Use of thienopyridone derivatives in treating adrenoleukodystrophy or adrenal myeloneuropathy

Abstract

The present invention relates to use of a thienopyridone derivative or a pharmaceutical composition containing the same in treating a hereditary neurodegenerative disease selected from adrenoleukodystrophy (ALD) and adrenomyeloneuropathy (AMN).

Description

Technical Field

[0001] The present invention relates to the use of thienopyridone derivatives in treating hereditary neurodegenerative diseases selected from adrenoleukodystrophy (ALD) and adrenomyeloneuropathy (AMN). Background Art

[0002] Adrenoleukodystrophy and adrenoleuroneuropathy are neurodegenerative genetic diseases. X-linked adrenoleukodystrophy (ALD) is a genetic disease linked to the X chromosome. Three main phenotypes are observed in affected males. The pediatric cerebral form usually appears between 4 and 8 years of age. It initially resembles attention deficit disorder or hyperactivity disorder. After the initial symptoms, progressive central demyelination occurs, accompanied by impaired cognition, behavior, vision, hearing and motor function, often leading to complete disability within 2 years.

[0003] The second phenotype, adrenomyoneuropathy (AMN), is the adult form of ALD, most commonly manifested in the last two decades as progressive paraparesis, sphincter disorders, sexual dysfunction, and often impaired adrenal cortical function. In addition, spinal cord dysfunction is common in AMN patients, which leads to initial symptoms including difficulty walking or changes in walking style. All symptoms are progressive over decades. Specifically, AMN can be divided into two general clinical forms: brain-involved AMN, in which both the spinal cord and brain are affected, and brain-uninvolved AMN, in which only the spinal cord is affected. The third phenotype is Addison's disease, with primary adrenal insufficiency occurring between the ages of 2 and adulthood, most commonly at the age of seven and a half, without signs of neurological abnormalities.

[0004] X-linked adrenoleukodystrophy (ALD) is caused by mutations in the ABCD1 gene. The ABCD1 gene provides instructions for producing adrenoleukodystrophy protein (ALDP). ALDP is located in the membrane of a cell structure called a peroxisome. Peroxisomes are small sacs within cells that process many types of molecules. ALDP brings a group of fats called very long-chain fatty acids (VLCFA) into the peroxisomes, where they are broken down. The mutations that cause adrenoleukodystrophy prevent about 75% of people with this condition from producing any ALDP. With little or no functional ALDP, VLCFA are not broken down and they accumulate in the body. This causes elevated levels of very long-chain fatty acids (VLCFA) and decreased oxidation of VLCFA in peroxisomes. Lipids containing VLCFA accumulate in all tissues; however, the brain, spinal cord, adrenal cortex, and Leydig cells of the testes have the greatest increase in VLCFA. The accumulation of these fats can be toxic to the adrenal glands and to the fatty insulation (myelin) that surrounds many nerves in the body. In the childhood cerebral form of ALD, not only do cells undergo demyelination, but there is also an inflammatory response, all of which damages the brain. This inflammatory process destroys myelin, inexorably leading to progressive deterioration to a vegetative state or death, usually within five years.

[0005] Adrenomyeloneuropathy (AMN) is the adult-onset form of adrenoleukodystrophy (ALD). Like ALD, AMN is characterized by mutations in the ABCD1 gene, resulting in impaired peroxisome function with VLCFA accumulation and demyelination. Unlike ALD, which is a rapidly progressive and fatal disease in early childhood, AMN is a slowly progressive adult-onset disease that causes dysfunction of the adrenal glands, spinal cord (myelopathy), and peripheral nerves (neuropathy).

[0006] There is no effective treatment for X-ALD; however, hematopoietic stem cell transplantation (HSCT) can slow the progression of ALD in some patients if performed at the first onset of neurologic symptoms. However, HSCT is also associated with significant morbidity and mortality.

[0007] The way these diseases are currently treated is essentially aimed at alleviating the symptoms that the patient has. For example, one of the symptoms that an AMN patient may have is adrenal insufficiency, so the focus would be on treating this adrenal insufficiency with steroid replacement therapy.

[0008] Recently, some authors reported the loss of AMPKα1 in ALD patients and proposed that metformin could be useful in treating these patients by activating AMPK (J. Singh et al., Journal of Neurochemistry, 138, 86-100, 2016). However, metformin is known to cause lactic acidosis as a side effect. In addition, its efficacy was observed at a dose of at least 100 μM in cultured fibroblasts and when administered orally at 100 mg / kg in ABCD2 KO mice.

[0009] Therefore, there remains a need for alternative compounds that would be useful for treating ALD and AMN at lower doses and / or with reduced side effects.

[0010] The inventors have shown that specific thienopyridone derivatives can meet this need. These compounds are broadly disclosed as AMPK activators in WO2014 / 001554, but to date they have not been suggested for use in the treatment of ALD and AMN. They have been shown to be direct activators of various AMPK isoforms containing the β1 subunit, which makes their efficacy in treating these conditions all the more surprising, as direct AMPK activators are known to provide metabolic effects that differ from those obtained with indirect AMPK activators such as metformin.

[0011] More specifically, the present inventors have discovered that these thienopyridone derivatives can restore the healthy phenotype or improve the phenotype of these diseases.

[0012] These thienopyridone derivatives act to reduce the accumulation of very long chain fatty acids (VLCFA). It was also found that when cells were cultured in the presence of thienopyridone derivatives of formula (I), the expression of an alternative protein (ABCD2) related to ALDP (with a sequence very close to ALDP) was induced. Since ALDP is missing in ALD and AMN, the induction of related proteins seems to be a potential mechanism by which thienopyridone derivatives of formula (I) can improve the disease phenotype in this model system. Therefore, these thienopyridone derivatives allow the reduction of fatty acid accumulation through the overexpression of this protein. Summary of the invention

[0013] The present invention relates to the treatment of adrenoleukodystrophy and / or adrenomyeloneuropathy with thienopyridone derivatives.

[0014] More specifically, the present invention relates to a thienopyridone derivative represented by formula (I):

[0015]

[0016] in:

[0017] R1 represents a hydrogen atom or a halogen atom,

[0018] R2 represents an indanyl or tetrahydronaphthyl group which may be substituted or unsubstituted by one or more (e.g. 2, 3, 4, 5, 6 or 7) groups selected from halogen atoms, alkyl groups, hydroxyl groups, alkoxy groups, amino groups, mono- or di-alkylamino groups, carboxyl groups, alkoxycarbonyl groups, mono- or di-alkylaminocarbonyl groups, formamide, cyano groups, alkylsulfonyl groups and trifluoromethyl groups,

[0019] R3 represents an aryl group which may be substituted or unsubstituted by one or more (e.g. 2, 3, 4 or 5) atoms or groups selected from halogen atoms, alkyl groups, hydroxyl groups, alkoxy groups, aralkyloxy groups, amino groups, mono- or di-alkylamino groups, carboxyl groups, alkoxycarbonyl groups, mono- or di-alkylaminocarbonyl groups, formamide, cyano groups, alkylsulfonyl groups and trifluoromethyl groups,

[0020] or a pharmaceutically acceptable salt and / or solvate thereof,

[0021] Or pharmaceutical compositions containing them, and use thereof in the treatment of ALD and AMN.

[0022] The present invention also relates to a method for treating ALD and / or AMN, which comprises administering an effective amount of the thienopyridone derivative as described above to a subject in need thereof, or administering a pharmaceutical composition comprising an effective amount of the thienopyridone derivative as described above and a pharmaceutically acceptable support to a subject in need thereof.

[0023] The present invention also relates to the use of the thienopyridone derivative as described above or a pharmaceutical composition comprising the same in the manufacture of a medicament for treating ALD and / or AMN. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Shown are the levels of hexacosanoids in AMN patient-derived lymphocytes treated with or without PXL770.

[0025] Six conditions were studied: healthy control / untreated AMN / AMN+PXL770(5μM) / AMN+PXL770(10μM) / AMN+PXL770(25μM) / AMN+PXL770(50μM).

[0026] Figure 2 Shown are the ABCD2 expression levels of AMN fibroblasts cultured in the presence or absence of PXL770.

[0027] Figure 3Shown are the levels of hexacosanoids in mixed glial cells of AMN mice treated with or without PXL770.

[0028] Three conditions were studied: wild type / ALD-KO / ALD-KO+PXL770 (25 μM).

[0029] Figure 4 Shown are the ABCD2 expression levels of ALD-KO mouse cerebral cortical mixed glial cells cultured in the presence or absence of PXL770.

[0030] Figure 5 Shown are the levels of hexacosanoids in ALD patient-derived lymphocytes treated with or without PXL770.

[0031] Six conditions were studied: healthy control / untreated AMN / AMN+PXL770(5μM) / AMN+PXL770(10μM) / AMN+PXL770(25μM) / AMN+PXL770(50μM).

[0032] Figure 6 Shown are the ABCD2 expression levels of ALD fibroblasts cultured in the presence or absence of PXL770.

[0033] Figure 7 Shown are the levels of hexadecanoic acid in the cerebral cortex of X-ALD mice treated with PXL770 compared to untreated X-ALD mice and wild-type mice.

[0034] Figure 8 Shown are the levels of hexacosanoids in the plasma of X-ALD mice treated with PXL770 compared to untreated X-ALD mice and wild-type mice.

[0035] Fig. 9 Shown are the levels of hexacosanoids in AMN patient-derived lymphocytes with or without metformin treatment.

[0036] Fig.10 A head-to-head comparison between PXL770 and metformin on hexacosanoid levels in AMN patient-derived fibroblasts is shown.

[0037] Fig.11 Shown are the ABCD2 expression levels of AMN and ALD fibroblasts cultured in the presence or absence of metformin.

[0038] Fig.12Shown are the ABCD2 expression levels of ALD-KO mouse cerebral cortical mixed glial cells cultured in the presence or absence of metformin.

[0039] Fig.13 Shown are the levels of hexacosanoids in the spinal cord of X-ALD mice treated with PXL770 compared to untreated X-ALD mice and wild-type mice.

[0040] Fig.14 Shown are the levels of hexacosanoid in AMN patient-derived fibroblasts when treated with various doses of the compounds of the invention.

[0041] Fig.15 Shown are the levels of hexacosanoids in AMN patient-derived fibroblasts when treated with various doses of a compound of the invention compared to when treated with metformin. DETAILED DESCRIPTION

[0042] definition

[0043] As used herein, the following terms, unless expressly stated otherwise, have the following meanings.

[0044] The term "halogen atom" refers to an atom selected from fluorine, chlorine, bromine and iodine atoms.

[0045] The term "alkyl group" refers to a straight or branched saturated chain of 1 to 5 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl or tert-butyl. Preferably, the alkyl group is a straight or branched saturated chain of 1 to 3 carbon atoms, such as methyl, ethyl, n-propyl or isopropyl groups.

[0046] The term "aryl group" refers to a C6-C18 aromatic group, such as a phenyl or naphthyl group, which may be optionally substituted by one or more atoms or groups selected from halogen atoms, alkyl groups, hydroxyl groups (OH), alkoxy groups, amino groups (NH2), mono- or di-alkylamino groups, carboxyl groups (COOH), alkoxycarbonyl groups, mono- or di-alkylaminocarbonyl groups, formamide (CONH2), cyano groups (CN), alkylsulfonyl groups and trifluoromethyl groups (CF3). More specifically, the aryl group may be substituted by fluorine, chlorine, bromine atoms, hydroxyl groups, methoxy groups, ethoxy groups, amino groups, dimethylamino groups, diethylamino groups, methyl groups, ethyl groups, n-propyl groups, n-butyl groups, isopropyl groups, sec-butyl groups, isobutyl groups, tert-butyl groups, carboxyl groups, methoxycarbonyl groups, ethoxycarbonyl groups, formamide groups, dimethylaminocarbonyl groups, methylaminocarbonyl groups, cyano groups, mesyl groups or trifluoromethyl groups.

[0047] The term "aralkyl group" refers to an alkyl group as defined above whose hydrogen atoms are replaced by aryl groups as defined above. An example of an aralkyl group is a benzyl group.

[0048] The term "alkoxy" group refers to an alkyl group as defined above which is linked to the rest of the molecule via an oxygen atom. Among the alkoxy groups that may be mentioned are the methoxy and ethoxy groups.

[0049] The term "aralkyloxy" group refers to an aralkyl group as defined above which is linked to the rest of the molecule via an oxygen atom. Among the aralkyloxy groups that may be mentioned is the benzyloxy group.

[0050] The term "alkylamino group" means an alkyl group as defined above which is linked to the rest of the molecule via a nitrogen atom. Among the alkylamino groups, mention may be made of the dimethylamino and diethylamino groups.

[0051] The term "alkoxycarbonyl group" refers to an alkoxy group as defined above which is attached to the rest of the molecule via a carbonyl group.

[0052] The term "alkylaminocarbonyl group" refers to an alkylamino group as defined above which is attached to the rest of the molecule via a carbonyl group.

[0053] The term "alkylsulfonyl" refers to an alkyl group as defined above which is linked to the rest of the molecule via a SO2 group. Among the alkylsulfonyl groups, mention may be made of the methylsulfonyl and ethylsulfonyl groups.

[0054] "Solvates" of compounds are understood within the context of the present invention to be adducts of inert solvent molecules onto the compounds which form due to their mutual attractive forces. Solvates are, for example, hydrates or alcoholates.

[0055] The present invention relates to the specific use of thienopyridone derivatives of formula (I) or pharmaceutically acceptable salts and / or solvates thereof:

[0056]

[0057] in:

[0058] R1 represents a hydrogen atom or a halogen atom,

[0059] R2 represents an indanyl or tetrahydronaphthyl group which may be substituted or unsubstituted by one or more (e.g. 2, 3, 4, 5, 6 or 7) groups selected from halogen atoms, alkyl groups, hydroxyl groups, alkoxy groups, amino groups, mono- or di-alkylamino groups, carboxyl groups, alkoxycarbonyl groups, mono- or di-alkylaminocarbonyl groups, formamide, cyano groups, alkylsulfonyl groups and trifluoromethyl groups,

[0060] R3 represents an aryl group which may be substituted or unsubstituted by one or more (e.g. 2, 3, 4 or 5) atoms or groups selected from halogen atoms, alkyl groups, hydroxyl groups, alkoxy groups, aralkyloxy groups, amino groups, mono- or di-alkylamino groups, carboxyl groups, alkoxycarbonyl groups, mono- or di-alkylaminocarbonyl groups, formamide, cyano groups, alkylsulfonyl groups and trifluoromethyl groups.

[0061] In a specific embodiment, at least one of the following conditions is met, preferably all of them are met:

[0062] R1 represents a halogen atom, especially a chlorine atom,

[0063] R2 is unsubstituted or substituted by 1 or 2 substituents containing at least one hydroxyl group,

[0064] R2 is a tetrahydronaphthyl group,

[0065] R3 represents a phenyl group which is unsubstituted or substituted by 1 or 2 substituents,

[0066] The compound of formula (I) is in the form of a salt, preferably a sodium salt or a potassium salt, more preferably a potassium salt,

[0067] The compound of formula (I) is in the form of a solvate, preferably a hydrate, more preferably a monohydrate.

[0068] More preferably, at least one of the following conditions is met, preferably all of them are met:

[0069] R1 represents a halogen atom, especially a chlorine atom,

[0070] R2 is substituted by 1 or 2 substituents containing at least one hydroxyl group,

[0071] R2 is a tetrahydronaphthyl group,

[0072] R3 represents an unsubstituted phenyl group,

[0073] The compound of formula (I) is in the form of a salt, preferably a sodium salt or a potassium salt, more preferably a potassium salt,

[0074] The compound of formula (I) is in the form of a solvate, preferably a hydrate, more preferably a monohydrate.

[0075] In another embodiment, at least one of the following conditions is met, preferably all of them are met:

[0076] R1 represents a halogen atom, especially a chlorine atom,

[0077] R2 is substituted by 1 or 2 substituents containing at least one hydroxyl group,

[0078] R2 is an indanyl group,

[0079] R3 represents a phenyl group which is unsubstituted or substituted by 1 or 2 substituents.

[0080] Examples of compounds of formula (1) are as follows:

[0081] 2-Chloro-4-hydroxy-3-indan-5-yl-5-phenyl-7H-thieno[2,3-b]pyridin-6-one

[0082] 2-Chloro-5-(4-fluorophenyl)-4-hydroxy-3-indan-5-yl-7H-thieno[2,3-b]pyridin-6-one

[0083] 2-Chloro-4-hydroxy-3-indan-5-yl-5-(3-methoxyphenyl)-7H-thieno[2,3-b]pyridin-6-one

[0084] 2-Chloro-4-hydroxy-3-indan-5-yl-5-(4-methoxyphenyl)-7H-thieno[2,3-b]pyridin-6-one

[0085] 3-(2-Chloro-4-hydroxy-3-indan-5-yl-6-oxo-7H-thieno[2,3-b]pyridin-5-yl)benzonitrile

[0086] 2-Chloro-4-hydroxy-3-indan-5-yl-5-(3-methylphenyl)-7H-thieno[2,3-b]pyridin-6-one

[0087] 2-Chloro-5-(4-fluorophenyl)-4-hydroxy-3-(4-hydroxyindan-5-yl)-7H-thieno[2,3-b]pyridin-6-one

[0088] 2-Chloro-5-(3-fluorophenyl)-4-hydroxy-3-(4-hydroxyindan-5-yl)-7H-thieno[2,3-b]pyridin-6-one

[0089] 2-Chloro-4-hydroxy-3-(4-hydroxyindan-5-yl)-5-phenyl-7H-thieno[2,3-b]pyridin-6-one

[0090] 2-Chloro-5-(2-fluorophenyl)-4-hydroxy-3-(4-hydroxyindan-5-yl)-7H-thieno[2,3-b]pyridin-6-one

[0091] 2-Chloro-4-hydroxy-3-(5-hydroxytetrahydronaphthalen-6-yl)-5-phenyl-7H-thieno[2,3-b]pyridin-6-one

[0092] 3-(2-Chloro-4-hydroxy-6-oxo-3-tetrahydronaphthalen-6-yl-7H-thieno[2,3-b]pyridin-5-yl)benzonitrile

[0093] 2-Chloro-3-(5-oxotetralin-6-yl)-5-phenyl-thieno[2,3-b]-4,6-dihydroxypyridine trisodium

[0094] 2-Chloro-4-hydroxy-5-phenyl-3-tetrahydronaphthalen-6-yl-7H-thieno[2,3-b]pyridin-6-one

[0095] 2-Chloro-5-(4-fluorophenyl)-4-hydroxy-3-(5-hydroxytetrahydronaphthalen-6-yl)-7H-thieno[2,3-b]pyridin-6-one

[0096] Disodium 2-chloro-3-(5-oxotetralin-6-yl)-6-oxo-5-phenyl-7H-thieno[2,3-b]-4-hydroxypyridine

[0097] 2-Chloro-4-hydroxy-3-(5-hydroxytetrahydronaphthalen-6-yl)-5-(3-methylphenyl)-7H-thieno[2,3-b]pyridin-6-one

[0098] 2-Chloro-4-hydroxy-3-(5-hydroxytetrahydronaphthalen-6-yl)-5-(4-methylphenyl)-7H-thieno[2,3-b]pyridin-6-one

[0099] 2-Chloro-5-(3-fluorophenyl)-4-hydroxy-3-(5-hydroxytetrahydronaphthalen-6-yl)-7H-thieno[2,3-b]pyridin-6-one

[0100] Sodium 2-chloro-3-(5-hydroxytetrahydronaphthalen-6-yl)-6-oxo-5-phenyl-7H-thieno[2,3-b]-4-hydroxypyridine

[0101] Potassium 2-chloro-3-(5-hydroxytetralin-6-yl)-6-oxo-5-phenyl-7H-thieno[2,3-b]-4-hydroxypyridine.

[0102] The compounds of formula (I) can generally be prepared as disclosed in WO 2014 / 001554.

[0103] Examples of such compounds include:

[0104] PXL770, which is the monohydrated potassium salt of 2-chloro-4-hydroxy-3-(5-hydroxytetralin-6-yl)-5-phenyl-7H-thieno[2,3-b]pyridin-6-one, corresponds to the structure of the following formula (Ia):

[0105]

[0106] 2-Chloro-5-(3-fluorophenyl)-4-hydroxy-3-(4-hydroxyindan-5-yl)-7H-thieno[2,3-b]pyridin-6-one, having formula (Ib):

[0107]

[0108] 2-Chloro-4-hydroxy-3-(4-indan-5-yl)-5-phenyl-7H-thieno[2,3-b]pyridin-6-one, having formula (Ic):

[0109]

[0110] PXL770 can be prepared according to a method comprising the following steps:

[0111] (A) reacting a compound of formula (II) with potassium carbonate in a solution comprising water and a solvent selected from n-butyl acetate and isopropanol:

[0112]

[0113] (B) forming a precipitate; and

[0114] (C) recovering the precipitate obtained in step (B), preferably by filtration.

[0115] The compound of formula (II) and its preparation method have been disclosed in patent application WO 2014 / 001554.

[0116] Alternatively, the compound of formula (II) can be obtained by an improved method comprising the following steps:

[0117] (a) reacting 6-acetyl-5-hydroxytetralin with an electrophilic benzyl source, preferably benzyl bromide, in the presence of a base;

[0118] (b) reacting the compound obtained in step (a) with ethyl cyanoacetate in the presence of hexamethyldisilazane and acetic acid;

[0119] (c) reacting the compound obtained in step (b) with sulfur in the presence of a base;

[0120] (d) optionally forming a salt, preferably a hydrochloride, of the compound obtained in step (c);

[0121] (e) reacting the compound obtained in step (c) or (d) with an electrophilic chlorine source, preferably N-chlorosuccinimide;

[0122] (f) reacting the compound obtained in step (e) with phenylacetyl chloride;

[0123] (g) reacting the compound obtained in step (f) with a base;

[0124] (h) reacting the compound obtained in step (g) with boron tribromide or boron trichloride, preferably boron trichloride; and

[0125] (i) optionally recovering the compound obtained in step (h).

[0126] Typically, step (B) may comprise a sub-step (b1) of heating the mixture obtained in step (A), preferably at a temperature close to the reflux of the mixture, followed by a sub-step (b2) of cooling the resulting mixture, for example at a temperature between -15° C. and 35° C. The expression “close to the reflux of the mixture” typically refers to a temperature between 90% and 100% of the boiling point of the solvent system (e.g., water / isopropanol or water / n-butyl acetate) in step (A).

[0127] A distillation step may be carried out between the heating sub-step and sub-step (b2), preferably under reduced pressure.

[0128] Step (B) allows for the formation of a crystalline precipitate, which formation may be facilitated or triggered by the addition of seeds to step (b2).

[0129] In a preferred embodiment, the 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.

[0130] The compound of formula (Ia) is thus obtained in solid form, for example in powder form, namely PXL770, which has the following XRPD (X-ray powder diffraction) peaks measured by a diffractometer using Cu K (α) radiation:

[0131]

[0132]

[0133] An object of the present invention is a method for treating a disease selected from adrenoleukodystrophy and adrenomyeloneuropathy, which comprises administering to a subject in need thereof an effective amount of a thienopyridone derivative of formula (I) or a pharmaceutical composition comprising an effective amount of a thienopyridone derivative of formula (I) and a pharmaceutically acceptable support.

[0134] The present invention also relates to the use of the thienopyridone derivative of formula (I) or a composition comprising the same in the manufacture of a medicament for treating adrenoleukodystrophy and / or adrenomyeloneuropathy.

[0135] The thienopyridone derivatives of formula (I) appear to treat ALD and / or AMN by reducing the accumulation of VLCFA. In fact, the literature has shown that VLCFA load increases with the severity of the disease, and reducing VLCFA can eliminate the inflammatory response. Therefore, reducing VLCFA overload, especially in the central nervous system, has the potential to stop or reverse the disease progression of ALD and / or AMN.

[0136] The inventors have found that the thienopyridone derivatives of formula (I) significantly reduce the VLCFA levels in fibroblasts and lymphocytes from ALD and AMN patients and in mixed glial cells of mice. The thienopyridone derivatives of formula (I) act on the recovery of ALDP (ABCD1) function by inducing overexpression of ABCD2, also known as ALDRP, which has significant sequence similarity to ALDP. The inventors have found that the thienopyridone derivatives of formula (I) induce overexpression of ABCD2, which compensates for the deficiency of ABCD1, thereby allowing the reduction of VLCFA accumulation.

[0137] The pharmaceutical compositions used according to the invention can be prepared by any conventional method. The thienopyridone derivatives of formula (I) can be converted into a suitable dosage form together with at least one solid, liquid and / or semi-liquid excipient or adjuvant and, if necessary, in combination with one or more other active ingredients.

[0138] The term "pharmaceutically acceptable support" refers to a carrier, adjuvant or excipient that is acceptable to subjects from a pharmacological / toxicological point of view, and to a pharmaceutical chemist from a physical / chemical point of view with respect to composition, formulation, stability, subject acceptance and bioavailability.

[0139] The term "carrier", "adjuvant" or "excipient" refers to any substance added to a pharmaceutical composition as a carrier, adjuvant and / or diluent to deliver a therapeutic agent to a subject, which is not a therapeutic agent itself, but is added to improve the handling or storage properties of the therapeutic agent, or to enable or facilitate the formation of dosage units of the composition into discrete products. The pharmaceutical composition of the present invention, alone or in combination, may contain one or more agents or media selected from dispersants, solubilizers, stabilizers, preservatives, etc.

[0140] The term "treatment" refers to the treatment, prevention and prophylaxis of a condition selected from adrenoleukodystrophy (ALD) and adrenomyeloneuropathy (AMN). The term "treatment" as disclosed herein refers to the prevention of a disease or at least one symptom thereof. This also means improving, preventing at least one measurable physical parameter associated with the disease being treated, which parameter is perceptible or unperceptible in the subject. The term "treatment" also refers to inhibiting or slowing the progression of the disease in the body, stabilizing physiologically perceptible symptoms, such as stabilizing physical parameters, or both. The term "treatment" also refers to delaying the onset of a disease or condition. In some specific embodiments, the compounds of the present invention are administered as a preventive measure. In this context, "prevention" refers to reducing the risk of at least one symptom associated with the disease.

[0141] The term "treatment" may include acting on the accumulation of very long chain fatty acids (VLCFA) with a thienopyridone derivative of formula (I) or a pharmaceutical composition comprising it. More specifically, the thienopyridone derivative of formula (I) reduces VLCFA accumulation, thereby eliminating or reducing inflammatory responses. As used herein, "treatment" also encompasses any treatment for central demyelination, adrenal insufficiency, or adrenal dysfunction. Therefore, the term "treatment" includes the treatment of symptoms associated with ALD and / or AMN.

[0142] The treatment comprises administering the thienopyridone derivative of formula (I) or the pharmaceutical composition of the present invention to a subject suffering from the declared disorder to cure, delay or slow down the progression, thereby improving the patient's condition.

[0143] In the context of the present invention, the term "subject" refers to a mammal, more particularly a human being. The subject to be treated of the present invention can be appropriately selected based on several criteria related to the disease, such as previous drug treatment, relevant pathology, genotype, exposure to risk factors, viral infection, and any other relevant biomarkers that can be evaluated by immunological, biochemical, enzymatic, chemical or nucleic acid detection methods.

[0144] In the case of ALD, the treatment is more particularly suitable for patients between the ages of 2 and 10. Since AMN is the adult form of ALD, the treatment is more particularly suitable for patients between the ages of 20 and 39.

[0145] The pharmaceutical composition may be administered in the form of dosage units, each dosage unit containing a predetermined effective amount of an active ingredient.

[0146] The pharmaceutical composition can be suitable for administration by 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 by, for example, combining the active ingredient with an excipient or adjuvant using all methods known in the pharmaceutical art. Preferably, the pharmaceutical composition of the present invention is suitable for oral administration.

[0147] Pharmaceutical compositions suitable for oral administration can be administered as discrete units, such as 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.

[0148] Thus, for example, in the case of oral administration in tablet or capsule form, the active ingredient components may be combined with an oral non-toxic and pharmaceutically acceptable inert excipient. Powders are prepared by pulverizing the compound to a suitable fine size and mixing it with a similarly pulverized pharmaceutical excipient, such as an edible carbohydrate, such as starch or mannitol. Flavoring agents, preservatives, dispersants and dyes may also be present.

[0149] Capsules can be produced by preparing a powder mixture as described above and filling a shaped gelatin shell with it. Before the filling operation, glidants and lubricants such as highly dispersed silicic acid, talc, magnesium stearate, calcium stearate or polyethylene glycol in solid form can be added to the powder mixture. Disintegrants or solubilizers such as agar, calcium carbonate or sodium carbonate can also be added to improve the availability of the drug after the capsule is taken.

[0150] In addition, if desired or necessary, suitable binders, lubricants and disintegrating agents and dyes can also be incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, sweeteners made from corn, natural and synthetic rubbers such as gum arabic, tragacanth or sodium alginate, carboxymethyl cellulose, polyethylene glycol, wax, etc. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, etc. Disintegrating agents include but are not limited to starch, methylcellulose, agar, bentonite, xanthan gum, etc. Tablets are prepared, for example, as described below: prepare a powder mixture, granulate the mixture or dry press, add lubricants and disintegrating agents, and compress the entire mixture to produce tablets. Powder mixture is prepared as follows: the compound crushed in a suitable manner is mixed with a diluent or alkali as described above, and optionally mixed with a binder such as carboxymethyl cellulose, alginate, gelatin or polyvinyl pyrrolidone, a dissolution delay agent such as paraffin, an absorption promoter such as a quaternary salt, and / or an absorbent such as bentonite, kaolin or dicalcium phosphate. The powder mixture can be granulated by wetting it with a binder such as syrup, starch paste, gum arabic, or a cellulose or polymer material solution and pressing it through a sieve. As an alternative to granulation, the powder mixture can be passed through a tablet press to produce unevenly shaped blocks, which are broken to form particles. The particles can be lubricated by adding stearic acid, stearate, talcum powder or mineral oil to prevent sticking to the tablet casting mold. Then the lubricated mixture is pressed to produce tablets. The compound of the present invention can also be combined with a free-flowing inert excipient, and then the granulation or dry pressing step is not performed, and the tablet is directly pressed to produce a tablet. There can be a transparent or opaque protective layer consisting of a shellac sealing layer, a sugar or polymer material layer and a wax gloss layer. Dyestuffs can be added to these coatings to allow for differentiation between different dosage units.

[0151] Pharmaceutical compositions suitable for oral administration may also be formulated by spray-drying of solid or liquid dispersions.

[0152] Oral liquids, such as solutions, syrups and elixirs, can be prepared in dosage unit form so that a given amount contains a predetermined amount of the compound. Syrups can be prepared by dissolving the compound in an aqueous solution with suitable flavoring agents, while elixirs are prepared using non-toxic alcoholic media. Suspensions can be prepared by dispersing the compound in a non-toxic medium. Solubilizers and emulsifiers such as ethoxylated isostearyl alcohol and polyoxyethylene sorbitol ethers, preservatives, flavor additives such as peppermint oil or natural sweeteners or saccharin, or other artificial sweeteners, etc. can also be added.

[0153] If desired, dosage unit formulations for oral administration can be encapsulated in microcapsules. The formulations can also be prepared in a manner to prolong or delay the release, for example, by coating or embedding particulate materials in polymers, waxes, etc.

[0154] The thienopyridone derivatives used according to the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles.Liposomes can be formed from a variety of phospholipids such as cholesterol, stearylamine or phosphatidylcholine.

[0155] "Effective amount" refers to the amount of the compound as defined above that prevents, eliminates or reduces the harmful effects of the disease being treated in humans. It should be understood that those skilled in the art can adjust the dosage according to the patient, pathology, mode of administration, etc. For example, the thienopyridone derivative of formula (I) can be administered once or twice a day, and the daily dose for human patients is 0.5 mg to 3000 mg, preferably 20 mg to 1000 mg, and more preferably 60 mg to 500 mg. It can be administered as a long-term drug for 4, 5, 6 or 7 days a week.

[0156] In a particular embodiment of the present invention, the thienopyridone derivative of formula (I) is administered in a dosage unit comprising 0.5 mg to 1500 mg, preferably 20 mg to 1000 mg, more preferably 60 mg to 500 mg of the thienopyridone derivative of formula (I).

[0157] The present invention will be described in more detail in the following examples, which are not intended to limit the scope of the present invention, which is defined by the claims.

[0158] Example

[0159] Embodiment 1: Synthesis of PXL770

[0160] abbreviation

[0161] a / a: The ratio of the peak area of ​​a given compound to the total peak area on a spectrum or chromatogram.

[0162] Analytical methods

[0163] XRPD

[0164] X-ray powder diffraction (XRPD) analysis was performed using a Panalytical Xpert Pro diffractometer equipped with a Cu (Kα radiation) X-ray tube and a Pixcel detector system. Samples were analyzed in transmission mode and sandwiched between low-density polyethylene films. XRPD patterns were classified, manipulated, and indexed using HighScore Plus 2.2c software.

[0165] TG / DTA

[0166] Thermogravimetric (TG) analysis was performed on a Perkin Elmer Diamond Thermogravimetric / Differential Temperature Analyzer (TG / DTA). The calibration standards were indium and tin. The samples were placed in an aluminum sample pan, inserted into a TG furnace and accurately weighed. The samples were heated from 30°C to 300°C at a rate of 10°C / min in a nitrogen stream. The furnace temperature was equilibrated at 30°C before analyzing the samples.

[0167] 1a) Synthesis of 1-(5-benzyloxytetralin-6-yl)ethanone (1)

[0168]

[0169] 6-Acetyl-5-hydroxytetralin (100 g, 1 eq.) was dissolved in acetonitrile (300 mL). After adding 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 a total of 74 hours, the solid was filtered off and washed with acetonitrile (200 mL), and the combined filtrate was evaporated.

[0170] Compound 1 was obtained as a syrup: m = 148.6 g, quantitative yield, purity 96.6% a / a.

[0171] 1b) Synthesis of ethyl 2-amino-4-(5-benzyloxytetralin-6-yl)thiophene-3-carboxylate (2)

[0172]

[0173] Acetic acid (70 mL) was heated to T = 65°C. HMDS (1.5 eq.) was added over 10 minutes. Then, 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 hours.

[0174] After cooling to room temperature, NaOH aqueous solution (1 M, 140 mL) and TBME (210 mL) were added. The layers were separated. The organic layer was washed with NaOH aqueous solution (1 M, 4×140 mL) until the pH of the aqueous phase was basic (pH=13). The organic layer was washed with HCl aqueous solution (1 M, 140 mL) and H2O (2×140 mL).

[0175] EtOH (240 mL), NaHCO3 (1.3 eq.) and sulfur (1.0 atomic equivalent) were added. After heating to reflux for 180 min, the reaction mixture was concentrated to 210 mL and co-evaporated with TBME (3×140 mL). After cooling to room temperature, the suspension was filtered and the solids were washed with TBME (70 mL). The combined filtrate was concentrated to 210 mL and distilled water was added dropwise at room temperature. A solution of 1.1 eq. of heptane was added. A precipitate was observed after inoculation. Heptane (350 mL) was added dropwise at room temperature. After stirring for 14 hours, the suspension was filtered. After washing with heptane (3×70 mL) and drying, compound 2 was recovered as a solid. m=83.2 g, yield 71%, purity 93.7% a / a.

[0176] 1c) 4-(5-Benzyloxytetralin-6-yl)-5-chloro-2-[(2-phenylacetyl)amino]thiophene -Synthesis of ethyl 3-formate (3)

[0177]

[0178] Compound 2 (17.69 g, 1 eq.) was dissolved in dichloromethane (140 mL). The resulting solution was cooled with ice / water. N-chlorosuccinimide (1.05 eq.) was added under stirring. The mixture turned black within a few minutes. After 1 hour, phenylacetyl chloride (1.25 eq.) was added.

[0179] After 1 hour at 0°C and 2 hours at room temperature, the mixture was evaporated to about 35 mL, EtOH (2×70 mL) was added, and evaporated again. The mixture was diluted with EtOH (35 mL) and cooled with ice / water. The product precipitated. The solid was filtered off and washed with cold EtOH (3×18 mL).

[0180] Compound 3 was obtained as a solid: m=20.99 g, yield 94.2%, purity 99.3% a / a.

[0181] 1d) 3-(5-Benzyloxytetralin-6-yl)-2-chloro-4-hydroxy-5-phenyl-7H-thieno Synthesis of [2,3-b]pyridin-6-one (4)

[0182]

[0183] Compound 3 (19.88 g, 1 eq.) was dissolved in methyltetrahydrofuran (120 mL), and the reaction mixture was cooled to a temperature between -16°C and -10°C (NaCl / ice). Potassium tert-butoxide (5 eq.) was added in four portions. The reaction mixture was then warmed to room temperature and stirred at room temperature for 65 minutes. 2N HCl (5 eq.) was added dropwise at T = 0-5°C (water / ice), and the resulting mixture was stirred vigorously. NaCl was added to obtain a 2.5-nitro-1H-pyrrolidone (1.5 eq.). (水溶液)(11%, 1×50 mL) and water (2×50 mL) were used to wash the organic phase. The organic phase was concentrated to a ~50% solution. Methyltetrahydrofuran (80 mL) was added and the resulting solution was concentrated to a ~50% solution. TBME (100 mL) was added and the resulting solution was concentrated to a ~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 about 50 mL, filtered, rinsed with mother liquor, washed with n-heptane (2×40 mL), and dried. Compound 4 was obtained as a granular solid. Yield 88%, purity 99.5% a / a.

[0184] 1e) 2-Chloro-4-hydroxy-3-(5-hydroxytetrahydronaphthalen-6-yl)-5-phenyl-7H-thieno[2,3-b]pyridine-6-yl Synthesis of Ketone (I)

[0185]

[0186] Compound 4 (15 g, 1 eq.) was dissolved in 75 mL of dichloromethane and cooled to T = -10 ° C / -15 ° C (with ice / NaCl). BCl3 (1.5 eq., solution: 1 mol / L dichloromethane solution) 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 × 45 mL). The organic phase was concentrated, solvent exchanged with toluene (3 × 90 mL) and diluted with toluene to a final volume of 90 mL toluene. The resulting mixture was heated to reflux and 15 mL of methanol was added. A light brown solution with a few particles was obtained. Seed crystals were added at T = 40 ° C, warmed to T = 52 ° C and cooled to room temperature. The resulting mixture was stirred overnight and then cooled with ice / NaCl (T = -10 ° C / -15 ° C) for 100 minutes. The precipitated product was filtered off, washed with toluene / heptane 1:2 v / v (15 mL) and heptane (15 mL) and dried to obtain crystals of compound (I): yield 87%, purity 99.0% a / a.

[0187] 1f) 2-Chloro-4-hydroxy-3-(5-hydroxytetrahydronaphthalen-6-yl)-5-phenyl-7H-thieno[2,3-b]pyridine-6-yl Synthesis of the monohydrated potassium salt of ketone (Ia)

[0188] Compound (I) is suspended in a water / isopropanol mixture (1 / 1, 5 parts of each solvent), and then 0.50 to 0.55 eq of potassium carbonate is added. The pH at the end of the addition of potassium carbonate is about 12 (pH test paper). After stirring at 50°C for 3 hours, the suspension thickens and the pH is about 8 (pH test paper). The temperature is raised to 80°C until a solution is obtained (10-15 minutes). If necessary, clarification can be performed at this point in the process. 7 parts of water are added, and the reaction mixture is then cooled to 40°C (a turbid solution is observed). The solvent is distilled under reduced pressure at 40°C (from 180 mbar to 40 mbar) until 7 parts of solvent remain in the reactor. Crystallization of potassium salt monohydrate can occur at this time. 4.2 parts of water are added, and the mixture is seeded with compound (I) (1 to 2% of seeds). The suspension is then cooled from 40°C to 5°C (5°C / hour) within 7 hours and maintained at 5°C for several hours. The suspension is filtered. The filter cake is washed twice with 1.42 parts of water. The collected solid was dried under vacuum at 40°C to give compound (Ia) in minimum 80% yield at the desired chemical purity (ie 98%+).

[0189] Embodiment 2: Characterization of PXL770

[0190] a) X-ray powder diffraction (XRPD) data of compound (Ia) showed that it was composed of crystalline material. The XRPD description of compound (Ia) is shown in Table 1.

[0191] Table 1:

[0192]

[0193]

[0194] b) TG / DTA analysis showed an initial weight loss of 1.1% from 30-100°C, followed by a larger weight loss of 3% from 117-160°C due to the loss of bound water. The second weight loss was accompanied by a large endotherm, and the combined weight loss of 4% was close to the theoretical weight loss of the monohydrate (3.75% w / w). The compound decomposed above 240°C.

[0195] Example 3 Very long-chain fatty acids (VLCFA) levels are reduced in AMN patient-derived lymphocytes treated with PXL770

[0196] Accumulation of very long chain fatty acids (VLCFA) in plasma and tissues, including the brain and spinal cord, is a hallmark of AMN disease. Literature has shown that VLCFA load increases with disease severity and that reducing VLCFA can abrogate the inflammatory response. Therefore, reducing VLCFA overload, especially in the central nervous system, has the potential to halt or reverse disease progression in AMN.

[0197] To show the efficacy of PXL770 in reducing very long chain fatty acids (VLCFA) in vitro, this study focused on evaluating the levels of the most prevalent VLCFA, hexadecanoic acid, in primary lymphocytes from healthy control cells and AMN human patients. The cell line was obtained from Coriell Cell Repositories. Lymphocytes were cultured in RPMI-1640 with 10% fetal bovine serum (FBS). The cultures were split at a ratio of 1:5. Lymphocytes from healthy patients were used as controls. All treatments were performed in complete medium containing fetal bovine serum (FBS, 15%). All cultured cells were maintained at 37°C in 5% CO2.

[0198] The VLCFA content was then measured as follows. The samples were adjusted to a final volume of 0.5–1 ml with LC-MS grade water and spiked with 10 ng of lignoceric acid-d4 as an internal standard. The samples were acidified to pH 3-4 with dilute hydrochloric acid and extracted three times with equal volumes of isooctane-ethyl acetate (9:1). The extracts were dried under nitrogen and the residues were reconstituted in methanol-water-ammonium acetate (75:25:10 mM).

[0199] Total fatty acids: After preparing samples containing internal standards for free fatty acids as described above, aqueous sodium hydroxide was added to a final concentration of 1 M. The mixture was incubated at 37°C in the dark under nitrogen for 3 hours. The samples were then acidified, extracted, and reconstituted as described above.

[0200] LC-MS analysis of fatty acids: The reconstructed fatty acid extract was subjected to HPLC on a Targa C8 column (2×10 mm) using a methanol-ammonium acetate aqueous solution (10 mM) solvent mixture. The column was eluted with a gradient of methanol (75% to 90%) at a flow rate of 0.25 ml / min for 8 minutes. The column eluent was directly introduced into a mass analyzer (QTRAP5500) and the published pseudo MRM method was used to monitor fatty acids. Under these conditions, VLCFAs were eluted between 5 and 8 minutes. Each fatty acid was quantified by the addition of an internal standard.

[0201] First, the evolution of hexacosanoids in lymphocytes was monitored in healthy control cells and primary lymphocytes derived from AMN human patients; second, PXL770 treatment was assigned to primary lymphocytes derived from AMN human patients in culture at different concentrations (5μM, 10μM, 25μM, 50μM) for one week and the levels of hexacosanoids were measured.

[0202] The results of this study are Figure 1This figure shows that when lymphocytes from patients with AMN are treated with different doses of PXL770, the level of hexacosanoids (in ng / 10 6 All tested doses of PXL770 significantly reduced the levels of hexacosanoids and at doses of 5 and 50 μM reached levels equivalent to those present in lymphocytes of healthy controls.

[0203] In conclusion, it was confirmed that PXL770 significantly reduced the levels of hexacosanoids, more generally the most prevalent VLCFA, in lymphocytes derived from AMN patients.

[0204] Example 4 : Overexpression of ABCD2 in AMN fibroblasts cultured in the presence of PXL770

[0205] Treatment research for AMN has also focused on the induction of the functionally redundant peroxisomal transporter adrenoleukodystrophy-related protein (ABCD2, also known as ALDRP). ABCD2 has significant sequence similarity to the gene ABCD1 (also known as ALDP) that is mutated / deleted in AMN and can therefore compensate for the loss of ABCD1 when overexpressed.

[0206] To demonstrate that the presence of PXL770 in AMN fibroblast cultures increased ABCD2 expression, Western blotting was performed to compare ABCD2 levels in AMN fibroblasts in the absence and presence of PXL770. More specifically, protein extraction was performed as follows.

[0207] The cells were washed with cold Tris-buffered saline (20 mM Trizma base and 137 mM NaCl, pH 7.5) and lysed in 1X SDS loading buffer (62.5 mM Trizma base, 2% [w / v] SDS, 10% glycerol), and after sonication and centrifugation at 15,000 g for 5 minutes, the supernatant was used for immunoblotting. The protein concentration of the samples was determined using a detergent-compatible protein assay reagent (Bio-Rad) using BSA as a standard. The samples were boiled for 3 minutes with 0.1 volume of a mixture of 10% β-mercaptoethanol and 0.5% bromophenol blue. Then, 40 μg of total cellular protein was separated by 8% or 12% polyacrylamide gel electrophoresis, electrotransferred, and blocked with Tris-buffered saline containing Tween 20 (TBST; 10 mM Trizma base, pH 7.4, 1% Tween 20, and 150 mM NaCl) with 5% skim milk. After incubation with antibodies against ABCD2, ABCD3 and β-actin at 4°C overnight, the membrane was washed with TBST and incubated with horseradish peroxidase-conjugated anti-rabbit or mouse IgG for 1 hour at room temperature. After washing the membrane with TBST buffer, detection was performed by autoradiography using ECL-plus (Amersham Biosciences).

[0208] The results of Western blot analysis were Figure 2 This figure allowed us to determine that when AMN fibroblasts were treated with PXL770 at concentrations of 5, 10, and 25 μM, there was an overexpression of ABCD2 protein in said cells compared to its levels in control fibroblasts and, more interestingly, compared to AMN fibroblasts not treated with PXL770.

[0209] It is known from the literature that overexpression of ABCD2 causes reduced VLCFA accumulation in cultured fibroblasts from AMN patients (Kemp S., Wei HM, Lu JF, Braiterman LT, McGuinness MC, Moser AB, Watkins PA and Smith KD (1998) Gene redundancy and pharmacological gene therapy: implications for X-linked adrenoleukodystrophy. Nat. Med. 4, 1261–1268).

[0210] Thus, PXL770 induces overexpression of ABCD2, which can compensate for the deficiency of ABCD1 and thereby allow one or both of these alternative proteins to contribute to the reduction of VLCFA accumulation.

[0211] Example 5 Very long-chain fatty acids (VLCFA) levels are reduced in mixed glial cells of AMN mice

[0212] The inventors conducted a study measuring VLCFA accumulation in mixed glial cells of AMN mice when not receiving any treatment and when treated with different doses of PXL770.

[0213] C57BL6 mouse breeding pairs were purchased from Jackson Laboratory (Jackson Laboratory) (Bar Harbor, ME) and maintained in the Henry Ford Health System (HFHS) animal facility. All animal procedures were approved by the HFHS Animal Review Committee, and all animals were humanely cared for in accordance with the HFHS experimental guidelines and the National Research Council's humane care standards (Guide for Care and Use of Laboratory Animals). Abcd1 knockout mice were raised and used to extract mixed glial cells. Cultures rich in primary astrocytes were prepared from the whole cortex of 1-day-old C57BL / 6 mice. In short, the cortex was quickly dissected out in ice-cold calcium / magnesium-free HBSS at pH 7.4 as described above. The tissue was minced, incubated in HBSS containing trypsin (2 mg / ml) for 20 min, washed twice in plating medium containing 10% FBS and 10 μg / ml gentamicin, and then disrupted by pipetting with a Pasteur pipette. The cells were then plated on 75-cm 2 The cells were cultured in flasks (Falcon, Franklin, NJ). After one day of incubation at 37°C in 5% CO2, the medium was completely replaced with culture medium (DMEM containing 10% FBS and 10 μg / ml gentamicin). The cultures were given and half-changed with fresh medium twice a week. All cultured cells were maintained at 37°C in 5% CO2. After 10 days, the confluent mixed glial cultures were used for the experiments outlined.

[0214] The levels of hexadecanoic acid in six groups of mixed glial cells were measured. These six groups included wild-type mixed glial cells, ALD-KO mixed glial cells (corresponding to glial cells derived from AMN mice, ALD is another name for ABCD1), and ALD-KO mixed glial cells treated with different concentrations of PXL770 (5μM, 10μM, 25μM, 50μM).

[0215] The results are as follows Figure 3 When ALD-KO mouse mixed glial cells were cultured in the presence of PXL770 (25 μM), the accumulation of hexacosanoids in these cells was reduced.

[0216] In conclusion, PXL770 plays a role in reducing the accumulation of hexacosanoids and more generally VLCFAs.

[0217] Embodiment 6: ABCD2 overexpression in mixed glial cells in the cerebral cortex of ALD-KO mice in the presence of PXL770

[0218] The same experimental protocol as in Example 4 was performed on the mixed glial cells of the cerebral cortex of ALD-KO mice. This model is mainly used for AMN.

[0219] Figure 4 As with the AMN patient-derived fibroblasts of Example 4, ABCD2 was overexpressed when ALD-KO mouse cerebral cortical mixed glial cells were treated with PXL770 compared with untreated ALD-KO mouse cerebral cortical mixed glial cells (ctl).

[0220] In conclusion, PXL770 upregulated ABCD2 levels in mixed glial cells in the cerebral cortex of ALD-KO mice.

[0221] Example 7 Very long-chain fatty acids (VLCFA) levels are reduced in ALD patient-derived lymphocytes treated with PXL770

[0222] After studying cells from patients with the most severe form of ALD (AMN), the present inventors repeated the same study method as in Example 1 to analyze the effect of PXL770 on cells from patients with X-ALD (severe inflammatory phenotype).

[0223] Figure 5 The results presented show that when lymphocytes from ALD patients were treated with increasing concentrations of PXL770, the levels of hexacosanoids in the cells were reduced to the same levels as those present in lymphocytes from healthy controls.

[0224] PXL770 significantly reduced VLCFA levels in lymphocytes derived from ALD patients.

[0225] Example 8 : Overexpression of ABCD2 in ALD fibroblasts cultured in the presence of PXL770

[0226] In the same manner as in Example 4, to demonstrate that the presence of PXL770 in ALD patient fibroblast cultures increased the expression of ABCD2, Western blotting was performed to compare the ABCD2 levels in ALD patient fibroblasts in the absence and presence of PXL770.

[0227] Western blot results Figure 6 These results indicate that when ALD fibroblasts are treated with PXL770, especially at a low concentration (5 μM), there is overexpression of the protein ABCD2 in the cells.

[0228] In conclusion, PXL770 can compensate for the deficiency of ABCD1 and thus contribute to the reduction of VLCFA accumulation.

[0229] Example 9 :In vivo experiments in X-ALD mice

[0230] Male ABCD1-KO mice (n=15) were treated with PXL770 (75 mg / kg) orally twice daily for 60 consecutive days. Untreated ABCD1-KO mice (n=15) and wild-type (WT) mice (n=12) were used as controls. The treated mice were sacrificed and the cerebral cortex and plasma were collected for VLCFA analysis. The analysis was performed according to the standardized protocol of the Lipidomics Core Facility, and the data are expressed as VLCFA μg / ml levels. Hexacosanoic acid (C26:0) was selected as a representative of VLCFA.

[0231] One-way ANOVA with Dunnett's multiple comparison test was performed to evaluate the effect of PXL770 on brain VLCFA (****p<0.0001, ***p<0.001). Kruskal-Wallis test with Dunn's multiple comparison test was performed to evaluate the effect of PXL770 on plasma VLCFA (****p<0.0001, ***p<0.01).

[0232] like Figure 7 and 8 As shown, PXL770 significantly reduced VLCFA accumulation in the brain cortex and plasma of X-ALD mice, respectively.

[0233] Example 10 Very long-chain fatty acids (VLCFA) levels are reduced in AMN and ALD patient-derived lymphocytes treated with metformin

[0234] The experiments described in Examples 3 and 7 were performed by the same laboratory using metformin and described by Singh et al. in Journal of Neurochemistry, 2016; 138, 86-100.

[0235] The results of these experiments are shown in Fig. 9 .

[0236] It was observed that 5 mM metformin reduced the VLCFA content in AMN and ALD patient-derived lymphocytes by -29% and -42%, respectively, after 7 days of treatment. Fig. 9 As shown, this was not sufficient to provide a normalization effect (VLCFA content was still higher than in controls).

[0237] In contrast, PXL770, when administered at a much lower concentration (5 μM), provided a normalizing effect in both AMN patients (see Example 3) and ALD patients (see Example 7).

[0238] Thus, this example demonstrates that PXL770 reduces VLCFA content in AMN and ALD patient-derived cells with greater efficacy and potency than metformin.

[0239] Embodiment 11 : Head-to-head comparison between PXL770 and metformin

[0240] To compare the efficacy of PXL770 and metformin in reducing very long chain fatty acids (VLCFA) in vitro, this study focused on evaluating the levels of the most prevalent VLCFA, hexadecanoic acid, in primary lymphocytes from healthy control cells and AMN human patients. The cell lines were obtained from Coriell Cell Repositories. Fibroblasts were cultured in DMEM with 15% FBS. The cultures were split at a ratio of 1:5. Fibroblasts from healthy patients were used as controls. All treatments were performed in complete medium containing fetal bovine serum (FBS, 15%). All cultured cells were maintained at 37°C in 5% CO2.

[0241] The VLCFA content was then measured as follows. The samples were adjusted to a final volume of 0.5–1 ml with LC-MS grade water and spiked with 10 ng of lignoceric acid-d4 as an internal standard. The samples were acidified to pH 3-4 with dilute hydrochloric acid and extracted three times with equal volumes of isooctane-ethyl acetate (9:1). The extracts were dried under nitrogen and the residues were reconstituted in methanol-water-ammonium acetate (75:25:10 mM).

[0242] Total fatty acids: After preparing samples containing internal standards for free fatty acids as described above, aqueous sodium hydroxide was added to a final concentration of 1 M. The mixture was incubated at 37°C in the dark under nitrogen for 3 hours. The samples were then acidified, extracted, and reconstituted as described above.

[0243] LC-MS analysis of fatty acids: The reconstructed fatty acid extract was subjected to HPLC on a Targa C8 column (2×10 mm) using a methanol-ammonium acetate aqueous solution (10 mM) solvent mixture. The column was eluted with a gradient of methanol (75% to 90%) at a flow rate of 0.25 ml / min for 8 minutes. The column eluent was directly introduced into a mass analyzer (QTRAP5500) and the published pseudo-MRM method was used to monitor fatty acids. Under these conditions, VLCFAs were eluted between 5 and 8 minutes. Each fatty acid was quantified by the addition of an internal standard.

[0244] Different patient groups were planned for this study. First, the evolution of hexacosanoids in fibroblasts was monitored in healthy control cells and primary lymphocytes derived from AMN human patients; second, PXL770 treatment was assigned to primary fibroblasts derived from AMN human patients in culture at different doses (PXL770: 0.1, 0.5, 1, 2, 3.5 and 5 μM; metformin: 100, 200, 300 and 400 μM) for one week and the levels of hexacosanoids were measured.

[0245] The results of this study are Fig.10 This figure shows that when fibroblasts from patients with AMN were treated with increasing doses of PXL770, the levels of hexacosanoids (in ng / 10 6 In addition, the potency and efficacy achieved with PXL770 were much higher than with metformin.

[0246] Example 12: ABCD2 Overexpression in AMN and ALD Patients' Fibroblasts Cultured in the Presence of Metformin

[0247] The experiments described in Examples 4 and 6 were performed by the same laboratory using metformin and described by Singh et al. in Journal of Neurochemistry, 2016; 138, 86-100.

[0248] The results of these experiments are shown in Fig.11 .

[0249] It was observed that a very high concentration of 5 mM of metformin only slightly increased ABCD2 protein expression in AMN and ALD patient-derived fibroblasts.

[0250] In contrast, PXL770 provided a much greater effect when administered at a much lower concentration (5 μM). Thus, PXL770 has greater efficacy and potency than metformin.

[0251] Embodiment 13: ABCD2 overexpression in mixed glial cells in the cerebral cortex of AMN-KO mice in the presence of metformin

[0252] The same experimental protocol as described in Example 6 was performed by the same laboratory using metformin and described by Singh et al. in Journal of Neurochemistry, 2016; 138, 86-100.

[0253] The results of these experiments are shown in Fig.12 .

[0254] It was observed that 100 μM of metformin induced a limited increase in ABCD2 protein levels in glial cells of ABCD1-KO mice.

[0255] In contrast, PXL770 provided a much greater effect when administered at a much lower concentration (5 μM). Thus, PXL770 has greater efficacy and potency than metformin.

[0256] Embodiment 14 :In vivo experiments in X-ALD mice

[0257] Male ABCD1-KO mice (n=8) were treated with PXL770 (75 mg / kg) orally twice daily for 90 consecutive days. Untreated ABCD1-KO mice (n=8) and wild-type (WT) mice (n=8) were used as controls. The treated mice were sacrificed and the spinal cords were collected for VLCFA analysis. The analysis was performed according to the standardized protocol of the Lipidomics Core Institute, and the data are expressed as VLCFA μg / ml levels. Hexacosanoic acid (C26:0) was selected as a representative of VLCFA.

[0258] like Fig.13 As shown, PXL770 significantly reduced VLCFA accumulation in the spinal cord of X-ALD mice.

[0259] Embodiment 15 Very long-chain fatty acids (VLCFA) levels are reduced in AMN patient-derived fibroblasts treated with various thienopyridones

[0260] The experiment described in Example 3 was repeated with various thienopyridone compounds, namely:

[0261] PXL770: Potassium 2-chloro-3-(5-hydroxytetrahydronaphthalen-6-yl)-6-oxo-5-phenyl-7H-thieno[2,3-b]4-hydroxypyridine,

[0262] PXL700: 2-chloro-4-hydroxy-3-indan-5-yl-5-(3-pyridyl)-7H-thieno[2,3-b]pyridin-6-one,

[0263] PXL702: 2-Chloro-4-hydroxy-3-(4-hydroxyindan-5-yl)-5-phenyl-7H-thieno[2,3-b]pyridin-6-one,

[0264] PXL695: 2-Chloro-5-(3-fluorophenyl)-4-hydroxy-3-(4-hydroxyindan-5-yl)-7H-thieno[2,3-b]pyridin-6-one,

[0265] Each was tested at concentrations ranging from 0.1 to 5 μM.

[0266] PXL700 has the following formula:

[0267]

[0268] One-way ANOVA with Dunnett's multiple comparison test was performed to evaluate the effect of PXL770 on brain VLCFA (****p<0.0001, ***p<0.001). Kruskal-Wallis test with Dunn's multiple comparison test was performed to evaluate the effect of PXL770 on plasma VLCFA (****p<0.0001, ***p<0.01).

[0269] like Fig.14 As shown, PXL770, PXL695 and PXL702 showed comparable reductions in VLCFA levels, whereas PXL700 (not included in the thienopyridones of the present invention) showed no effect or a very limited effect.

[0270] These compounds were further compared with 400 μM metformin at a concentration of 5 μM. Fig.15 As shown, metformin also showed no effect or very limited effect.

Claims

1. Use of a compound of formula (II) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating adrenoleukodystrophy and / or adrenal myeloneuropathy, 2. Use of a compound of formula (Ia) in the preparation of a medicament for the treatment of adrenoleukodystrophy and / or adrenal myeloneuropathy, 3. Use of a compound selected from the following formula (Ib), (Ic) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating adrenoleukodystrophy and / or adrenal myeloneuropathy, 4. The use according to any one of claims 1 to 3, wherein the medicament is in the form of a dosage unit comprising 0.5 mg to 1500 mg of the compound.

5. The use according to any one of claims 1 to 3, wherein the medicament is for oral administration.

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