DHEA-derived steroids

Abstract

The present invention relates to novel DHEA-derived steroids of formula (I) and their use against arteriosclerosis and / or atherosclerosis. Furthermore, the present invention relates to pharmaceutical compositions and medical uses thereof, wherein R1 is a C7-C 13 It is an alkyl.

Description

[Technical field]

[0001] The present invention relates to novel steroids for use against arteriosclerosis and / or atherosclerosis, as well as pharmaceutical compositions and medical uses thereof. [Background technology]

[0002] Atherosclerosis is an inflammatory disease involving hardening of the arteries that can lead to a variety of outcomes, including chronic kidney disease (CKD), heart attack, stroke, or thrombosis. According to the World Health Organization (WHO), atherosclerosis is a major cause of mortality and morbidity (31%) worldwide. The progression of atherosclerosis is associated with the development of calcification of the heart valves, called calcific aortic valve disease (CAVD). Several studies have demonstrated that CAVD involves lipid accumulation, inflammation, and calcification within the heart valve tissue [Lusis AJ et al., 2004; Mohler ER, 2004; Speer MY et al., 2004; Mohler ER, 2001; Chester A. 2011]. Currently, CAVD is a significant problem because no pharmacological drugs are available for aortic valve calcification [Yutzey KE et al., 2014].

[0003] Bekesi et al. found that sex steroids and cortisol significantly reduced the release of superoxide anions from human neutrophils [Bekesi et al., 2000].

[0004] Brancaleone V. et al. evaluated H2S biosynthesis in isolated rat aortic rings after androgen receptor stimulation and found that H2S biosynthesis in rat aorta was regulated by androgen hormones but was not induced by the female hormones 17-beta-ostradiol or progesterone. They concluded that androgens can exert a protective effect on cardiovascular and metabolic function by inducing various beneficial effects mediated by H2S [Brancaleone et al., 2015].

[0005] Naftolin F et al. showed that estradiol-inducible neural cell adhesion molecule (nCAM) sialylase is present in vascular endothelial cells, and pretreatment of human arterial endothelial cells with estradiol, testosterone, dehydroepiandrosterone, and dihydrotestosterone all induced endothelial cell sialylation, reduced monocyte capture, and protected against atherogenesis and its sequelae [Naftolin F et al., 2016].

[0006] Early studies in the 1990s showed that dehydroepiandrosterone (DHEA) inhibits iron-dependent lipid peroxidation, promotes the production of superoxide dismutase in hepatic mitochondria, and prevents the development of atherosclerosis [Schauer JE et al., 1990; Aragno M. et al., 1993].

[0007] Magyar et al. studied the role of sulfation-desulfation of DHEA / DHEAS (dehydroepiandrosterone sulfate) by steroid sulfotransferase and sulfatase in adult male Sprague-Dawley rats in vivo and found that DHEAS had a more pronounced effect on total scavenging capacity (TSC) than DHEA [Magyar et al., 2011].

[0008] Hochberg RB et al. already studied and reviewed the role of steroid fatty acid esters in the early 1990s and reported that androsterone esters were found in human breast tumors and breast cyst fluids [Hochberg et al., 1991].

[0009] A decade later, Loria, Roger M. suggested that 5-androstene-3β,17α-diol and possibly its esters are inhibitors of tumor growth. Otherwise, the literature on such ester derivatives is sparse [Loria, Roger M., U.S. Patent Application Publication No. 2001 / 0014675].

[0010] Song, Ching et al. [Song et al., 2001] teach that autoxidized cholesterol sulfates are antagonist ligands for liver X receptors and suggest that LXR agonists may have therapeutic potential for atherosclerosis because they can attenuate the activity of these antagonists. However, the authors found that the nonsulfated forms of these compounds, as well as many other steroid sulfates, including 5-androstene-3β,17α-diol, had no significant effect, in contrast to 5α,6α-epoxycholesterol.

[0011] It should be mentioned that the administration of steroids is contraindicated in human medicine since, in addition to their antioxidant properties, steroids also have strong endocrine effects that are clearly undesirable in the case of anti-atherosclerotic drugs.

[0012] Furthermore, the inventors have found that in animal studies, neither DHEA nor DHEAS (dehydroepiandrosterone sulfate) has any real demonstrable effect on atherosclerosis, and both compounds have been found to be toxic and to induce mortality.

[0013] Unexpectedly, the inventors found that narrow-range alkanoyl esters of DHEAS inhibit ApoE in atherogenic diet-fed subjects. - / - We found that DHEA-derived steroids prevent and treat atherosclerotic plaque formation and calcification in mice. Thus, DHEA-derived steroids are potential novel therapeutics to prevent and / or reverse atherosclerosis and valvular calcification. Summary of the Invention

[0014] compound The present invention relates to a compound represented by formula (I) [ka] (Wherein, R1 is C7 to C 13 wherein R is an alkyl group; or a salt or solvate thereof, preferably a pharma- ceutically acceptable salt thereof.

[0015] Preferably, C7 to C 13 The alkyl is an n-alkyl, i.e., a straight chain alkyl.

[0016] Preferably, R1 is C9 to C 13 It is an alkyl.

[0017] Particularly preferably, R1 is a C7 to C 11 It is an alkyl.

[0018] Particularly preferably, R1 is C 11 ~C 13 It is an alkyl.

[0019] In a further embodiment, R1 is C-C 12 Or C9~C 12 It is an alkyl.

[0020] In a further embodiment, R1 is C-C 11 Or C9~C 11 It is an alkyl.

[0021] In one embodiment, R is C 10 ~C 12 It is an alkyl.

[0022] More particularly preferably, R1 is C 11 It is an alkyl.

[0023] In a preferred embodiment, R1 is any alkyl as defined above. Preferably, R1 is n-alkyl.

[0024] Highly preferably, R1 is C 11 It is n-alkyl, i.e., the compound is 17-lauroyloxy-5-androstene-3-sulfate.

[0025] Preferably the salt is a sodium or potassium salt, especially a sodium salt.

[0026] Preferably, the alkyl is an n-alkyl, ie, a straight chain alkyl.

[0027] Preferably, the salt is a sodium salt. Preferably, the solvate is a water solvate.

[0028] In one embodiment, the configuration at carbon 3 is 3β.

[0029] In one embodiment, the configuration of carbon 17 is 17β.

[0030] In a preferred embodiment, the configuration is 3β,17β.

[0031] In one embodiment, the configuration at carbon 3 is 3α.

[0032] In one embodiment, the configuration of carbon 17 is 17α.

[0033] In one embodiment, any of the compounds of general formula (I) has the general formula (Is1): [ka]

[0034] The present invention relates to any of the compounds according to the present invention for use in treating arteriosclerosis, particularly atherosclerosis, in a subject.The subject is a vertebrate having a blood circulatory system (cardiovascular system or vascular system), for example a mammalian subject as defined herein.The mammalian subject is preferably a human subject.

[0035] The present invention relates to any of the compounds of the present invention for use in the prevention or reducing the risk of atherosclerosis.

[0036] The present invention relates to any of the compounds of the present invention for use in preventing or reducing the formation or attenuating the formation of or reducing the risk of atherosclerotic lesions in arteries.

[0037] The present invention relates to any of the compounds of the present invention for use in preventing or reducing the formation of atherosclerotic plaque in an artery, or attenuating the formation of or reducing the risk of such plaque.

[0038] The present invention relates to any of the compounds of the present invention for use in the treatment of atherosclerosis.

[0039] The present invention relates to any of the compounds of the present invention for use in the therapeutic treatment of atherosclerosis.The present invention relates to any of the compounds of the present invention for use in the reversal of atherosclerosis or the reversal of the formation of atherosclerotic lesions in arteries.

[0040] The present invention relates to any of the compounds of the present invention for use in reversing atherosclerosis or reversing the formation of atherosclerotic plaque in an artery.

[0041] Preferably, the present invention relates to any of the following conditions (including one or more of the following conditions): - thrombosis, - coronary heart disease or coronary artery disease, including narrowing of the coronary arteries, - Myocardial infarction or heart attack, - stroke, in particular ischemic and / or hemorrhagic stroke, - thrombosis, for example thrombosis formed on atherosclerotic plaques, - Angina pectoris, - Peripheral arterial disease, - aortic calcification, e.g. calcific aortic valve disease (CAVD), - Atherosclerosis in renal dysfunction, e.g. chronic kidney disease (CKD), - Atherosclerosis in neurodegenerative diseases, e.g. Alzheimer's disease, The present invention relates to any of the compounds provided above for use in preventing or reducing the risk of the onset or formation of

[0042] Preferably, the present invention relates to any of the following conditions (including one or more of the following conditions): - thrombosis, - coronary heart disease or coronary artery disease, including narrowing of the coronary arteries, - As a result of a myocardial infarction or heart attack, - stroke, in particular ischemic and / or hemorrhagic stroke, - thrombosis, for example thrombosis formed on atherosclerotic plaques, - Angina pectoris, - Peripheral arterial disease, - aortic calcification, e.g. calcific aortic valve disease (CAVD), - Atherosclerosis in renal dysfunction, e.g. chronic kidney disease (CKD), - Atherosclerosis in neurodegenerative diseases, e.g. Alzheimer's disease, The present invention relates to any of the compounds provided above for use in the treatment or reversal of the formation of

[0043] The present invention also relates to any of the compounds provided above for use in reducing the amount of reactive oxygen species (ROS) and / or inhibiting the production of ROS, thereby reducing oxidative stress and preventing the onset of or alleviating an oxidative stress disease.

[0044] Preferably, in the method of treatment, preferably in the method of prevention, the compound is of formula (I), 13 any of the preferred compounds thereof, or any compound for use in the treatment or prevention.

[0045] Pharmaceutical Compositions The present invention also relates to a compound of the present invention, which is represented by the formula (I) (wherein R1 is a C7-C 13 or any of the preferred compounds, and pharma- ceutically acceptable salts thereof, preferably the sodium salts thereof, and a pharma- ceutically acceptable carrier or excipient.

[0046] Preferably, C7 to C 13 The alkyl is an n-alkyl, i.e., a straight chain alkyl.

[0047] Preferably, R1 is C9 to C 13 It is an alkyl.

[0048] Particularly preferably, R1 is a C7 to C 11 It is an alkyl.

[0049] Particularly preferably, R1 is C 11 ~C 13 It is an alkyl.

[0050] In a further embodiment, R1 is C-C 12 Or C9~C 12 It is an alkyl.

[0051] In a further embodiment, R1 is C-C 11 Or C9~C 11 It is an alkyl.

[0052] In one embodiment, R is C 10 ~C 12 It is an alkyl.

[0053] More particularly preferably, R1 is C 11 It is an alkyl.

[0054] In a preferred embodiment, R1 is any alkyl as defined above. Preferably, R1 is n-alkyl.

[0055] Highly preferably, R1 is C 11 It is n-alkyl, i.e., the compound is 17-lauroyloxy-5-androstene-3-sulfate.

[0056] Preferably, the salt is a sodium salt. Preferably, the salt is a potassium salt.

[0057] Preferably, the alkyl is an n-alkyl, ie, a straight chain alkyl.

[0058] Preferably, the salt is a sodium salt. Preferably, the solvate is a water solvate.

[0059] In one embodiment, the configuration at carbon 3 is 3β.

[0060] In one embodiment, the configuration of carbon 17 is 17β.

[0061] In a preferred embodiment, the configuration is 3β,17β.

[0062] In one embodiment, the configuration at carbon 3 is 3α.

[0063] In one embodiment, the configuration of carbon 17 is 17α.

[0064] The present invention relates to a pharmaceutical composition comprising a compound according to the invention, said composition also comprising a pharma- ceutically acceptable carrier or excipient.Preferably, the compound of the present invention is a compound for use as defined herein, preferably as defined above in the compound section.

[0065] The present invention relates to a pharmaceutical composition according to the present invention for use in a subject for preventing a condition as taught herein, said composition also comprising a pharma- ceutically acceptable carrier or excipient.

[0066] Pharmaceutically acceptable carriers or excipients are well known to those skilled in the art.

[0067] Preferably the pharmaceutical composition is for or suitable for enteral administration, such as oral or parenteral, such as intravenous, or topical administration to a patient. Oral administration is preferred.

[0068] The oral pharmaceutical composition may be formulated, for example, as pills, tablets, tabs, coated tablets, film tablets, capsules, powders, granules, sustained release formulations, suspensions or drops.

[0069] Intravenous pharmaceutical compositions can be formulated, for example, as liquid solution preparations for injection or infusion and can be administered by any conventional intravenous administration means, for example, via an intravenous line (cannula) or injection.

[0070] The pharmaceutical compositions can be formulated for topical administration, for example as drops, sprays, aerosols, ointments, creams, pastes, syrups, lotions or gels, which can be useful for administration near the site of an atherosclerotic lesion or plaque.

[0071] Thus, the pharmaceutical compositions can be formulated for systemic administration, such as oral and intravenous compositions, or for local administration, such as topical or other locally administered compositions, such as sublingual, buccal, and the like.

[0072] Most preferably, the pharmaceutical composition is in a form suitable for oral administration, most preferably in the form of a tablet.

[0073] In a preferred embodiment, the pharmaceutical composition of the present invention is formulated in a dosage form for daily doses as defined below according to the method for treatment, preferably once, twice or three times a day. Preferably, the dosage form is for oral administration.

[0074] In preferred embodiments, the dosage form comprises a dose of at least 0.5 mg, 1 mg, 2 mg, 5 mg, 10 mg, 15 mg, 20 mg or 30 mg, particularly in human patients.

[0075] In preferred embodiments, the dosage form comprises a dose of up to 500 mg, 200 mg, 150 mg or 100 mg, 80 mg, 50 mg or 40 mg, especially in human patients.

[0076] In a preferred embodiment, the dosage form comprises a dose, especially in a human patient, of between 0.5 and 500 mg, between 1 and 500 mg, or between 5 and 200 mg, or in particular between 10 and 100 mg, or between 20 and 200 mg, or between 5 and 100 mg, or more in particular between 10 and 80 mg, or between 20 and 150 mg, or between 1 and 30 mg, or between 1 and 40 mg, or between 1 and 60 mg, or between 5 and 20 mg, or between 5 and 40 mg, or between 5 and 60 mg, or between 10 and 40 mg.

[0077] In another preferred embodiment, the dosage for humans is 10-40 mg / day.

[0078] In another preferred embodiment, the dosage for humans is between 0.01 mg / kg and 5 mg / kg or between 0.01 mg / kg and 1 mg / kg, preferably between 0.01 mg / kg and 0.5 mg / kg, in particular between 0.01 mg / kg and 0.4 mg / kg, or between 0.01 mg / kg and 0.3 mg / kg, or between 0.01 mg / kg and 0.05 mg / kg, or between 0.05 mg / kg and 0.5 mg / kg. In these dosages, per kg refers to per kilogram of body weight.

[0079] In another preferred embodiment, the dosage for mice is between 1 μg / day and 200 μg / day, in particular 4 μg / day to 100 μg / day or 4 μg / day to 20 μg / day or 20 μg / day to 100 μg / day.

[0080] In another preferred embodiment, the dosage for mice is between 0.1 mg / kg and 10 mg / kg, in particular between 0.1 mg / kg and 5 mg / kg, or between 0.1 mg / kg and 1 mg / kg, or between 0.5 mg / kg and 5 mg / kg, or between 1 mg / kg and 5 mg / kg. In these dosages, per kg refers to per kilogram of body weight.

[0081] In one embodiment, the dose is a dose that corresponds to any of the above doses calculated for another animal based on appropriate guidelines (see, e.g., USDepartment of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (2005), Guidance for Industry - Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers).

[0082] Methods for Treatment The present invention also relates to a method for treatment, wherein the pharmaceutical composition is administered to a subject or patient in an effective dose.

[0083] According to the present invention, the routes of administration listed above in relation to the pharmaceutical composition can be used in the method for treatment of the present invention to administer the composition to a subject. The subject is a vertebrate having a blood circulatory system (cardiovascular or vascular system), such as a mammalian or human subject as defined herein.

[0084] In preferred embodiments, particularly in human patients, the daily dosage is at least 0.5 mg, 1 mg / day, 5 mg / day, 10 mg / day, 20 mg / day or 30 mg / day.

[0085] In preferred embodiments, particularly in human patients, the daily dosage is up to 500 mg / day, 200 mg / day, 150 mg / day or 100 mg / day or 80 mg / day.

[0086] In a preferred embodiment, the daily dosage, especially in a human patient, is between 0.5 and 500 mg / day, between 1 and 500 mg / day, or between 5 and 200 mg / day, or in particular between 10 and 100 mg / day, or between 20 and 200 mg / day, or between 5 and 100 mg / day, or more in particular between 10 and 80 mg / day, or between 20 and 150 mg / day, or between 1 and 30 mg, or between 1 and 40 mg, or between 1 and 60 mg, or between 5 and 20 mg, or between 5 and 40 mg, or between 5 and 60 mg, or between 10 and 40 mg.

[0087] In another preferred embodiment, the dosage for humans is 10-40 mg / day.

[0088] In another preferred embodiment, the dosage for humans is between 0.01 mg / kg and 5 mg / kg or between 0.01 mg / kg and 1 mg / kg, preferably between 0.01 mg / kg and 0.5 mg / kg, in particular between 0.01 mg / kg and 0.4 mg / kg, or between 0.01 mg / kg and 0.3 mg / kg, or between 0.01 mg / kg and 0.05 mg / kg, or between 0.05 mg / kg and 0.5 mg / kg. In these dosages, per kg refers to per kilogram of body weight.

[0089] In another preferred embodiment, the dosage for mice is between 1 μg / day and 200 μg / day, in particular 4 μg / day to 100 μg / day or 4 μg / day to 20 μg / day or 20 μg / day to 100 μg / day.

[0090] In another preferred embodiment, the dosage for mice is between 0.1 mg / kg and 10 mg / kg, in particular between 0.1 mg / kg and 5 mg / kg, or between 0.1 mg / kg and 1 mg / kg, or between 0.5 mg / kg and 5 mg / kg, or between 1 mg / kg and 5 mg / kg. In these dosages, per kg refers to per kilogram of body weight.

[0091] In one embodiment, the dose is a dose that corresponds to any of the above doses calculated for another animal based on appropriate guidelines (see, e.g., USDepartment of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (2005), Guidance for Industry - Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers).

[0092] In one embodiment, an effective dose refers to administration of an amount that detectably or effectively elevates H2S levels in a control or patient.

[0093] The present invention relates to a method for the treatment of atherosclerosis.

[0094] In the methods of the invention, a compound of the invention, as disclosed herein, is administered to a subject, such as a mammalian subject, preferably a human subject.

[0095] In one embodiment, the method for treatment is a method for preventing or reducing the risk of atherosclerosis, or for preventing or reducing the formation of atherosclerotic lesions or plaques in arteries, or for mitigating or reducing the risk of the formation of such plaques.

[0096] In one embodiment, the method for treatment is a method for reversal of atherosclerosis or reversal of the formation of atherosclerotic lesions or plaques in arteries.

[0097] Preferably, the present invention relates to a method of treatment as taught herein, wherein said compound or composition is administered to prevent, or reduce the risk of, the onset or formation of any of the conditions listed above in the compounds section, or Methods of treatment as taught herein relate to methods wherein said compounds or compositions are administered for the treatment or reversal of the formation of any of the conditions listed above in the Compounds section.

[0098] In one embodiment, prior to commencing said treatment method, the mammalian or human subject has been diagnosed with atherosclerosis.

[0099] In one embodiment, prior to commencing said treatment method, the mammalian or human subject has been diagnosed as being at risk for atherosclerosis.

[0100] Such diagnostic methods are well known to those of skill in the art, as taught below.

[0101] Process for preparation The present invention also relates to a process for the preparation of a compound of the present invention, comprising the following steps: I. converting 3-hydroxy-17-oxo-5-androstene by acylation to obtain 3-acetoxy-17-oxo-5-androstene; II. Converting 3-acetoxy-17-oxo-5-androstene to 3-acetoxy-17-hydroxy-5-androstene by reduction; III. 3-Acetoxy-17-hydroxy-5-androstene, preferably C8-C 14 -Alkanoyloxyhalides, preferably C8-C 14 -Alkanoyloxy chloride is used to acylate 3-acetoxy-17-C8-C 14 converting the 5-alkanoyloxy-5-androstene into IV. 3-Acetoxy-17-C8~C 143-Hydroxy-17-C8-C 14 converting the 5-alkanoyloxy-5-androstene into V. 3-Hydroxy-17-C8~C 14 -Alkanoyloxy-5-androstene is sulfonated to give 17-C8~C 14 3. converting 5-alkanoyloxy-5-androstene-3-sulfate to VI. Optionally 17-C8~C 14 and converting the 5-alkanoyloxy-5-androstene-3-sulfate into a salt, in particular a pharma- ceutically acceptable salt thereof, preferably the sodium salt.

[0102] In a preferred embodiment, R1 is a C 13 Particularly preferably, R1 is a C7 to C 11 Particularly preferably, R1 is C 11 ~C 13 It is an alkyl.

[0103] Preferably, R1 is n-alkyl.

[0104] In another preferred embodiment, R1 is C9-C 13 It is an alkyl.

[0105] In a further embodiment, R1 is C-C 12 Or C9~C 12 It is an alkyl.

[0106] In a further embodiment, R1 is C-C 11 Or C9~C 11 It is an alkyl.

[0107] In one embodiment, R is C 10 ~C 12 It is an alkyl.

[0108] More particularly preferably, R1 is C 11It is an alkyl.

[0109] In a preferred embodiment, R1 is any alkyl as defined above. Preferably, R1 is n-alkyl.

[0110] In a preferred embodiment, in acylation step III, 10 ~C 14 17-C in step V of the process. 10 ~C 14 5-Alkanoyloxy-5-androstene-3-sulfate is obtained.

[0111] In a particularly preferred embodiment, in acylation step III, 12 -alkanoyloxy-halide is used, whereby in step V of the process 17-C8 12 5-Alkanoyloxy-5-androstene-3-sulfate is obtained.

[0112] In a particularly preferred embodiment, in acylation step III, 12 ~C 14 -alkanoyloxy-halide is used, thereby forming 17-C in step V of the process 12 ~C 14 5-Alkanoyloxy-5-androstene-3-sulfate is obtained.

[0113] In a highly preferred embodiment, in acylation step III, 12 -alkanoyloxy-halide is used, thereby forming 17-C in step V of the process 12 5-Alkanoyloxy-5-androstene-3-sulfate is obtained.

[0114] In one embodiment, the acylation step I is carried out by reacting 3-hydroxy-17-oxo-5-androstene with acetic anhydride under suitable conditions, for example in an aprotic non-polar solvent, for example dichloromethane, preferably at a temperature T=0° C., preferably for 2-4 hours.

[0115] In one embodiment, reduction step II involves reacting 3-acetoxy-17-oxo-5-androstene with a reducing agent, such as a hydride, particularly NaBH4, under suitable conditions, such as in a protic polar solvent, such as methanol, preferably at a temperature T < 0 °C, more preferably -10 °C < T < 0 °C, even more preferably -8 °C < T < -2 °C, and preferably for 1 to 5 hours, preferably 2 to 3 hours.

[0116] In one embodiment, acylation step III involves reacting 3-acetoxy-17-hydroxy-5-androstene with each C8 - C 14 -alkanoyloxy-halide, preferably C8 - C 14 -alkanoyloxy-chloride, under suitable conditions, such as in an aprotic solvent, preferably in a mixture of a nonpolar solvent and an aromatic solvent, preferably in a mixture of dichloromethane and pyridine, preferably at room temperature or at a temperature of 20 °C < T < 30 °C, preferably for less than 1 day, or 2 to 24 hours, preferably 4 to 16 hours.

[0117] In one embodiment, hydrolysis step IV involves hydrolyzing 3-acetoxy-17-C8 - C 14 -alkanoyloxy-5-androstene under basic conditions, preferably in a protic polar solvent, such as a mixture of methanol and water, using an alkali hydroxide, such as sodium hydroxide, at a temperature of 10 °C < T < 30 °C, preferably at a temperature of 15 °C < T < 25 °C, preferably for less than 1 day, or 4 to 24 hours, preferably 6 to 20 hours.

[0118] In one embodiment, sulfation step V involves 3-hydroxy-17-C8 - C 14-Alkanoyloxy-5-androstene is sulfated preferably in a mixture of a nonpolar solvent and an aromatic solvent, preferably in a mixture of dichloromethane and pyridine, with a sulfating agent, preferably chlorosulfonic acid, preferably at a temperature of T < 0 °C, more preferably at -10 °C < T < 0 °C, even more preferably at -8 °C < T < -2 °C, preferably for 1 to 12 hours, preferably for 2 to 8 hours.

[0119] In a preferred embodiment, the appropriate salt is formed by using a metal salt-forming reagent, preferably an alkali metal salt, for example, preferably in water containing the corresponding alkali carbonate or bicarbonate, preferably by adding the metal salt-forming reagent to the sulfation reaction mixture after the sulfation reaction has proceeded for a sufficient time. Optionally, the salt is formed in a multiphase, for example, a three-phase reaction, diluted with a nonpolar volatile solvent, such as diethyl ether, the salt is filtered, purified, and optionally recrystallized.

[0120] In a preferred embodiment, the process of the present invention comprises the following steps: - Converting 3-hydroxy-17-oxo-5-androstene (10) (DHEA) to 3-acetoxy-17-oxo-5-androstene (1) by acylation, - Converting 3-acetoxy-17-oxo-5-androstene (1) to 3-acetoxy-17-hydroxy-5-androstene (2) by reduction, - Converting 3-acetoxy-17-hydroxy-5-androstene (2) to 3-acetoxy-17-lauroyloxy-5-androstene (3) by acylation, - Converting 3-acetoxy-17-lauroyloxy-5-androstene (3) to 3-hydroxy-17-lauroyloxy-5-androstene (4) by hydrolysis, - Converting 3-hydroxy-17-lauroyloxy-5-androstene (4) to 17-lauroyloxy-5-androstene-3-sulfate, preferably the sodium salt of 17-lauroyloxy-5-androstene-3-sulfate (5) by sulfation.

[0121] Abbreviation ApoE - / - Apolipoprotein E-deficient mice C6 17-Hexanoyloxy-5-androstene-3-sulfate sodium salt C16 17-palmitoyloxy-5-androstene-3-sulfate sodium salt CAD (also CHD) Coronary Artery Disease CAVD Calcific Aortic Valve Disease CHD (also CAD) Coronary Heart Disease CKD Chronic Kidney Disease DCM Dichloromethane DHEA Dehydroepiandrosterone DHEA-S or DHEAS Dehydroepiandrosterone Sulfate H2S Hydrogen Sulfide LEAD Lower limb obstructive arterial disease MB Methylene Blue PAD Peripheral Arterial Disease ROS reactive oxygen species S2 17-Lauroyloxy-5-androstene-3-sulfate sodium salt (DHEA-derived steroid or steroid 2) TSC Total Scavenger Capacity

[0122] definition "Arteriosclerosis" is a condition in which the arterial walls thicken, harden, and lose elasticity. As used herein, arteriosclerosis is considered a disease.

[0123] "Atherosclerosis" is a specific type of arteriosclerosis in which abnormal substances build up in the inner lining of the artery wall, forming "atherosclerotic lesions," which are made up of fats, cholesterol, and other substances in and on the artery wall that restrict blood flow.

[0124] "Atherosclerotic lesions" are abnormalities in the inner wall of arteries and have several types (Types I-VI) based on their size, composition, and severity. Less advanced atherosclerotic lesions do not cause symptoms.

[0125] "Atherosclerotic plaque" or atheroma is an atherosclerotic lesion that is an abnormal accumulation of material in the inner lining of an arterial wall, particularly one that contains dense extracellular lipids, which can cause symptoms in a subject with atherosclerosis.

[0126] A "subject" as used herein refers to an individual of an animal species that has a blood circulation and is susceptible to developing arteriosclerosis or atherosclerosis, preferably a vertebrate, more preferably a mammalian species or an avian species, especially a mammalian species, and very preferably the individual is a primate, a hominidae or a human.

[0127] A "patient" is a subject receiving, or intended to receive, medical or veterinary observation, supervision, diagnosis or treatment.

[0128] "Treatment" refers to the process or method of administering a pharmaceutical composition or medicament to a subject (or patient) receiving medical or veterinary assistance, either directly or indirectly, with the goal of improving the condition of the subject or patient or restoring health, or maintaining health or a stable condition to avoid deterioration. As used herein, treatment of a subject includes restoring or maintaining normal function of an organ or tissue, preferably at least partially restoring or maintaining health (medical or veterinary treatment). Treatment generally refers to the administration of an effective amount of a compound or composition as described herein. Treatment may relate to or include medical or veterinary treatment and cosmetic treatment, especially medical or veterinary treatment.

[0129] "Therapeutic treatment" (or treatment) is a treatment intended to improve the condition or restore the health of a subject or patient. For example, as used herein, treatment includes the reversal of atherosclerosis or the reversal of the formation of atherosclerotic lesions in arteries.

[0130] "Prophylactic treatment" (or prevention) is treatment intended to prevent or avoid the onset or worsening of a disease or condition. For example, as used herein, prevention includes reducing the formation of, or slowing the formation of, or reducing the risk of, atherosclerotic lesions in arteries.

[0131] A "pharmaceutical composition" or "composition" of the present invention is a composition of matter comprising at least one biologically active agent of the present invention suitable for increasing H2S levels in a subject, thereby preventing the formation of atherosclerotic lesions or plaques. Treatment includes administration of an effective amount of the agent. The composition may also comprise additional biologically active agents, e.g., useful in combination therapy. Additionally, the composition may comprise pharma- ceutical carriers and / or excipients, including formulation agents well known in the art. A pharmaceutical is a "pharmaceutical composition" that is marketed or to be marketed under a marketing authorization issued or to be issued by the respective authorities of a specified geographical area.

[0132] The terms "effective amount" and "therapeutically effective amount" are intended to define the amount of a therapeutic agent required to alleviate or prevent to some extent one or more of the symptoms of a condition.

[0133] As used herein, the term "alkyl," alone or in combination, refers to a straight or branched chain saturated hydrocarbon group of a length indicated by the number of carbon atoms. In a broader sense, alkyl may include substituents, such as halogen substituents. In a narrower sense, alkyl does not include substituents.

[0134] As used herein, the term "alkanoyl" refers to a group containing a double-bonded oxygen atom and an alkyl group having the formula RC=O or R-(CO)-, where R represents an alkyl group linked by a single bond to a carbon atom of the group, which may be linked to another portion of an organic molecule by a single covalent bond. In organic chemistry, alkanoyl groups are commonly derived from carboxylic acids.

[0135] As used herein, the terms "halogen" and "halo" include fluorine, chlorine, bromine, and iodine, and fluoro, chloro, bromo, and iodo, respectively.

[0136] As used herein, "sulphation" (or "sulfation") refers to the process by which sulfate esters are formed in organic molecules.

[0137] As used herein, the term "solvate" refers to a crystalline form that contains a compound of the invention, or a pharma- ceutically acceptable salt thereof, and either a stoichiometric or non-stoichiometric amount of a solvent, such as, for example, water. A reference to a compound of the invention includes all physical forms of that compound, unless a particular form, salt, or solvate thereof is specified.

[0138] The terms "comprises" or "comprising" or "including" are herein to be construed as having a non-exhaustive meaning, allowing for additional features or method steps or components to be added to or involved in anything that includes the recited features or method steps or components. "Comprising" may be replaced with "including" where required by the practice of a given language variant, or may be limited to "consisting essentially of" where other members or components are not essential to the practice of the invention.

[0139] The indefinite articles "a" or "an" can, where the context allows, refer to either the singular or the plural. [Brief description of the drawings]

[0140] [Figure 1] DHEA-derived steroids inhibit atherosclerosis. (A) Representative Oil Red O staining of aortas isolated from ApoE- / - mice fed an atherogenic diet and treated with DHEA-derived steroids (N=9) or control (tap water, N=13) for 8 weeks was previously demonstrated. (B) Quantification of atherosclerotic plaque size was performed by ImageJ software. Graph shows mean ± SEM of tap water (N=13) and DHEA-derived steroids (N=9) mice aortas. Differences in means were analyzed by Student's t-test. ***P<0.001. [Diagram 2] DHEA-derived steroids reduce the expansion of the extracellular matrix. (A) Hematoxylin and eosin (left panel, ×400 magnification); von Kossa staining (middle and right panels) were performed on aortic valves of mice fed a high-fat diet and tap water (upper panel; N=13) and mice fed a high-fat diet treated with DHEA-derived steroids (lower panel; N=9). Von Kossa immunohistochemistry was shown at two different magnifications (×400 magnification and ×1000 magnification). (B) Quantification of mineralized areas of mouse heart valves was performed by ImageJ software. The graph shows the mean ± SEM of tap water (N=13) and DHEA-derived steroid (N=9) mouse aortas. Differences in the means were analyzed by Student's t-test. ***P<0.001 [Diagram 3]DHEA-derived steroids increase the H2S content of mouse plasma The Zn2+-precipitable sulfide content was measured in plasma of tap water (N=4) and DHEA-derived steroids (N=4) treated mice under alkaline conditions and normalized to the protein content of the samples. The graph shows the mean ± SEM of the H2S content of tap water (N=4) and DHEA-derived steroids (N=4) mice plasma. Differences between the means were analyzed by Student's t-test. *P<0.05. [Figure 4A] DHEA, DHEA-S, C6, and C16 do not inhibit atherosclerosis Representative Oil Red O staining of aortas isolated from ApoE− / − mice fed an atherogenic diet and treated with DHEA (N=11), DHEA-S (N=7), C6 (N=3), and C16 (N=3) for 8 weeks was previously demonstrated. [Figure 4B] DHEA, DHEA-S, C6, and C16 do not inhibit atherosclerosis Quantification of atherosclerotic plaque size was performed by ImageJ software. Graphs show the mean ± SEM of control (N = 13), S2 (N = 9), DHEA (N = 5), DHEA-S (N = 6), C6 (N = 3), and C16 (N = 3) mouse aortas. Differences in mean values ​​were analyzed by one-way ANOVA with Dunnett's post-hoc test. ***P < 0.001; ns: not significant. [Figure 4C]DHEA, DHEA-S, C6, and C16 do not inhibit atherosclerosis Survival rates of control, S2, DHEA, DHEA-S, C6, and C16 mouse groups are shown. Control mice and mice treated with S2, C6, and C16 survived (did not die) during the experiment. However, 45% of mice treated with DHEA and 22% of mice treated with DHEA-S died before the end of the experiment. Mice treated with DHEA started to die earlier during treatment than mice treated with DHEA-S. The first animal treated with DHEA died on day 50, whereas the first animal treated with DHEA-S died on day 55 from the treatment regimen (i.e., from the start of treatment with S2, DHEA, DHEA-S, C6, or C16). Comparison of survival curves was analyzed by Mantel-Cox test. **P<0.01 [Figure 5A] DHEA and DHEA-S fail to reduce extracellular matrix mineralization in aortic valves from ApoE knockout mice. Hematoxylin and eosin (left panel, ×50 magnification); von Kossa (middle (×50 magnification) and right panel (×250 magnification)) staining was performed on aortic valves from mice fed a high-fat diet and tap water (first row; N=13), high-fat diet-fed mice treated with DHEA-derived S2 steroids (second row; N=9), high-fat diet-fed mice treated with DHEA (third row, N=11), and high-fat diet-fed mice treated with DHEA-S (fourth row, N=7). [Figure 5B] DHEA and DHEA-S fail to reduce extracellular matrix mineralization in aortic valves of ApoE knockout mice. Quantification of mineralized areas of mouse heart valves was performed by ImageJ software. Graphs show the mean ± SEM of tap water (N = 13), DHEA-derived steroids (N = 9), DHEA (N = 8), and DHEA-S (N = 9) mouse aortas. Differences in means were analyzed by Student's t-test. ***P < 0.001; **P < 0.01; ns: no significant difference. [Figure 6A]DHEA-derived S2 steroids reverse atherosclerosis and aortic valve calcification Representative Oil Red O staining of aortas isolated from ApoE- / - mice fed an atherogenic diet for 6 weeks followed by a chopped diet and treatment with DHEA-derived S2 steroids (N=3) or control (tap water, N=3) for 4 weeks was previously demonstrated. [Figure 6B] DHEA-Derived S2 Steroids Reverse Atherosclerosis and Aortic Valve Calcification Representative Oil Red O staining of aortas isolated from ApoE- / - mice fed an atherogenic diet for 4 weeks followed by a chopped diet and treated with DHEA-derived S2 steroids (N=5) or control (tap water, N=3) for 8 weeks was previously demonstrated. [Figure 6C] DHEA-derived S2 steroids reverse atherosclerosis and aortic valve calcification Quantification of atherosclerotic plaque size measured in the aorta shown in panel A was performed with ImageJ software. Graph shows mean ± SEM of tap water (N=3) and DHEA-derived steroid (N=3) mouse aortas. Differences in means were analyzed by Student's t-test. *P<0.05. [Figure 6D] DHEA-derived S2 steroids reverse atherosclerosis and aortic valve calcification. Quantification of plaque size in the experiment shown in panel B was performed with ImageJ software. Graphs show the mean ± SEM of tap water (N=3) and DHEA-derived steroid (N=5) mouse aortas. Differences in means were analyzed by Student's t-test. **P<0.01. [Figure 6E] DHEA-Derived S2 Steroids Reverse Atherosclerosis and Aortic Valve Calcification Von Kossa staining (x50 and x250 magnification) was performed on aortic valves from mice fed a high-fat diet for 6 weeks followed by 4 weeks on a chopped diet and tap water (N=3) or S2 steroid therapy in tap water (N=3). [Figure 6F]DHEA-derived S2 steroids reverse atherosclerosis and aortic valve calcification. Quantification of mineralized areas of cardiac valves of mice shown in panel E was performed by ImageJ software. Graph shows mean ± SEM of aortas of tap water (N=3) and S2 steroid (N=3) mice. Differences in means were analyzed by Student's t-test. *P<0.05. [Figure 7A] NMR recording of the compound of the present invention NMR recording of C6 steroid (17-hexanoyloxy-5-androstene-3-sulfate sodium salt) [Figure 7B] NMR recording of the compounds of the present invention NMR recording of S2 steroid (17-lauroyloxy-5-androstene-3-sulfate sodium salt) [Figure 7C] NMR recording of the compound of the present invention NMR recording of C16 steroid (17-palmitoyloxy-5-androstene-3-sulfate sodium salt) DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0141] Arteriosclerosis and atherosclerosis are diseases in which the arterial walls thicken, harden and lose their elasticity, which can impede blood flow. These conditions can lead to more serious diseases. The causes of arteriosclerosis and atherosclerosis appear to be lipid retention and denaturation, oxidation by free radicals (oxidative stress), which cause chronic inflammation at susceptible sites in the wall. Early lesions, including fatty streaks in arteriosclerosis, can evolve into fibrous atherosclerotic plaques that are prone to rupture and cause thrombosis or stenosis [Insull, W 2009]. Risk factors for atherosclerosis and its thrombotic complications include hypertension, smoking, and diabetes mellitus, and chronic inflammation is a risk factor in itself, and the role of the immune system is essential.

[0142] Type I lesions contain atheromatous lipoproteins that cause an increase in macrophages and the formation of scattered macrophage foam cells. Type II lesions, also called fatty streaks, are formed by a layer of lipid-carrying macrophage foam cells and smooth muscle cells. Type III lesions contain scattered extracellular lipid droplets and particles that disrupt the cohesion of some intimal smooth muscle cells. Type IV lesions also contain extracellular lipids that are confluent and larger than those in type III lesions (the latter creating a transition state between type II and type IV lesions). Type IV lesions and more severe lesions are also called atherosclerotic plaques or atheromas and may already produce symptoms. Type V lesions usually have a lipid core and also contain a layer or layers of fibrous connective tissue, while type VI lesions contain fissures, hematomas, and thrombi (type VI lesions). Type V lesions have variants, e.g., type Vb lesions are mostly calcified, and type Vc lesions are composed mainly of fibrous connective tissue with little or no lipid or calcium accumulation. Plaques can rupture and cause thrombus [Stary H C. et al., 1995].

[0143] The role of free radicals has been investigated in relation to arteriosclerosis and atherosclerosis.

[0144] Free radicals are molecules that contain an unpaired electron in the outer atomic orbital. They search for their own pair and receive electrons from their surroundings, thus starting a chain reaction of creating new molecules with unpaired electrons. Free radicals are therefore highly reactive and can damage surrounding biological materials such as proteins, carbohydrates, lipids, and nucleic acids. This leads to the development of free radical-mediated diseases such as atherosclerosis.

[0145] Previous studies of the radical neutralizing, antioxidant, also known as scavenging, effects of steroid hormones have demonstrated that several steroids, such as sex hormones and cortisol, are effective antioxidants in isolated human neutrophil granulocytes. In addition to their antioxidant effects, these steroids have also been shown in the art to have potent endocrine effects, thus making their prophylactic administration in human medicine out of the question.

[0146] Starting with dehydroepiandrosterone (DHEA), a very weak androgen, as the initial molecule, the inventors have surprisingly found that it possesses antioxidant properties when certain substituents are attached to the 3rd and 17th carbon atoms of the sterane backbone. In addition to preventing the formation of atherosclerotic lesions and plaques in animal models, the DHEA-derived steroids of the present invention reverse previously formed atherosclerotic lesions and plaques in animals.

[0147] Thus, the DHEA-derived steroids of the present invention are novel antiatherogenic drugs that can be used to prevent the formation of lipid derivatives in atherosclerotic lesions and plaques, and to inhibit and reverse calcification of heart valves.

[0148] Using organic chemistry methods, the inventors have placed a sulfate group at the 3rd position and a lauroyloxy hydrocarbon chain at the 17th position. Thus, the inventors have created a series of novel steroid compounds, namely 17-alkyloxy-5-androstene-3-sulfates, which have been shown to exhibit high antioxidant activity in isolated human neutrophil granulocytes without substantial endocrine effects. In a preferred embodiment, the 17-alkyl is n-alkyl.

[0149] The chemical name of the highly preferred novel compound is 17-lauroyloxy-5-androstene-3-sulfate (or 5-androstenediol-17-lauroyl-3-sulfate).

[0150] In certain embodiments, the compound is selected from the group consisting of 17β-lauroyloxy-5-androstene-3β-sulfate, 17β-lauroyloxy-5-androstene-3α-sulfate, 17α-lauroyloxy-5-androstene-3β-sulfate, and 17α-lauroyloxy-5-androstene-3α-sulfate.In a preferred embodiment, the compound is 17β-lauroyloxy-5-androstene-3β-sulfate.

[0151] In certain embodiments, the sodium salt is used. 31 H 51 It is NaO6S.

[0152] The chemical steps are as follows: 1: DHEA, 2: 3-acetoxy-17-oxo-5-androstene, 3: 3-acetoxy-17-hydroxy-5-androstene, 4: 3-acetoxy-17-lauroyloxy-5-androstene, 5: 3-hydroxy-17-lauroyloxy-5-androstene, 6: 17-lauroyloxy-5-androstene-3-sulfate sodium salt. See below for details.

[0153] The preferred compounds have a hydrocarbon chain with 12 carbon atoms at the 17th position. We investigated whether compounds with shorter hydrocarbon chains (6 carbon atoms: 17-hexanoyloxy-5-androstene-3-sulfate sodium salt, see compound C6) or longer hydrocarbon chains (16 carbon atoms: 17-palmitoyloxy-5-androstene-3-sulfate sodium salt, see compound C16) have an atherosclerosis-suppressing effect. In animals treated with these compounds, the size and number of plaques were similar to control mice (although in the case of compound C16 there may have been a very small, non-significant effect). However, 17-lauroyloxy-5-androstene-3-sulfate sodium salt (see compound S2) was found to be very effective in both preventing and reversing atherosclerosis. This means that the obvious effect of the newly synthesized compounds of the present invention is linked to the range of 6 to 16 carbon atoms. Thus, the chain length of the alkanoyl group is at least 8 carbon atoms, i.e., in general formula (I), R1 is C7, i.e., in general formulas (I') and (V), n is 6, and at most 14 carbon atoms, i.e., in general formula (I), R1 is C13, i.e., in general formulas (I') and (V), n is 12.

[0154] Preferably, the chain length of the alkanoyl group is at least 10 carbon atoms, i.e., in general formula (I), R1 is C9, i.e., in general formula (I') and (V), n is 8, and up to 14 carbon atoms, i.e., in general formula (I), R1 is C13, i.e., in general formula (I') and (V), n is 12.

[0155] In certain embodiments, the chain length of the alkanoyl group is at least 11 carbon atoms, i.e., in general formula (I), R1 is C10, i.e., in general formula (I') and (V), n is 9, and up to 13 carbon atoms, i.e., in general formula (I), R1 is C12, i.e., in general formula (I') and (V), n is 11.

[0156] In a highly preferred embodiment, the chain length of the alkanoyl group is 12 carbon atoms, ie, R1 is C11 in general formula (I), ie, n is 10 in general formulas (I') and (V).

[0157] An exemplary compound of the invention used in the experiments below is 17-lauroyloxy-5-androstene-3-sulfate, preferably the sodium salt.

[0158] The compounds have the general formula (I'). [ka] (Wherein, R1 is (CH2) n -CH3, Here, n is an integer from 6 to 12, Preferably, n is 6, 8, 9, 10, 11 or 12; More preferably, n is 6, 8, 10 or 12, or n is 8, 10 or 12; Highly preferably, n is 10.

[0159] Thus, very preferably, R1 is (CH2) 10 -CH3.

[0160] In one embodiment, the configuration of the third carbon is S(3β).

[0161] In a further embodiment, the configuration at the third carbon is R(3α).

[0162] Preferably, the configuration of the third carbon is S(3β).

[0163] Alternatively, the IUPAC numbering of steranes may be used herein, but positions 3 and 17 are the same as those given by IUPAC and in the present numbering.

[0164] During preparation of the compound, a mixture of beta and alpha configurations about carbon 3 may be present. In one embodiment, the mixture may be a racemic mixture. In one embodiment, there may be an excess of either the beta or alpha configuration about carbon 3.

[0165] In the pharmaceutical composition, a mixture of beta and alpha configurations about carbon 3 may be present. In one embodiment, the mixture may be a racemic mixture. In one embodiment, there may be an excess of either the beta or alpha configuration about carbon 3.

[0166] It should be noted that the 3β configuration is a specific configuration.

[0167] For example, the configuration of the compound may be as shown in general formula (Is1'). [ka]

[0168] In one embodiment, the configuration at the 17th carbon is R.

[0169] In a further embodiment, the 17th carbon has an S configuration.

[0170] In one embodiment, the configuration of the 17th carbon is beta (17β).

[0171] In a further embodiment, the configuration of the 17th carbon is alpha (17α).

[0172] In one embodiment, the stereochemistry of the compound is selected from the group consisting of 17β,3β, 17β,3α, 17α,3β, and 17α,3α, preferably 17β,3β.

[0173] During preparation of the compound, a mixture of beta and alpha configurations about carbon 17 may be present. In one embodiment, the mixture may be a racemic mixture. In one embodiment, there may be an excess of either the beta or alpha configuration about carbon 17.

[0174] In a preferred embodiment, the configuration is 3β,17β, as shown in formula (Is2).

[0175] For example, the configuration of the compound may be as shown in general formula (Is2). [ka]

[0176] In the pharmaceutical composition, there may be a mixture of beta and alpha configurations about carbon 17. In one embodiment, the mixture may be a racemic mixture. In one embodiment, there may be an excess of either the beta or alpha configuration about carbon 17.

[0177] It should be noted that the 17β configuration (present in DHEA) is a specific configuration.

[0178] The newly synthesized compound(s) were tested ex vivo in isolated human neutrophil granulocytes and also in an in vivo animal model. We selected apoE-deficient mice fed a lipid-rich diet to be used as an atherosclerosis model. The treatment lasted for 8 weeks. Control animals received normal drinking water and lipid-rich food, while the treatment group, fed the same diet, received 100 micrograms of the new compound synthesized by us per animal per day. The compound was dissolved in drinking water. At the end of the test period, the size and number of atherosclerotic plaques formed on the inner surface of the aortic arch and aorta, the calcification of the heart valves, and the hydrogen sulfide levels in the serum were determined. We found that the number and size of plaques, as well as the degree of calcification, were significantly lower in treated animals, and the concentration of hydrogen sulfide, a powerful antioxidant, was significantly higher. The latter could explain the mechanism of action.

[0179] In a further part of this study, we investigated whether the compounds of the present invention also have obvious therapeutic effects. In the above animal model, we developed actual atherosclerosis in 8 weeks, and fed the animals with S2 compound (100 micrograms per animal per day) for another 8 weeks. Control mice received pure drinking water. At the end of the experiment, we measured the size and number of sclerotic plaques, the calcification of the heart valves, and the amount of hydrogen sulfide. In animals treated with the compounds of the present invention, the size and number of plaques and calcification were significantly less than in control animals, and the concentration of hydrogen sulfide was significantly higher. This means that the compounds of the present invention have not only preventive effects but also therapeutic effects.

[0180] Without wishing to be bound by theory, the inventors have investigated the possible mechanisms behind the biological and medical effects.

[0181] Previous studies have found that elevated H2S (I.) scavenges the atherogenic free radical superoxide, (II.) limits the formation of pro-oxidative and pro-inflammatory lipid mediators and the subsequent endothelial responses triggered by these species, and (III.) prevents cardiac valve calcification by regulating osteoblast-like differentiation of valvular interstitial cells [Bekesi G et al., 2000; Bekesi G et al., 2004; Potor L et al., 2018; Sikura KE et al., 2019].

[0182] A systematic study found that neutrophils incubated with corticosterone and 18-hydroxy-deoxycorticosterone showed a significant decrease in superoxide production, whereas the inventors found a significant increase in superoxide production in the presence of 11beta-hydroxyprogesterone. Furthermore, a non-significant decrease was observed after incubation with cholesterol 3-sulfate, and an increase was observed when 11-hydroxyandrostenedione was used [Bekesi G et al., 2004].

[0183] Hydrogen sulfide (H2S), along with nitric oxide and carbon monoxide, is the newest member of the endogenous gasotransmitter family [Wang R. 2002]. Potor et al. also reported that ApoE-induced hypercalcemia in mice fed a high-fat diet. - / - showed that H2S exerts an anti-atherosclerotic effect by inhibiting lipid peroxidation in mice. Furthermore, Potor et al. showed that exogenous H2S inhibited lipid oxidation in human atheroma and human hemorrhagic lesions [Potor L et al., 2018]. Recently, Sikura and Potor et al. reported that ApoE-induced hypercalcemia in mice fed a high-fat diet. - / - In mice, inhibition of heart valve calcification by H2S was observed [Sikura KE et al., 2019].

[0184] It was found that the increase in H2S levels in mouse plasma by DHEA-derived steroid treatment contributed to this effect. In view of this, we wished to determine whether the basic chemical structures of the compounds of the present invention (DHEA and its sulfate DHEAS) have atherosclerosis-inhibiting properties, and found that in animal models, these basic compounds did not reduce the progression of atherosclerosis and increased the mortality rate of the animals (see Figure 4C).

[0185] In conclusion, the present invention provides evidence that the DHEA-derived steroids of the present invention are potent preventive and therapeutic agents for arteriosclerosis, atherosclerosis and atherosclerosis-related diseases.

[0186] The present invention is useful in the treatment of a number of conditions and diseases.

[0187] Atherosclerosis-related diseases Atherosclerosis is a pathological process in arteries in which the buildup of plaque causes the inside of the artery to narrow. Arteries throughout the body, such as the coronary, cerebral, iliac, and femoral arteries, and the aorta, can be affected, resulting in a variety of diseases depending on the artery or arteries affected. (National Research Council (US) Committee on Diet and Health. (1989). Diet and Health: Implications for Reducing Chronic Disease Risk. National Academy Press, Washington DC, 1989)

[0188] Damage to the coronary arteries leads to CHD (coronary heart disease, also known as coronary artery disease = CAD), which is one of the most common and serious manifestations of atherosclerosis. The term coronary heart disease includes many syndromes, such as sudden cardiac death, myocardial infarction, stroke, heart attack, and angina pectoris.

[0189] Sudden cardiac arrest or sudden cardiac death occurs when the heart's electrical system malfunctions and becomes erratic. Blood flow to the body and brain is reduced, the affected person loses consciousness and may die if emergency treatment is not started immediately.

[0190] Myocardial infarction (or heart attack) occurs when blood flow to a part of the heart is reduced or stopped, causing ischemic necrosis of the myocardium. The necrotic tissue is replaced by connective tissue. The subsequent clinical outcome depends on the amount and location of myocardium lost.

[0191] Stenosis of the coronary arteries may not cause infarction, but may cause ischemic pain, especially on exercise - this condition is called angina pectoris. This condition indicates the presence of severe disease and a high risk of myocardial infarction.

[0192] A stroke occurs when the flow of oxygen-rich blood to a part of the brain is blocked. Without oxygen, brain cells begin to die and brain tissue becomes damaged. The two main types of stroke are ischemic (due to lack of blood) and hemorrhagic (due to bleeding). A blood clot forming on an atherosclerotic plaque in a cerebral artery can reduce or interrupt blood flow to a part of the brain, causing ischemic necrosis. Symptoms can be paralysis on the contralateral side, impaired speech, vision, hearing, and memory.

[0193] Peripheral arterial disease (PAD) is the abnormal narrowing of arteries other than those supplying the heart and brain (e.g., abdominal aorta, iliac arteries, and femoral arteries). It occurs when atherosclerosis of the arteries and its complications cause temporary arterial insufficiency in the lower limbs or ischemic necrosis of the limbs. PAD includes several clinical syndromes of limb arterial insufficiency, which are characterized by pain, inflammation, and ischemic damage to soft tissues due to partial or complete occlusion of major arteries. PAD is often used interchangeably with lower extremity occlusive arterial disease (LEAD) when it affects the arteries of the lower limbs of the body.

[0194] Atherosclerosis may also be associated with other diseases.

[0195] Calcification can occur in atherosclerotic vascular lesions and in the aortic valve. Calcific aortic valve disease (CAVD) is a slowly progressive disease. Aortic stenosis has many forms, ranging from mild valve thickening that does not obstruct blood flow (aortic sclerosis) to severe calcification with impaired leaflet motion (aortic stenosis) [Lerman et al., 2015]. CAVD is the most common heart valve disease, and so far there is no medical treatment that can slow the progression of CAVD [Hulin et al., 2018].

[0196] Chronic kidney disease (CKD) may also be associated with atherosclerosis. CKD patients are at increased risk for atherosclerotic cardiovascular disease [Kon et al., 2014]. Accelerated atherosclerosis progression has been observed in the early stages of renal dysfunction [Olechnowicz-Tietz et al., 2013]. A greater hallmark of atherosclerotic cardiovascular disease in CKD is the severity of the disease, which reflects increased inflammatory mediators and vascular calcification associated with renal-derived hyperparathyroidism, which is unique to patients with CKD [Mathew et al., 2017].

[0197] Atherosclerosis may also be associated with vascular disease of the eye. Retinal arteries can become blocked when blood clots or fatty deposits clog the arteries. Such blockages are more likely to occur if the eye has atherosclerosis [Medical Encyclopedia, Retinal artery occlusion. Retrieved from https: / / medlineplus.gov / ency / article / 001028.htm.]. When the central retinal artery becomes diseased, i.e. the artery narrows, the blood supply to the retina is reduced, which can cause vision loss. Vascular hypertension can also occur in association with atherosclerosis, which can lead to retinal vascular occlusion.

[0198] Atherosclerosis may also play a role in neurodegenerative diseases such as Alzheimer's disease. According to Roher et al. [Roher et al., 2003], atherosclerosis-induced cerebral hypoperfusion contributes to the clinical and pathological symptoms of Alzheimer's disease. Alzheimer's disease is a progressive brain disorder that causes loss of brain cells, leading to memory loss and other declines in thinking abilities.

[0199] H2S-related diseases H2S has been shown to have various effects on various systems, including the digestive, nervous, cardiovascular, respiratory, renal, and hepatic systems [Singh, SB, & Lin, HC 2015]. Hydrogen sulfide reduces arterial blood pressure, limits atherosclerotic plaque formation, and promotes angiogenesis in ischemic tissues. H2S is a pro-angiogenic substance that can restore the function of ischemic tissues and has been shown to have beneficial effects in the treatment of cerebral artery occlusion and post-ischemic cardiac remodeling as shown in animal models [Kanagy, NL, Szabo, C., & Papapetropoulos, A. 2017].

[0200] Hydrogen sulfide reduces the amount of reactive oxygen species (ROS) by inhibiting their production, directly scavenging them, and increasing the expression of antioxidant enzymes. Thus, H2S can reduce oxidative stress, thereby combating oxidative stress-related changes in blood vessels, such as hypertension, atherosclerosis, and vascular diabetic complications [Kanagy, supra].

[0201] The present inventors have demonstrated that the DHEA-derived steroid S2 according to the present invention inhibits ApoE - / - Treatment of mice has been shown to increase serum H2S levels, and therefore, it can be reasonably predicted to have beneficial effects not only in the treatment of atherosclerosis-related diseases, but also in the treatment of other oxidative stress-related disorders or conditions associated with reduced H2S levels.

[0202] According to a review by Zhang et al. (2013) [Zhang et al., 2013], H2S is also involved in aging by inhibiting free radical reactions. Furthermore, H2S has been shown to have therapeutic potential in age-related diseases [Kanagy, op. cit.].

[0203] The antioxidant effects of hydrogen sulfide have been demonstrated in relation to atherosclerosis: H2S slowed the progression and / or reduced the severity of the disease.

[0204] Diagnosis of Atherosclerosis and Related Diseases Patients diagnosed with atherosclerosis are preferably treated for atherosclerosis, or patients at risk for arteriosclerosis or atherosclerosis are administered prophylactic treatment.

[0205] Diagnosis of atherosclerosis includes measuring a localized drop in blood pressure (near the site of an atherosclerotic lesion). Alterations in blood flow may also be heard by a specialist through a stethoscope, or a weak or absent pulse below an area of ​​an artery narrowed by an atherosclerotic lesion may also be a sign of atherosclerosis.

[0206] Diagnosis of atherosclerosis may also include the following diagnostic tests: Blood tests; ankle-brachial index, which compares blood pressure in the ankle with blood pressure in the arm; Electrocardiogram (ECG); Stress tests (or exercise stress tests) in which information about the heart is collected during physical activity; In addition, imaging modalities: Ultrasound or Doppler ultrasound, a non-invasive method that allows the differentiation of multiple plaque components; Intravascular ultrasound, which is invasive and requires radiation exposure and iodine contrast for catheter positioning, is useful for characterizing plaque tissue; Optical coherence tomography, which is also invasive and requires exposure to radiation and iodine contrast for catheter positioning, provides high-resolution imaging of plaque features; Computed tomography (CT angiography) scans, which can distinguish between calcified and noncalcified plaques; MRI or magnetic resonance angiography (MRA), which is essentially limited to large caliber vessels and does not allow assessment of plaque components [Libby, 2019].

[0207] Typically, treatment methods involve diagnosis and once signs or symptoms of atherosclerosis(s) are established, treatment can be initiated.

[0208] Atherosclerosis begins decades before clinical consequences become apparent. With regard to arteriosclerosis and the risk of atherosclerosis, it is desirable to initiate preventive measures in patients at high risk of developing these diseases.

[0209] Risk factors typically include obesity, type II diabetes mellitus, elevated low-density lipoprotein (LDL) cholesterol levels, metabolic syndrome, family history of atherosclerosis or related diseases, all of which are examples of increased risk factors. With regard to lifestyle management, sedimentary lifestyle, smoking, cholesterol-rich diet, etc. are further risk factors.

[0210] Although diagnostic guidelines vary from country to country and may change over time, almost all of them recommend an early assessment of an individual's risk of future cardiovascular disease (CVD) [Libby, 2019]. It is within the skill of the physician to determine what preventive measures can be initiated if there is a risk of developing arteriosclerosis and / or atherosclerosis.

[0211] The dose for prophylactic treatment may be lower and administered for a longer period of time than the dose for therapeutic treatment.

[0212] When atherosclerosis develops and cannot be completely reversed, treatment can have a therapeutic character that strives to at least partially reverse the lesions or plaques that have formed, and a prophylactic character that avoids the formation of further lesions or plaques.

[0213] In certain cases, the treating physician may determine that long-term treatment is necessary, particularly if risk factors support this decision and / or complete reversal of symptoms has not been achieved. Age is an additional risk factor and long-term treatment may be justified, for example, for patients over 40 or 50 years of age, particularly over 55 or 60 years of age.

[0214] Long-term treatment can mean administration of a compound or pharmaceutical composition of the invention for at least 6 months, or for at least 1 year, 2 years, 5 years, or lifelong.

[0215] Pharmaceutical Compositions In the present invention, stereoisomers, enantiomers, or diastereomers may be present in the pharmaceutical composition. Preferred compositions are enriched in the active isomer. Various isomers are disclosed above and in the examples.

[0216] Pharmaceutically acceptable salts can be obtained by treating the compound with the corresponding base or the salt of the cation that is added to the composition.

[0217] The compounds of the present invention can exist in unsolvated forms as well as solvated forms with pharma- ceutically acceptable solvents, such as water. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of the present invention.

[0218] With respect to pharma- ceutically acceptable carriers and excipients, the term acceptable means compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0219] For oral administration, the active ingredient may be provided in the forms described in the "Brief Description of the Invention" section.

[0220] In general, the preparation of compositions containing pharma- ceutically acceptable carriers or excipients can be carried out as described, for example, in Gennaro, AR et al., Remington: The Science and Practice of Pharmacy (20th Edition., Lippincott Williams & Wilkins, 2000, see especially Part 5: Pharmaceutical Manufacturing), as a standard reference.

[0221] More specifically, oral lipid-based drug delivery systems can be prepared as described, for example, in the review article Kalepu, S. Oral lipid-based drug delivery systems - an overview. Acta Pharmaceutica Sinica B, 2013 3(6)361-372.

[0222] Solubilizing oral and injectable formulations is taught, for example, by Strickley, RG Solubilizing Excipients in Oral and Injectable Formulations. Pharmaceutical Research, Vol. 21, No. 2, February 2004.

[0223] Pharmaceutical compositions of the invention may be provided in unit dose or multi-dose containers.

[0224] For oral preparations, solid dosage units such as pills, tablets, etc. may be provided as described above.

[0225] Injectable solutions can be presented, for example, in predetermined quantities, for example in sealed vials and ampoules and can be stored in a dry crystalline form or in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, such as water or saline, prior to use.

[0226] The compounds of the invention are present in the pharmaceutical compositions in a purity suitable for administration to humans or animals. Purification techniques include, for example, recrystallization as shown in the examples and / or chromatographic purification as is well known in the art.

[0227] The invention is further illustrated by the following non-limiting examples. EXAMPLES

[0228] Materials and Methods All reagents were purchased from Sigma-Aldrich (Darmstadt, Germany) at ACS grade or higher purity, with the exceptions including DHEA specified below. Steroid compounds were dissolved in tap water and then sonicated. DHEA-derived steroids were produced by the inventors as described in the Examples. Reagents used in the synthesis were obtained from Reanal, Budapest, Hungary ("alt" quality).

[0229] mouse All animal experiments were approved by the guidelines of Directive 2010 / 63 / EU of the European Parliament on the protection of animals used for scientific purposes. Animal experiments performed in this study were approved by the Scientific and Research Ethics Committee of the Scientific Council of Health of the Hungarian Government under registration number DE MAB / 157-5 / 2010 and are reported according to the ARRIVE guidelines. - / -Mice (The Jackson Laboratory; B6.129P2 Apoetm1Unc / J; RRID:IMSR_JAX:002052) were kept at the University of Debrecen under specific pathogen-free conditions in accordance with the guidelines of the Institutional Ethics Committee. At 8 weeks of age, standard chow was replaced with an atherogenic diet (15% fat, 1.25% cholesterol, ssniff Spezialdiaten GmbH, Soest, Germany) to induce the formation of atherosclerotic plaques. Mice were randomly divided into two groups, and in parallel with the atherogenic diet, mice received tap water or DHEA-derived steroids in tap water (100 μg steroids / mouse / day). The DHEA-derived steroids were replaced daily. Aortas were harvested after 8 weeks of treatment. All mice were euthanized by performing a predictable and controllable slow-loading compression CO2 asphyxiation method. Composition of atherosclerotic diet (high fat diet): Crude nutrients (%): Crude protein 19%, Crude fat 15.2%, Crude fiber 3.4%. Crude ash 6.3%, Starch 25.6%, Sugars 11.2%. Additives (per kg): Vitamin A 15,000 IU, Vitamin D3 1,000 IU, Vitamin E 110 mg, Vitamin K3 5 mg, Vitamin C 0 mg, Copper 13 mg.

[0230] ApoE - / - Quantification of aortic pathology in mice The aorta was dissected from the aortic arch to the iliac bifurcation. Then, the adipose tissue was carefully removed under a stereomicroscope (M125; Leica Biosystems Nussloch GmbH). The atherosclerotic lesions were revealed by en face Oil Red O (Sigma-Aldrich, Lot. O0625;) staining. 0.07 g of ORO powder was dissolved in 25 mL of methanol and mixed for 10 min with a magnetic stirrer. Finally, 10 mL of 1 mol / L NaOH was added to the ORO solution, which was filtered. Images were taken with a camera (Nikon D3200; Nikon Corp., Japan), and the entire aortic plaque area was analyzed with ImageJ1.49 software.

[0231] immunohistochemistry Immunohistochemistry of the aortic root was performed on formalin-fixed paraffin-embedded tissue sections. 4 μm slides were then deparaffinized using xylol and ethanol. Serial sections were stained with Gill's hematoxylin solution (Merck) followed by eosin counterstain or von Kossa staining. For digital documentation, stained specimens were scanned with a Mirax Midi scanner (3D Histech, Budapest, Hungary). Macrophotographs of the arteries were taken with a Nikon D3200 camera (Nikon Corp.; Minato, Tokyo, Japan).

[0232] Determination of sulfide levels from tissues by zinc precipitation assay Sulfide levels were measured by the zinc precipitation method developed by Gilboa-Garber [Gilboa-Garber N. 1971] and improved by AD Ang et al. [Ang AD et al., 2012]. Human carotid arteries were homogenized under liquid nitrogen in 7.4 pH PBS and sonicated. After sonication, samples were centrifuged at 12000G for 15 min and lipid-free clear supernatant was collected. 200 μL of sample was mixed with 350 μL of 1% zinc acetate and 50 μL of 1.5 mol / L sodium hydroxide and incubated on a shaker for 60 min. After the incubation step, the resulting zinc sulfide was pelleted by centrifugation at 2000G for 5 min. The supernatant was then removed and the pellet was washed with 1 mL of distilled water under vigorous vortexing followed by centrifugation at 2000G for 5 min. The supernatant was then aspirated and the pellet was reconstituted in 160 μL of distilled water and mixed with 40 μL of premixed dye (20 μL of 20 mmol / L dimethyl-p-phenylenediamine dihydrochloride (NNDP) in 7.2 mol / L hydrochloric acid (HCl) and 20 μL of 30 mmol / L iron(III) chloride (FeCl3) in 1.2 mol / L HCl). After 10 min, the absorbance of the produced methylene blue (MB) was measured at 667 nm in a spectrophotometer. Since 1 mol / L MB was formed from 1 mol / L sulfide during the reaction, the concentration was calculated using the extinction coefficient of MB (30200 M -1 cm -1) Samples were normalized to protein concentration.

[0233] Experimental Unit "N" represents the number of tissue samples used in each group. "n" indicates the number of independent replicates.

[0234] statistical analysis Data were analyzed by GraphPad Prism 5.02 software (GraphPad Software Inc., 7825 Fay Avenue, Suite 230 La Jolla, CA 92037; RRID: SCR_002798). All statistical data are expressed as mean ± SEM. When data groups passed normality and equal variance tests, Student's t-test or one-way ANOVA was performed followed by Bonferroni post-hoc test, as indicated in the figure brief. P<0.05 was considered significant.

[0235] Quantification of DHEAS in biological samples DHEAS can be quantitatively determined in biological samples, for example as described by Sanchez-Guijo et al. [Sanchez-Guijo et al., 2015] and in the papers they reference.

[0236] Preparation of the Compounds of the Invention Name of compound prepared: A) (n=4): 17-Hexanoyloxy-5-androstene-3-sulfate sodium salt B) (n=10): 17-Lauroyloxy-5-androstene-3-sulfate sodium salt C) (n=14): 17-Palmitoyloxy-5-androstene-3-sulfate sodium salt

[0237] Molecular formula and molecular weight: A):n=4;C 25 H 39 NaO6S;MW:490.6 B): n = 10; C 31 H 51 NaO6S;MW:574.8 C): n = 14; 35 H 59 NaO6S;MW:630.9

[0238] Structural formula: [ka] Solubility: Ethanol-water, methanol-water mixtures Note: During the synthesis, the reaction was monitored by thin layer chromatography (TLC). 1 Identification by NMR and IR spectroscopy

[0239] Reaction Pathway Preparation of 17-lauroyloxy-5-androstene-3-sulfate sodium salt (compound S2) [Table A-1] [Table A-2] [Table A-3]

[0240] Synthesis of sodium 17-lauroyloxy-5-androstene-3-sulfate I. 3-Acetoxy-17-oxo-5-androstene (1) In a round-bottom flask equipped with a magnetic stirrer, add 25 cm 3 of dichloromethane (DCM) and 5.8 g (20 mmol) of DHEA were weighed in. The solution was cooled to T=0° C. and diluted with 10 cm 3 A mixture of 100 μl of dichloromethane, 3.1 g of acetic anhydride, and 200 μl of concentrated H2SO4 was added dropwise. The mixture was stirred for 3 hours (t=3 hours) and then 50 cm 3Pour into 5% (w / v) NaHCO3 solution (T = 5 °C) and stir for 10 min; then pour in a further 50 cm 3 of DCM was added. The organic phase was washed with water, then with 20% (w / v) NaCl solution and dried over Na2CO3. The solvent was removed in vacuo (Rotadest). The product was purified by elution with 50 cm 3 of methanol and 50 cm 3 of water for 15 min and then cooled to T=0° C.

[0241] The white crystals of 3-acetoxy-17-oxo-5-androstene (compound I) were filtered, washed with a small amount of cold solvent, then with water, and then dried in a vacuum desiccator at T=50° C.

[0242] Yield: = 5.2g, R = 78.8%

[0243] II. 3-Acetoxy-17-hydroxy-5-androstene (2) Compound I (4.96 g) and 180 cm 3 of methanol was added to a flask equipped with a magnetic stirrer and cooled to T=-5°C. To this cooled solution, 0.7 g of sodium borohydride was added in portions over a period of about 30 minutes. The mixture was then allowed to react for an additional 2.5 hours, after which the reaction mixture was diluted with 100 cm 3 The mixture was mixed with 1M NaHCO3 (approximately 25 cm 3 ) to pH 6, and then 50 cm 3 The product was filtered and diluted with 2 x 15 cm 3 of water:methanol (1:1) mixture and 2 × 25 cm 3 Washed with water.

[0244] After drying in vacuum (T=50° C.), 4.1 g of product was obtained (R=82.0%).

[0245] III. 3-Acetoxy-17-lauroyloxy-5-androstene (3) In a flask equipped with a magnetic stirrer, add 20 cm 3 DCM, 15cm 3of pyridine, 3.35 g (10 mmol) of 3-acetoxy-17-hydroxy-5-androstene (compound II) are weighed in, followed by addition of 3.1 g (14 mmol) of lauric acid chloride (lauroyl chloride) and 10 cm3 at T = 20-25 ° C over 20-30 minutes. 3 A mixture of DCM was added.

[0246] The mixture was then allowed to react for an additional 5.0 hours (t=5.0 hours), and then the mixture was poured into 50 cm 3 Dilute with DCM and store at T = 5-10 °C for 50 cm 3 The separated organic phase was washed twice with 10% hydrochloric acid, dried over sodium sulfate, and then the solvent was removed in a rotary evaporator (Rotadest) under reduced pressure in a water bath at T=55° C.

[0247] The crude product was stirred at T = 55-60 ° C for 40 cm 3 of water:MeOH (1:1), then cooled to T = 5 °C, and the crystals were then filtered and poured into 2 × 10 cm of the aforementioned solvent. 3 Washed with.

[0248] After drying in vacuum (T=50° C.), 4.3 g of compound III was obtained (R=83.4%).

[0249] IV. 3-Hydroxy-17-lauroyloxy-5-androstene (4) 3.1 g (6.0 mmol) of the compound III obtained in the previous step was dissolved in 120 cm 3 of methanol and then 5.4 cm 3 of 1 M NaOH (5.4 mmol) was added and the mixture was stirred for 12 h. 3 of water was added and the reaction mixture was acidified to pH 6 with 2M hydrochloric acid. The precipitated product was filtered and 20cm 3 of water:MeOH (1:1) mixture and then 2 × 20 cm 3 The wet material was washed with 50 cm 3 Dissolve in chloroform and 2 x 20 cm 3The mixture was extracted with water (by shaking), then the solution was dried over Na2SO4, and then the solvent was removed with Rotadest. The product was dried in a vacuum oven (T=50° C.). 2.30 g of compound IV was obtained (R=80.0%).

[0250] V. 17-Lauroyloxy-5-androstene-3-sulfate sodium salt (5) In a flask equipped with a magnetic stirrer, add 25 cm 3 DCM, 10cm 3 of pyridine and 1.42 g (3 mmol) of 3-hydroxy-17-lauroyloxy-5-androstene (compound IV) were weighed in. The reaction mixture was cooled to T=-5° C. and then 0.420 g (250 μl) of chlorosulfonic acid (chlorosulfuric acid) was added in 5 cm 3 of cold dichloromethane was added to the mixture. After 4.0 hours of reaction, 20 cm 3 of 5% sodium bicarbonate solution and 20 cm 3 of water was added to the mixture at room temperature, and then the mixture was stirred vigorously for 30 min.

[0251] The three-phase reaction mixture was then added to a 50 cm 3 After diluting with 100 ml of diisopropyl ether and stirring for 15 minutes, the mixture was filtered. The filtered crude product was dried in a vacuum dryer (T = 50 ° C) and then stirred at T = 50-55 ° C for 30 minutes. After cooling, the target product was filtered and separated into 2 × 10 cm 3 and then dried again in vacuum.

[0252] The dried product was stirred for 50 cm at T = 40 °C. 3 The mixture was suspended in DCM, cooled, filtered, and then dried over 15 cm 3 DCM twice more. Dry again in vacuum oven. Obtain 1.1g product, R=63.9%.

[0253] The NMR spectrum of the compound is shown in FIG. 7B.

[0254] Two other related compounds (compounds C6 and C16) were also obtained via the outlined reaction pathway, in which case intermediate II was first acylated with hexanoyl chloride or palmitoyl chloride, respectively.

[0255] Synthesis of 17-hexanoyloxy-5-androstene-3-sulfate sodium salt (compound C6) Acylation Preparation of 3-acetoxy-17-hexanoyloxy-5-androstene Molecular formula:C 27 H 42 O4MW:430.6 In a flask equipped with a magnetic stirrer, add 25 cm 3 of DCM (or chloroform), 20 cm 3 of pyridine and 4.0 g (12 mmol) of 3-acetoxy-17-hydroxy (II) intermediate were weighed in. Then, this was cooled with cold water while 10 cm 3 2.16 g (16 mmol) of hexanoyl chloride mixed with the solvent was added dropwise at T=20° C. over about 30 min.

[0256] The mixture was allowed to react for 3 hours, after which the reaction mixture was poured into 50 cm 3 Dilute with the solvent and stir (vigorously!) at T = 5-10 °C for about 30 min at V = 150 cm 3 of 10% aqueous hydrochloric acid, then after stirring for 15 min the phases were separated, the organic phase was washed twice with water and once with 10% saline, then dried over sodium sulfate and the solvent was removed under reduced pressure on a Rotadest.

[0257] The product was dissolved in 50 cm at T ≈ 55 °C. 3 Water:MeOH (1:1) mixture, cooled to T≈5° C. and filtered. The filter cake of crystals was further washed with 2×15 cm3 of solvent.

[0258] After drying in vacuum (T=50° C.), 4.1 g of product was obtained (R=79.3%).

[0259] 1) Hydrolysis Synthesis of 3-hydroxy-17-hexanoyloxy-5-androstene Molecular formula:C 25 H 40 O3MW:388.5 Add 200 cm 3 of methanol and 3.9 g (9 mmol) of the diacyl derivative prepared in the previous step were weighed in and added at T=22° C. Then, with vigorous stirring for about 30 minutes, 8.2 cm 3 After stirring for 4 hours, the reaction mixture was diluted with 100 cm 3 of water and 20cm 3 After stirring for 15 minutes, the pH was adjusted to pH = 6.5-7.0 with 5% NaHCO3.

[0260] The product was filtered and poured into a 2 x 25 cm 3 of water:MeOH (1:1) mixture, then 25 cm 3 Washed with water.

[0261] The wet material is placed in a 50cm 3 Dissolve in chloroform and 2 x 25 cm 3 The extract was extracted by shaking with water, then dried over sodium sulfate, after which the solvent was removed under reduced pressure on a Rotadest with a water bath at T≈55° C. Finally, it was dried in a vacuum oven (T=50° C.).

[0262] 2.6 g of product was obtained (R=74.2%).

[0263] 2) Sulfation and salt formation (compound C6) In a flask equipped with a magnetic stirrer, add chloroform (25 cm 3 ) was added to the flask, and 2.34 g (6 mmol) of 3-hydroxy-17-hexanoyloxy-5-androstene was dissolved in the flask. 3 The reaction mixture was cooled to T=0-(-5)°C and a solution of 880 mg of chlorosulfuric acid and T<0°C in chloroform was added dropwise over about 20-30 minutes.

[0264] The mixture was then reacted for an additional 4.0 h at T ≈ 5 °C and then added to 35 cm 3 of 5% sodium bicarbonate solution and 20 cm 3 of water, and 50 cm 3 of diisopropyl ether was slowly added. The heterogeneous reaction mixture was stirred for 15 minutes and the solid product was filtered and then dried in a vacuum oven (T=50° C.). This material was then poured into a 20 cm 3 The mixture was stirred with water for 30 minutes at T=50-55°C.

[0265] After cooling, the desired product was filtered and separated using a 2×10 cm 3 It was washed with water and then dried again in a vacuum dryer.

[0266] Add 50 cm of dry crystalline material. 3 of DCM at T=40° C. for 30 min, which is then cooled and filtered, 3 The mixture was washed with a solvent and then dried again.

[0267] 1.8 g of 17-hexanoyloxy-5-androstene-3-sulfate sodium salt was obtained (R=61.2%).

[0268] The NMR spectrum of the compound is shown in FIG. 7A.

[0269] Synthesis of 17-palmitoyloxy-5-androstene-3-sulfate sodium salt (compound C16) 1) Acylation Synthesis of 3-acetoxy-17-palmitoyloxy-5-androstene Molecular formula:C 37 H 62 O4MW:570.8 In a flask equipped with a magnetic stirrer, 15 cm 3 of chloroform and 10 cm 3 3.0 g (9 mmol) of 3-acetoxy-17-hydroxy-5-androstene was dissolved in a mixture of 100 ml of pyridine and 170 ml of 170 ml of pyridine. 3A mixture of chloroform and 3.3 g (12 mmol) of palmitoyl chloride was added.

[0270] After stirring for an additional 5.0 hours, the reaction mixture was added to 25 cm 3 of chloroform, and then, with vigorous stirring, this was heated at T=5-10°C and V=100cm 3 The mixture was dried over sodium sulfate and the solvent was removed under reduced pressure on a Rotadest (water bath, T≈50° C.).

[0271] The crystallized product was placed in a 50 cm 3 of water:MeOH (1:1) mixture and cooled to T<10° C., which was then filtered into a 2×10 cm 3 The mixture was washed with a solvent of 1000 ml.

[0272] After drying in vacuum (T=50° C.), 3.9 g of product was obtained (R=75.9%).

[0273] 2) Hydrolysis Preparation of 3-hydroxy-17-palmitoyloxy-5-androstene Molecular formula:C 35 H 60 O3MW:528.7 300cm 3 3.42 g (6.0 mmol) of the diacyl compound was weighed into 10 ml of methanol, and the suspension was first heated to T = 55 °C and then rapidly cooled to T ≈ 20-22 °C. Then, over about 30 min, 5.4 cm 3 c = 1.0 M sodium hydroxide and 10 cm 3 A mixture of 100 ml of methanol was added dropwise.

[0274] The reaction mixture was stirred at room temperature for 36.0 hours. During this extended period, a conversion of 85-90% can be achieved. Reacting for an additional period would not be prudent as it would generate unwanted by-products, and the remaining approximately 15% starting material would not interfere with the final sulfation reaction.

[0275] After hydrolysis, 100cm 3 of water and 20cm 3 c = 0.4 M hydrochloric acid was added to the reaction mixture. Then, about 80% of the methanol was distilled on a Rotadest under reduced pressure in a water bath at T ≈ 55 °C. The suspension was cooled, filtered and the solid product was collected in a 2 × 20 cm 3 of water:MeOH (1:1) mixture and then 2 × 20 cm 3 Washed with water.

[0276] After drying in vacuum, 3.0 g, approximately 85% product was obtained (approximately 2.55 g active material; R: ≈ 80%).

[0277] 3) Sulfation and salt formation (compound C16) For normal equipment, 15cm 3 of chloroform, 2.0 cm 3 of pyridine and 2.4 g (about 4 mmol) of the 17-hydroxy derivative were weighed in. The mixture was cooled to T = 0 - (-5) °C and then, over a period of 20 - 30 min, 10 cm 3 A mixture of chloroform and 590 mg of chlorosulfuric acid at T≈0° C. was added dropwise.

[0278] The mixture is then stirred for 5 h at T≈5° C. and then added with 25 cm 3 of 5% sodium bicarbonate solution and 20 cm 3 of water, and 40 cm 3 of diisopropyl ether was slowly added. The three-phase mixture was stirred for 15 min, then the product was filtered and then dried in a vacuum oven (T=50° C.).

[0279] The crude product was then poured into 20 cm 3 of water at T = 50-55 ° C for 30 minutes, and then cooled. After that, the product was filtered and separated into 2 × 10 cm 3 The product was dried in a vacuum oven (T=50° C.). The dried crystalline material was poured into a 50 cm 3 of DCM for 30 min at T=40° C., then cooled and filtered into 2×15 cm 3 The mixture was washed with a solvent of 1000 ml.

[0280] After drying in vacuum, 1.5 g of 17-palmitoyloxy-5-androstene-3-sulfate sodium salt was obtained (R=59%).

[0281] The NMR spectrum of the compound is shown in FIG. 7C.

[0282] result DHEA-derived steroids suppress atherosclerotic plaque formation in mice To investigate whether DHEA-derived steroids have an atherosclerosis-suppressing effect, we performed experiments using an atherosclerotic animal model. Mice that had consumed DHEA-derived steroids were sacrificed after 8 weeks, and the aorta was dissected from the aortic arch to the iliac bifurcation. Lipid staining revealed the formation of atherosclerotic lesions. As shown by the Anphas Oil Red O staining of the whole aorta in Figure 1A, administration of DHEA-derived steroid solution significantly increased ApoE expression compared to mice that had consumed tap water (control). - / - Atherogenic diet-induced atherosclerotic plaque formation in mice was inhibited (Figure 1A). - / - Quantification of Oil Red O staining of whole aortas in mice demonstrated significantly smaller areas of atherosclerotic lesions in the DHEA-derived steroid group (N = 9) compared to the control group (N = 13) (Figure 1B).

[0283] DHEA-derived steroids prevent heart valve calcification ApoE fed a high-fat diet - / - Mice exhibited expanded extracellular matrix deposition in the aortic valve, as demonstrated by von Kossa staining. - / -Calcified nodules appearing on the aortic valve of the mouse heart are caused by a high-fat diet (Fig. 2A; upper center and upper right panels). To demonstrate the anti-calcification effect of DHEA-derived steroids in vivo, DHEA-derived steroids were added to the drinking water of mice. Following this, valvular calcification was evaluated. We concluded that DHEA-derived steroid treatment significantly inhibited the development of calcified nodules on the aortic valve (Fig. 2A, lower center and lower right panels). Analysis of the aortic valve calcified nodule area showed that ApoE mice drinking tap water had a significantly higher calcified nodules than mice drinking tap water. - / - Mice treated with DHEA-derived steroids showed 91.2% lower mineralized area compared to control mice ( Fig. 2B ).

[0284] DHEA-derived steroid treatment increases H2S levels in mouse serum Our research group recently revealed that H2S has a strong ability to inhibit atherosclerotic plaque formation and calcification through alleviating inflammation, preventing LDL oxidation, and scavenging reactive oxygen species [Potor L et al., 2018; Sikura KE et al., 2019]; and to reverse aging-related pathologies [Perridon BW et al., 2016; Zhan JQ et al., 2018; Hou CL et al., 2016]. Therefore, we have demonstrated that H2S has a strong ability to inhibit atherosclerotic plaque formation and calcification through alleviating inflammation, preventing LDL oxidation, and scavenging reactive oxygen species [Potor L et al., 2018; Sikura KE et al., 2019]; and to reverse aging-related pathologies [Perridon BW et al., 2016; Zhan JQ et al., 2018; Hou CL et al., 2016]. - / - Mice treated with steroids derived from DHEA - / - The presence of H2S was measured in mouse plasma obtained from mice. We found that the H2S concentration in DHEA-derived steroid-treated mouse plasma was approximately 2.4-fold higher compared to control mouse plasma (Figure 3).

[0285] DHEA, DHEA-S, C6 and C16 fail to inhibit atherosclerotic plaque formation in mice To investigate the specific inhibitory effect of S2 steroids in atherosclerosis, we carried out animal experiments using the base molecule of S2 steroids derived from DHEA. Furthermore, to further investigate the characteristics of S2, we used dehydroepiandrosterone sulfate (DHEA-S) to investigate the sulfide side chain of S2. Finally, we also investigated the carbon side chain length of S2 in atherosclerosis using 17-hexanoyloxy-5-androstene-3-sulfate sodium salt (C6) and 17-palmitoyloxy-5-androstene-3-sulfate sodium salt (C16) steroids. We found that none of the DHEA (N=11), DHEA-S (N=7), C6 (N=3), and C16 (N=3) molecules could inhibit the formation of atherosclerotic plaques in mice (Figures 4A and B). Furthermore, the inventors found significant mouse mortality in experiments with DHEA (45.5%) and DHEA-S (28.6%) (FIG. 4C).

[0286] DHEA and DHEA-S failed to prevent aortic valve calcification in ApoE knockout mice We investigated the effects of DHEA and DHEA-S on cardiac valve calcification in mice. We found that neither DHEA nor DHEA-S was able to inhibit aortic valve mineralization compared to S2 steroids (Figures 5A and B).

[0287] The extent of established atherosclerotic lesions and aortic valve calcification in ApoE knockout mice is reduced by use of the DHEA-derived steroid S2 From a pharmacological point of view, to the best of our knowledge, there are no drugs on the market that reverse atherosclerosis and vascular calcification. Current drugs only slow the progression of atherosclerosis. Therefore, we investigated the possibility of S2 steroid treatment to reverse already formed atherosclerotic lesions and calcification. In this experiment, we fed mice on an atherogenic diet for 4 or 6 weeks to develop atherosclerotic lesions and calcification. We then changed the atherogenic diet to a chopped diet and added S2 steroids to the mice's water for 4 and 8 weeks. The control group was given tap water only. Surprisingly, we observed significantly fewer atherosclerotic lesions in the case of S2 therapy (Figure 6A-D). We also observed significantly less extracellular matrix mineralization with S2 therapy (Figure 6E and F). [Industrial Applicability]

[0288] Despite the improving trends of the past few years, diseases that develop on the basis of atherosclerosis (myocardial infarction, stroke, lower limb arterial occlusion, Alzheimer's disease, ocular circulatory disorders) still lead the statistics of morbidity and mortality worldwide. Given that billions of people suffer from these diseases, the successful medical innovation of our new compound could result in a drug used for both prevention and treatment without endocrine side effects. The human, scientific, professional and above all economic impacts are hardly estimable.

[0289] This study demonstrates that DHEA-derived S2 steroids are novel antiatherogenic drugs that can be used to prevent the formation of lipid derivatives in atherosclerotic plaques and inhibit cardiac valve calcification. Based on this study, DHEA-derived steroids (S2) induce an increase in H2S, which (I.) scavenges the atherogenic free radical superoxide, (II.) limits the formation of pro-oxidative and pro-inflammatory lipid mediators and the subsequent endothelial responses triggered by these species, and (III.) prevents cardiac valve calcification by controlling osteoblast-like differentiation of valvular interstitial cells.

[0290] In conclusion, this study provides evidence that DHEA-derived steroids are potent preventive and therapeutic agents for atherosclerosis and aortic valve calcification.

[0291] (References) JPEG2025506725000011.jpg188147 JPEG2025506725000012.jpg225147 JPEG2025506725000013.jpg153147

Claims

1. Formula (I) 【Chemistry 1】 (In the formula, R １ is C ７ ~C １３ wherein R is an alkyl group; or any salt or solvate thereof, preferably a pharma- ceutically acceptable salt thereof.

2. R １ But, C ９ ~C １３ Alkyl, C ７ ~C １１ Alkyl, C １１ ~C １３ 2. The compound of claim 1, wherein each alkyl is selected from the group consisting of: alkyl, and preferably each alkyl is a straight chain alkyl.

3. R １ The compound according to claim 1 or 2, wherein is a straight chain alkyl.

4. R １ But, C １１ 4. The compound according to claim 1, wherein the aryl group is an alkyl group.

5. A compound according to any one of claims 1 to 4, wherein the compound is 17-lauroyloxy-5-androstene-3-sulfate, preferably the salt is the sodium salt.

6. 10. A compound according to any one of claims 1 to 5 for use in the treatment of arteriosclerosis, in particular atherosclerosis.

7. 7. A compound for use according to claim 6, particularly for use in the therapeutic treatment of arteriosclerosis, preferably atherosclerosis, wherein the formation of atherosclerotic plaques in the arteries of a subject is reversed.

8. 7. A compound for use according to claim 6 for use in the prevention of arteriosclerosis, preferably atherosclerosis, in particular for use in preventing or reducing or attenuating the formation of atherosclerotic lesions in arteries or atherosclerotic plaques in arteries.

9. 6. A compound according to any one of claims 1 to 5 for use in reducing the amount of reactive oxygen species (ROS) and / or inhibiting the production of ROS, thereby reducing oxidative stress and preventing the onset of or alleviating an oxidative stress disease.

10. One or more of the following conditions: - thrombosis, - coronary heart disease or coronary artery disease, including stenosis of the coronary arteries, - myocardial infarction or heart attack or its consequences, stroke, in particular ischemic and / or hemorrhagic stroke, Thrombosis, for example thrombosis formed on atherosclerotic plaques, - Angina pectoris, - peripheral arterial disease, aortic calcification, e.g. calcific aortic valve disease (CAVD), - atherosclerosis in renal dysfunction, e.g. chronic kidney disease (CKD), - Atherosclerosis in neurodegenerative diseases, A compound for use according to any one of claims 6 to 9 for use in the treatment or prevention of.

11. A compound selected from the group consisting of the compounds according to any one of claims 1 to 5, or a pharma- ceutically acceptable salt thereof. and a pharma- ceutically acceptable carrier or excipient.

12. 12. A pharmaceutical composition or medicament according to claim 11, wherein the administration is oral, preferably the pharmaceutical composition is in the form of a tablet for oral administration.

13. 12. The pharmaceutical composition or medicament of claim 11, wherein the pharmaceutical composition is administered intravenously to a subject or patient.

14. 14. A pharmaceutical composition or medicament according to any one of claims 11 to 13, wherein the pharmaceutical composition is formulated as or comprises a daily dose of 1 to 500 mg / day, preferably 5 to 200 mg / day.

15. 15. A pharmaceutical composition or medicament according to any one of claims 11 to 14 for use in a condition as defined in any one of claims 6 to 10.

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