometer, using Cu K(alpha) radiation:

In the following description, "the thienopyridone derivative" is intended to mean both the base compound of formula (I), its solvates, its salts and the solvates of its salts, including PXL770.

In the present invention, the thienopyridone derivative or a pharmaceutical composition comprising the same are used in the treatment of specific cardiovascular diseases, namely heart failure with preserved ejection fraction, heart failure with reduced ejection fraction, chronic heart failure, diabetic cardiomyopathy, AL-amyloid cardiomyopathy, cardiac ischemia, myocardial ischemia, acute heart failure, acute myocardial infarction, angina, or doxorubicin- induced cardiotoxicity.

The pharmaceutical composition used according to the invention may be prepared by any conventional method. The thienopyridone derivative can be converted into a suitable dosage form together with at least one solid, liquid and/or semi-liquid excipient or adjuvant and, if desired, in combination with one or more further active ingredients.

The term "pharmaceutically acceptable support" refers to carrier, adjuvant, or excipient acceptable to the subject from a pharmacological/toxicological point of view and to the manufacturing pharmaceutical chemist from a physical/chemical point of view regarding to composition, formulation, stability, subject acceptance and bioavailability. The term "carrier", "adjuvant", or "excipient" refers to any substance, not itself a therapeutic agent, that is added to a pharmaceutical composition to be used as a carrier, adjuvant, and/or diluent for the delivery of a therapeutic agent to a subject in order to improve its handling or storage properties or to enable or facilitate formation of a dosage unit of the composition into a discrete article. The pharmaceutical compositions of the invention, either individually or in combination, can comprise one or several agents or vehicles chosen among dispersants, solubilisers, stabilisers, preservatives, etc.

The terms "treatment", "treating" and "treat" refer to therapy, prevention and prophylaxis of cardiovascular diseases or at least one of its symptoms. This also means an improvement, prevention of at least one measurable physical parameter associated with the disease being treated, which is discernible or not in the subject. The term "treatment" or "treating" further refers to inhibiting or slowing the progression of the disease, physically, stabilization of a discernible symptom, physiologically, for example, stabilization of a physical parameter, or both. The term "treatment" or "treating" also refers to delaying the onset of the disease. In some particular embodiments, the compound of the invention is administered as a preventive measure. In this context, "prevention" or "preventing" refers to a reduction in the risk of developing at least one of the symptoms related to the disease.

The term "treating " can thus include increasing cardiac output, reducing diastolic dysfunction, increasing ventricular perfusion and reducing ventricular hypertrophy with the thienopyridone derivative or a pharmaceutical composition comprising the same.

The treatment involves the administration of the thienopyridone derivative or a pharmaceutical composition of the invention to a subject having declared cardiovascular diseases to cure, delay, or slow down the progress, thus improving the condition of patients.

Within the context of the invention, the term "subject" means a mammal and more particularly a human. The subjects to be treated according to the invention can be appropriately selected on the basis of several criteria associated with the disease.

Pharmaceutical compositions can be administered in the form of dosage units which comprise a predetermined effective amount of active ingredient per dosage unit.

Pharmaceutical compositions can be adapted for administration via any desired suitable method, for example by oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) methods. Such compositions can be prepared using all processes known in the pharmaceutical art by, for example, combining the active ingredient with the excipient(s) or adjuvant(s). Preferably, the pharmaceutical composition according to the invention is adapted for oral administration.

Pharmaceutical compositions adapted for oral administration can be administered as separate units, such as, for example, capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or foam foods; or emulsions, such as oil-in- water liquid emulsions or water-in-oil liquid emulsions. Thus, for example, in the case of oral administration in the form of a tablet or capsule, the active ingredient component can be combined with an oral, non-toxic and pharmaceutically acceptable inert excipient. Powders are prepared by comminuting the compound to a suitable fine size and mixing it with a pharmaceutical excipient comminuted in a similar manner, such as, for example, an edible carbohydrate, such as, for example, starch or mannitol. A flavour, preservative, dispersant and dye may likewise be present. Capsules may be produced by preparing a powder mixture as described above and filling shaped gelatine shells therewith. Glidants and lubricants, such as, for example, highly disperse silicic acid, talc, magnesium stearate, calcium stearate or polyethylene glycol in solid form, can be added to the powder mixture before the filling operation. A disintegrant or solubiliser, such as, for example, agar-agar, calcium carbonate or sodium carbonate, may likewise be added in order to improve the availability of the medicament after the capsule has been taken.

In addition, if desired or necessary, suitable binders, lubricants and disintegrants as well as dyes can likewise be incorporated into the mixture. Suitable binders include starch, gelatine, natural sugars, such as, for example, glucose or beta-lactose, sweeteners made from maize, natural and synthetic rubber, such as, for example, acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. The lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. The disintegrants include, without being restricted thereto, starch, methylcellulose, agar, bentonite, xanthan gum and the like. The tablets are formulated by, for example, preparing a powder mixture, granulating or dry pressing the mixture, adding a lubricant and a disintegrant and pressing the entire mixture to give tablets. A powder mixture is prepared by mixing the compound comminuted in a suitable manner with a diluent or a base, as described above, and optionally with a binder, such as, for example, carboxymethylcellulose, an alginate, gelatine or polyvinylpyrrolidone, a dissolution retardant, such as, for example, paraffin, an absorption accelerator, such as, for example, a quaternary salt, and/or an absorbent, such as, for example, bentonite, kaolin or dicalcium phosphate. The powder mixture can be granulated by wetting it with a binder, such as, for example, syrup, starch paste, acadia mucilage or solutions of cellulose or polymer materials and pressing it through a sieve. As an alternative to granulation, the powder mixture can be run through a tableting machine, giving lumps of non-uniform shape which are broken up to form granules. The granules can be lubricated by addition of stearic acid, a stearate salt, talc or mineral oil in order to prevent sticking to the tablet casting moulds. The lubricated mixture is then pressed to give tablets. The compound according to the invention can also be combined with a free-flowing inert excipient and then pressed directly to give tablets without carrying out the granulation or dry-pressing steps. A transparent or opaque protective layer consisting of a shellac sealing layer, a layer of sugar or polymer material and a gloss layer of wax may be present. Dyes can be added to these coatings in order to be able to differentiate between different dosage units.

Pharmaceutical compositions adapted for oral administration can also be formulated by spray drying of a solid or liquid dispersion.

Oral liquids, such as, for example, solution, syrups and elixirs, can be prepared in the form of dosage units so that a given quantity comprises a prespecified amount of the compound. Syrups can be prepared by dissolving the compound in an aqueous solution with a suitable flavour, while elixirs are prepared using a non-toxic alcoholic vehicle. Suspensions can be formulated by dispersion of the compound in a non-toxic vehicle. Solubilisers and emulsifiers, such as, for example, ethoxylated isostearyl alcohols and polyoxyethylene sorbitol ethers, preservatives, flavour additives, such as, for example, peppermint oil or natural sweeteners or saccharin, or other artificial sweeteners and the like, can likewise be added.

The dosage unit formulations for oral administration can, if desired, be encapsulated in microcapsules. The formulation can also be prepared in such a way that the release is extended or retarded, such as, for example, by coating or embedding of particulate material in polymers, wax and the like.

The thienopyridone derivative used according to the invention can also be administered in the form of liposome delivery systems, such as, for example, small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from various phospholipids, such as, for example, cholesterol, stearylamine or phosphatidylcholines. By "effective amount" it is meant the quantity of the compound as defined above which prevents, removes or reduces the deleterious effects of the treated disease in humans. It is understood that the administered dose may be adapted by those skilled in the art according to the patient, the pathology, the mode of administration, etc. For instance, the thienopyridone derivative may be administered once or twice a day at a daily dose of 0.5mg to BOO mg for a human patient, preferably from 20 mg to 1000 mg, more preferably from 60 mg to 500 mg. It can be administered 4, 5, 6 or 7 days a week as a long-life medication. In a particular embodiment of this invention, the thienopyridone derivative is administered as dosage units which comprise from 0.5 mg to 1500 mg, preferably from 20 mg to 1000 mg, more preferably from 60 mg to 500 mg of the thienopyridone derivative.

The invention will also be described in further detail in the following examples, which are not intended to limit the scope of this invention, as defined by the attached claims.

EXAMPLES

Example 1: Synthesis of PXL770

Analytical methods

XRPD

X-Ray Powder Diffraction (XRPD) analyses were performed using a Panalytical Xpert Pro diffractometer equipped with a Cu (K alpha radiation) X-ray tube and a Pixcel detector system. The samples were analysed in transmission mode and held between low density polyethylene films. XRPD patterns were sorted, manipulated and indexed using HighScore Plus 2.2c software.

TG/DTA

Thermogravimetric (TG) analyses were carried out on a Perkin Elmer Diamond Thermogravimetric/Differential Temperature Analyser (TG/DTA). The calibration standards were indium and tin. Samples were placed in an aluminium sample pan, inserted into the TG furnace and accurately weighed. The samples were heated from 30-300°C in a stream of nitrogen at a rate of 10°C/minute. The temperature of the furnace was equilibrated at 30°C prior to the analysis of the samples. la) Synthesis of l-(5-benzyloxytetralin-6-yl)ethanone (1)

6-Acetyl-5-hydroxytetralin (100 g, 1 eq.) was dissolved in acetonitrile (BOO imL). After addition of K2CO3 (1.1 eq.) and benzyl bromide (1.05 eq.), the suspension was heated (76°C). After 48 hours, benzyl bromide (0.1 eq) was added. After overall 74 hours, the solid was filtered off and washed with acetonitrile (200 mL), and the combined filtrates were evaporated. Compound 1 was obtained as a syrup: m = 148.6 g, quantitative yield, 96.6% a/a purity. lb) Synthesis of ethyl 2-amino-4-(5-benzyloxytetralin-6-yl)thiophene-3-carboxylate (2)

Acetic acid (70 mL) was heated to T = 65°C. HMDS (1.5 eq.) was added over 10 min. Afterwards, a solution of compound 1 (69.5 g, 1 eq.) and ethyl cyanoacetate (1.5 eq.) in acetic acid (140 mL) was added. The resulting mixture was stirred at T = 65°C for 24 h.

After cooling to room temperature, aqueous NaOH (1 M, 140 mL) and TBME (210 mL) were added. The layers were separated. The organic layer was washed with aqueous NaOH (1 M, 4 x 140 mL) until the pH of the aqueous phase was basic (pH = 13). The organic layer was washed with aqueous HCI (1M, 140 mL) and H2O (2 x 140 mL). EtOH (240 mL), NaHCC>3 (1.3 eq.) and sulfur (1.0 atom eq.) were added. After heating to reflux for 180 min, the reaction mixture was concentrated to 210 mL and co-evaporated with TBME (3 x 140 mL). After cooling to room temperature, the suspension was filtered and the solid was washed with TBME (70 mL). The combined filtrates were concentrated to 210 mL and HCI in dioxane (1.1 eq.) was added dropwise at room temperature. After seeding, precipitation was observed. Heptane (350 mL) was added dropwise at room temperature. After stirring for 14 h, the suspension was filtered. After washing with heptane (3 x 70 mL) and drying, compound 2 was recovered as a solid m = 83.2 g, 71% yield, 93.7% a/a purity. lc) Synthesis of ethyl 4-(5-benzyloxytetralin-6-yl)-5-chloro-2-[(2-phenylacetyl)aminol thiophene-3-carboxylate (3)

solution was cooled with ice/water. Under stirring, N-chlorosuccinimide (1.05 eq.) was added. The mixture became dark over a few minutes. After 1 h, phenylacetyl chloride (1.25 eq.) was added.

After 1 hour at 0 °C and 2 hours at room temperature, the mixture was evaporated down to ca. 35 mL and EtOH (2 x 70 mL) was added, and evaporated down again. The mixture was diluted with EtOH (35 mL) and cooled with ice/water. The product precipitated. The solid was filtrated and washed with cold EtOH (3 x 18 mL).

Compound 3 was obtained as a solid: m = 20.99 g, 94.2 % yield, 99.3 % a/a purity.

Id) Synthesis of 3-(5-benzyloxytetralin-6-yl)-2-chloro-4-hvdroxy-5-phenyl-7H-thieno[2,3- blpyridin-6-one (4) Compound 3 (19.88 g, 1 eq.) was solubilized in methyltetrahydrofuran (120 mL), and the reaction mixture was cooled to a temperature between -16°C and -10 °C (NaCI/lce). Potassium tert-butoxide (5 eq.) was added in four portions. Then, the reaction mixture was warmed up to room temperature, and stirred for 65 min at room temperature. A dropwise addition of 2N HCI (5 eq.) was carried out at T = 0-5°C (water/ice) and the resulting mixture was stirred vigorously. The organic phase was washed with NaCI_(aq) (11%, 1 x 50 mL) and water (2 x 50 mL). The organic phase was concentrated to ~50% solution. Methyltetrahydrofuran (80 mL) was added, and the resulting solution was concentrated to ~50% solution. TBME (100 mL) was added, and the resulting solution was concentrated to ~50% solution (this step was repeated 3 times). Then, TBME (25 mL), seeds of compound 4 and n-Heptane (20 mL) were added and the resulting solution was stirred at room temperature overnight. The mixture was concentrated to ca. 50 mL, filtrated, rinsed with mother liquor and washed with n-Heptane (2 x 40 mL) and dried. Compound 4 was obtained as a granular solid. Yield 88 %, 99.5 % a/a purity. le) Synthesis of 2-chloro-4-hvdroxy-3-(5-hvdroxytetralin-6-yl)-5-phenyl-7H-thieno[2,3- blpyridin-6-one (I)

Compound 4 (15 g, l eq.) was dissolved in 75 mLof dichloromethane and was cooled to T = -10°C/-15°C (with ice/NaCI). BCU (1.5 eq., solution: 1 mol/L in dichloromethane) was added dropwise and the resulting mixture was stirred at room temperature for 15 hours. The resulting mixture was cooled with ice/water, and water (75 mL) was added. The resulting mixture was stirred vigorously and the organic phase was extracted with water/MeOH (9:1 v/v, 5 x 45 mL.). The organic phase was concentrated, a solvent swap was carried out with toluene (3 x 90 mL) and diluted with toluene to reach a final volume of 90 mL of toluene. The resulting mixture was heated to reflux and 15 mL of methanol was added. A brownish solution with few particles was obtained. Seeds were added at T = 40 °C, warmed to T = 52°C and cooled to room temperature. The resulting mixture was stirred overnight, and then was cooled with ice/NaCI (T = -10°C/-15°C) for 100 minutes. The precipitated product was filtrated, washed with toluene/heptane 1:2 v/v (15 mL) and heptane (15 mL) and dried. Crystals of compound (I) were obtained: 87 % yield, 99.0 % a/a purity.

If) Synthesis of the monohydrate potassium salt of 2-chloro-4-hvdroxy-3-(5-hvdroxytetralin-6- yl)-5-phenyl-7H-thieno[2,3-blpyridin-6-one (la)

Compound (I) was suspended in water/isopropanol mix (1/1, 5 parts of each solvents) then 0.50 to 0.55 eq of potassium carbonate was added. The pH was about 12 (pH indicator paper) at the end of the addition of potassium carbonate. After 3 hours of stirring at 50°C, the suspension was thicker and the pH was about 8 (pH indicator paper). The temperature was raised to 80 °C until a solution was obtained (10-15 minutes). A clarification can be done at this point of the process if required. 7 parts of water were added and the reaction mixture was then cooled to 40°C (turbid solution observed). The solvent was distilled under reduce pressure (from 180mbar to 40mbar) at 40°C until 7 parts of solvents remained in the reactor. Crystallization of potassium salt monohydrate may occur here. 4.2 parts of water were added and the mixture was seeded with compound (I) (1 to 2% of seeds). The suspension was then cooled down from 40°C to 5°C in 7 hours (5°C/hour) and kept at 5°C for several hours. The suspension was filtered. The cake was washed twice by 1.42 parts of water. The collected solid was dried at 40°C under vacuum given minimum 80% yield of Compound (la), at required chemical purity (i.e. 98%+). Example 2: Characterization of PXL770 a) X-ray powder diffraction (XRPD) data of compound (la) indicated that it was composed of a crystalline material. The XRPD description of compound (la) is shown in Table 1. Table 1

b) TG/DTA analysis showed an initial weight loss of 1.1% from 30-100°C, followed by larger weight loss of 3% from 117-160°C due to loss of bound water. The second weight loss was accompanied by a large endotherm and the combined weight losses of 4% approximate the theoretical weight loss for a monohydrate (3.75% w/w). The compound decomposed above

240°C.

Example 3: Biological tests

PXL770 effects were assessed in rat models of: - Heart failure with preserved ejection fraction

- Chronic heart failure (heart failure with reduced ejection fraction)

1. ZSF-1 rat model of heart failure with preserved ejection fraction (HFpEF)

ZSF-1 rat is a Leptin-resistant, obese, hypertensive Zucker diabetic fatty/Spontaneously hypertensive heart failure FI hybrid (ZSF-1) rat. This rat develops HFpEF phenotype between week 10 and 20 of natural aging (Hamdani et al.2013). HFpEF development is characterized by progressive left ventricle (LV) diastolic dysfunction, concentric LV remodelling and hypertrophy (Leite et al.2015) and is evident from elevated LV filling pressures with preserved LV systolic function, increased lung weight because of pulmonary congestion and increased LV stiffness (Handami et al.2015).

The goal of the study was to evaluate the effects of PXL770 on the onset and the progression of the cardiovascular dysfunction and the development of HFpEF in ZSF-1 rat.

1.1 Experimental design

Obese ZSF1 rats and Lean ZSF-1 were 12-week-old at the time of treatment initiation.

The following groups were constituted:

Lean rats (untreated; n=6-9)

ZSF-1 rats (untreated; n=5-14)

ZSF-1 treated with PXL770 (n=6-17)

PXL770 was administer by oral gavage at the dose of 150 mg/kg twice a day for 8 days or 90 days.

The PXL770 suspension was prepared at the concentrations used in the study (150 mg/kg) in the vehicle, carboxy methylcellulose 0.5% / tween 80 (98/2).

1.2 Investigated parameters

1.2.1 Cardiac function (Echocardiography)

Transthoracic Doppler echocardiographic studies were performed in randomly selected rats of each group. For this purpose, rats were anesthetized with methohexital (50 mg^-1 kg^-1, IP), the chest shaved and echocardiograms were performed with an echograph (Vivid 7 Ultrasound GE) system equipped with a 8-10 Mhz transducer, as described previously (Mulder et al. 2004). In brief, a two-dimensional short axis view of the left ventricle (LV) was obtained at the level of the papillary muscle, in order to record M-mode tracings. LV end-diastolic and end-systolic diameters were measured according to the American Society of Echocardiography leading-edge method from at least 3 consecutive cardiac cycles (Sahn et al. 1978). Measurements were performed by one observer blinded to prior results and treatment groups.

In addition, LV outflow velocity was measured by pulsed-wave and cardiac output was calculated as aortic velocity-time integral X [n x (LV outflow diameter/2)2] X heart rate.

Measurement: at Ds and Dgo after treatment initiation. 1.2.2 Myocardial perfusion (MRI)

Basal myocardial perfusion was assessed using a Bruker Biospec 4.7 Tesla MRI, as previously described (Merabet et al. 2012). Briefly, after induction of anesthesia, the animals were positioned prone on an actively decoupled and warming pad where hot water circulation was used to maintain physiological temperature. After standard adjustments, scout images were acquired to determine the short-axis plane for the perfusion imaging sequence. After optimization of the RF signal, the perfusion sequence was run allowing determination of myocardial tissue perfusion by Arterial Spin Labeling (ASL) technique, in which, the blood in the arteries upstream from the imaging volume was magnetically "labelled". As a consequence, image intensity changes occur depending on the blood supply to the tissue in the imaged slice. Upon subtraction of an image acquired without spin labelling, the background signal from static spins was removed and the difference image used to quantify perfusion. The difference of the inverse of the apparent T1 images then yields a measure of the regional Cardiac Blood Flow (rCBF) according to rCBF = L (1/Tlsel-l/Tlnonsel), where L is the blood-tissue partition coefficient.

Measurement: Dgo after treatment initiation

1.2.3 Left ventricular hemodynamic assessment (LV catheterization)

LV hemodynamics were assessed as previously described (Fang et al. 2012) in rats of each group at Dgo. For this purpose, rats were anesthetized with methohexital (50 mg/kg, IP) and a 2F miniaturized combined conductance catheter-micromanometer (model SPR-819, Millar Instruments) connected to a pressure-conductance unit (MPCU-200, Millar) were advanced retrogradely via the carotid artery into the LV. Pressure/volume loops were recorded at baseline and during loading by gently occluding the abdominal aorta with a cotton swab.

Data were stored and analyzed using Millar conductance data acquisition and analysis software. The following parameters were measured/calculated:

Left ventricular end-systolic and end-diastolic pressures (indices of left ventricular hemodynamic loading conditions),

Left ventricular end-systolic pressure-volume relation (index of contraction),

Left ventricular end-diastolic pressure-volume relation, time constant of relaxation tau (indices of relaxation). 1.2.4 Exercise testing (equivalent of 6min walk test in patient)

In order to determine whether an eventual improvement of cardiovascular function is associated with an improvement of an amelioration of exercise capacity, the latter was determined in randomly selected animals of each group at Ds and Dgo. In brief, the animals were accustomed with the motorized treadmill (Bioseb, Paris France) at a speed of 2.4 m-min ¹ at a 5° incline. The speed was increased by 2.4 m-min ¹ every 1 min to a maximum speed of

14.4 m-min ¹. A mild electrical shock (frequency current at 3.0 Hz at 1.6 mA with a voltage of a 115 mV) was provided when the animals cannot maintain the set pace. Fatigue was considered to occur when a rat starts to lower its hindquarters and raises its snout, resulting in a significantly altered gait, to the point of not being able to remain on the treadmill. When this degree of fatigue is noted, and the animal has difficulty remaining on the treadmill belt (regardless of the delivery of the electrical shock), the animal was taken off the treadmill, and the run time was recorded to the nearest second (Yamaguchi et al. 1999).

1.2.5 Cardio-Pulmonary morphology assessment

After determination of cardiac hemodynamics, the hearts were taken out, dissected and left ventricular weight was determined, while pulmonary weight was used as an indicator of pulmonary congestion.

1.3 Statistical analysis

All results will be given as mean ± SEM.

In order to evaluate the effect of the pathology, all parameters obtained in untreated obese ZSF-1 and Lean rats were compared by Student's unpaired two-tailed t-test.

In order to evaluate the effects of PXL770, all parameters obtained in 8- and 90-day PXL770- treated ZSF-1 rats were compared with age-matched untreated ZSF-1 rats using Student's unpaired two-tailed t-test.

1.4 Results

1.4.1 LV remodeling and function

LV systolic diameter was increased while LV diastolic diameter was similar in untreated ZSF-1 rats compared to time-matched lean rats, resulting in a significant decrease in LV fractional shortening. PXL770 did never modify LV diastolic diameter, but reduced LV systolic diameter after 8 days and 90 days of treatment (reaching statistical significance at Dgo) resulting in an increased LV fractional shortening (Figure 1).

Stroke volume and cardiac output, determined by echocardiography, were similar at 90 days in both Lean and ZSF-1 animals (Figure 1). PXL770 increased LV stroke volume and cardiac output after 90 days of treatment (Figure 1).

Moreover, LV end-systolic pressure and LV end-systolic pressure-volume relation (LVESPVR), determined by LV catheterization at the end of the study, were similar in both Lean and ZSF- 1 rats, while LV end-diastolic pressure, Tau (index of LV relaxation) and LV end-diastolic pressure-volume relation (LVEDPVR) were increased in ZSF-1 animals compared to Lean animals (Figure 2). These results suggest that LV diastolic function is reduced, but global and systolic LV function are preserved in ZSF-1 rats.

Administration of PXL770 resulted in a significant increase in LV end-systolic pressure-volume relation, while LV end-diastolic pressure, Tau and LV end-diastolic pressure-volume relation were reduced compared with untreated ZSF-1 animals leading to an improvement of diastolic function (Figure 2).

1.4.2 Myocardial tissue perfusion

Myocardial perfusion was decreased in 24 weeks-old untreated ZSF-1 rats compared to age- matched Lean rats. Administration of PXL770 for 90 days significantly prevented the decrease in myocardial tissue perfusion (Figure 3).

1.4.3 Exercise tolerance test

Exercise capacity was reduced in ZSF1 animals when compared with time matched Lean animals. Administration of PXL770 for 8 or 90 days resulted in an increase of running distance, even if this effect did not reach statistical significance (Figure 4).

1.4.4 LV and pulmonary remodeling

LV weight and pulmonary weight tended to be increased in ZSF1 rats compared to Lean rats. PXL770 decreased significantly LV and pulmonary weights compared to untreated ZSF1 rats (Table 2). Table 2

LV and pulmonary weights measured at day 90

Lean ZSRL ZSF1 + PXL770

LV weight (g) 1.218 ± 0.027 1.269 ± 0.023 1.168 ± 0.016 †

Pulmonary weight (g) 1.481 ± 0.066 1.548 ± 0.023 1.452 ± 0.037 † n= 11-17, ^† p<0.05 vs ZSFl 2. Rat model of chronic heart failure (CHF)

Wistar rats were subjected to a complete occlusion of the left anterior descending coronary artery. Within 2 months, they developed chronic heart failure (heart failure with reduced ejection fraction) characterized by decrease in cardiac output, LV dilatation, LV hypertrophy and systolic and diastolic dysfunctions. The goal of this study was to assess the effect of a short-term treatment with PXL770 on cardiac output and LV myocardial perfusion.

2.1 Experimental design

In 11 week-old Wistar rats, myocardial infarctions were induced by complete occlusion of the proximal left anterior descending coronary artery, as previously described (Mulder et al. 2004). The animals were kept for 2 months to allow the development of chronic heart failure. Two months after myocardial infarction, the following groups were constituted:

- Chronic heart failure rats, n=6, treated with PXL770 at the dose of 150 mg/kg bid by oral gavage for 10 days (echocardiography assessment). - Chronic heart failure rats, n=3, treated with PXL770 at the dose of 150 mg/kg bid by oral gavage for 5 days (MRI assessment).

2.2 Investigated parameters

2.2.1 Cardiac function (Echocardiography) See § 1.2.1 for method.

Measurement: before (Do) PXL770 administration as well as 1, 5 and 10 days (Di, Ds, and Dio, respectively) after. 2.2.2 Myocardial perfusion (MRI)

See § 1.2.1 for method.

Measurement: Myocardial perfusion was determined before (Do) and during a 5 days active treatment period, i.e. at Di and Ds. After 5 days, treatment was interrupted and myocardial perfusion was re-examined at Dio.

2.2.3 Statistical Analysis

All results will be given as mean ± SEM. Echocardiographic data were compared by paired t-test.

2.3 RESULTS

2.3.1 Cardiac function

PXL770 increased stroke volume at Dl, D5 and DIO when compared to DO, reaching statistical significance at Dl and D5. After 10 days of PXL770 treatment, stroke volume was increased compared to untreated heart failure rat. This increase in stroke volume resulted in a moderate, statistically non-significant increase in cardiac output, since heart rate was slightly reduced. After 10 day-PXL770 treatment, cardiac output was increased compared to untreated rat (Figure 5) .

2.3.2 LV myocardial tissue perfusion

PXL770 increased myocardial perfusion at Di and Ds when compared to Do. Myocardial tissue perfusion returned to baseline value five days after interruption of treatment, i.e. Dio (Figure 6).

No statistical analysis was made due to the limited number of animals (n=3)

3. Conclusion

Results of PXL770 in these two heart failure rat models showed the potential of PXL770 to improve cardiovascular dysfunctions in heart failure with preserved ejection fraction and to improve cardiac function and left ventricular perfusion in chronic heart failure following myocardial infarction. Based on PXL770 results observed in the two different heart failure rat models, PXL770 showed abilities to increase cardiac output, reduce diastolic dysfunction, increase ventricular perfusion and reduce ventricular hypertrophy. Based on these properties, beneficial effects are expected in various other cardiovascular diseases (Timn and Tyler, 2020; Li et al.2017; Salt and Hardie 2017) namely:

Diabetic cardiomyopathy Cardiac ischemia Acute heart failure Doxorubicin-induced cardiotoxicity.

REFERENCES

Background

⁽¹⁾ A. Ma et al., Journal of Lipid Research 58, 1536-1547 (2017)

⁽²⁾ V.W. Dolinsky et al., Biochimica et Biophysica acta 1832, 1723-1733 (2013)

⁽³⁾ E.S. Buhl et al., Diabetes 51, 2199-2206 (2002)

<⁴> T. Li et al., Cell. Mol. Life Sci. 74, 1413-1429 (2017)

⁽⁵⁾ Y-C Lai et al., DOI: 10.1161/CIRCULATIONAHA.115.018935 (2016)

⁽⁶⁾ R.W. Myers, Science 357, 507-511 (2017)

Experimental part

Hamdani N, Franssen C, Lourengo A, et al. Myocardial titin hypophosphorylation importantly contributes to heart failure with preserved ejection fraction in a rat metabolic risk model. Circ Heart Fail 2013;6:1239-49.

Hamdani N, Franssen C, Lourengo A, Falcao-Pires I, Fontoura D, Leite S, Plettig L, Lopez B, Ottenheijm CA, Becher PM, Gonzalez A, Tschope C, Diez J, Linke WA, Leite-Moreira AF, Paulus WJ. Myocardial titin hypophosphorylation importantly contributes to heart failure with preseved ejection fraction in a rat metabolic risk model. Circ Heart Fail. 2013;6:1239-1249.

Li T, Jiang S, Yang Z, Ma Z, Yi W, Wang D, Yang Y. Targeting the energy guardian AMPK: another avenue for treating cardiomyopathy. Cell. Mol. Life Sci. 2017 ;74:1413-1429 Leite S, Oliveira-Pinto J, Tavares-Silva M, Abdellatif M, Fontoura D, Falcao-Pires I, Leite- Moreira AF, Lourengo AP. Echocardiography and invasive hemodynamics during stress testing for diagnosis of heart failure with preserved ejection fraction: an experimental study. Am J Physiol Heart Circ Physiol. 2015;308:H1556-H1563.

Merabet N, Bellien J, Glevarec E, Nicol L, Lucas D, Remy-Jouet I, Bounoure F, Dreano Y, Wecker D, Thuillez C, Mulder P. Soluble epoxide hydrolase inhibition improves myocardial perfusion and function in experimental heart failure. J Mol Cell Cardiol. 2012;52:660-666 Mulder P, Barbier S, Chagraoui A, Richard V, Henry JP, Lallemand F, Renet S, Lerebours G, Mahlberg-Gaudin F, Thuillez C. Long-term heart rate reduction induced by the selective i(f) current inhibitor ivabradine improves left ventricular function and intrinsic myocardial structure in congestive heart failure. Circulation. 2004;109:1674-1679 Sahn, D.J., et al. (1978) Recommendations regarding quantitation in M-mode echocardiography: results of a survey of echocardiographic measurements. Circulation 58, 1072-1083

Salt I and Hardie D. AMP-Activated Protein Kinase - A Ubiquitous Signalling Pathway with Key Roles in the Cardiovascular System. Circ Res. 201726; 120(11): 1825-1841.

Timn K and Tyler D. The Role of AMPK Activation for Cardioprotection in Doxorubicin-Induced Cardiotoxicity. Cardiovascular Drugs and Therapy; 2020

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Claims

1. A thienopyridone derivative of Formula (I):

or its pharmaceutically acceptable salts and/or solvates, or a pharmaceutical composition comprising the same, for use in the treatment of cardiovascular diseases selected from the group consisting of: heart failure with preserved ejection fraction, heart failure with reduced ejection fraction, chronic heart failure, diabetic cardiomyopathy, AL-amyloid cardiomyopathy, cardiac ischemia, myocardial ischemia, acute heart failure, acute myocardial infarction, angina, and doxorubicin-induced cardiotoxicity.

2. The thienopyridone derivative of claim 1 or a pharmaceutical composition comprising the same for use according to claim 1, wherein said compound is to be administered once or twice a day at a daily dose of 0.5mg to 3000 mg for a human patient, preferably from 20 mg to 1000 mg, more preferably from 60 mg to 500 mg.

3. The thienopyridone derivative of claim 1 or a pharmaceutical composition comprising the same for use according to claim 1 or 2, wherein said compound or pharmaceutical composition is effective for increasing cardiac output, reducing diastolic dysfunction, increasing ventricular perfusion and/or reducing ventricular hypertrophy.