Compounds, pharmaceutical compositions containing their and medical use thereof for treating or preventing

Abstract

The present disclosure provides a compound capable of exhibiting an effect similar to that of 2-Cys-peroxidase reductase (2-Cys-Prx) in vivo, and a pharmaceutical use thereof, in which the compound has an excellent pharmacological effect and reduced side effects, such as reduced cytotoxicity. The compounds and pharmaceutically acceptable salts thereof of the present disclosure are useful in the treatment or prevention of vascular diseases, particularly ischemic coronary artery diseases, arteriosclerosis, vascular restenosis or pulmonary arterial hypertension. The compounds of the present invention and pharmaceutically acceptable salts thereof are particularly useful in the treatment or prevention of pulmonary arterial hypertension. The invention also provides methods of making the compounds of the disclosure.

Description

Technical Field

[0001] The present disclosure relates to compounds that exhibit 2-Cys-peroxidase reductase (peroxiredoxin) (2-Cys-Prx) peroxidase mimicking activity and pharmaceutically acceptable salts thereof. The present disclosure also relates to pharmaceutical compositions for preventing or treating vascular diseases. That is, the present disclosure also relates to the medical uses of the compounds disclosed herein and pharmaceutically acceptable salts thereof. The present disclosure also relates to methods for preparing compounds that exhibit 2-Cys-Prx activity mimicking effects and pharmaceutically acceptable salts thereof. Background Art

[0002] Arterial vascular disease refers to a state in which fatty substances (plaques) containing cholesterol, phospholipids, calcium, etc. accumulate in the endothelium of blood vessels, leading to inflammation, loss of elasticity and narrowing of arteries, impaired blood supply or increased pressure, and ultimately leading to rupture or detachment of blood vessels. In particular, blockage or occlusion of arterial vessels reduces blood supply and leads to lack of nutrition and oxygen, which is the main cause of vascular disease. Vascular diseases generally include cardiovascular diseases such as arteriosclerosis, heart failure, hypertensive heart disease, arrhythmia, myocardial infarction and angina pectoris, as well as cerebrovascular diseases such as stroke and peripheral vascular disease.

[0003] The method for solving this vascular occlusion includes arterial transplantation by surgical methods, and percutaneous angioplasty, which uses balloon stents to expand blood vessels. Restenosis refers to the situation in which the stenosis of the blood vessel diameter is greater than 50% in the angiography followed up after angioplasty. Although the incidence of restenosis has decreased with the improvement of stent materials, in patients who have undergone angioplasty (balloon dilatation and stent implantation), restenosis still occurs at a rate of about 30%. Although the mechanism of restenosis is not yet clear, it is known that due to damage to vascular endothelial cells during surgery, growth factors and cytokines are locally secreted, and vascular smooth muscle proliferation and migration are induced, thereby narrowing the arterial cavity and causing restenosis. Therefore, the proliferation of smooth muscle cells has recently been proposed as a major clinical problem that limits the efficiency of angioplasty. Therefore, a modified stent that releases drugs that inhibit the proliferation of vascular smooth muscle cells has been developed and used clinically. However, the drugs currently used for this purpose prevent the proliferation of the intimal layer by the cytotoxic mechanism of killing vascular smooth muscle cells, so the drugs have the limitation of toxicity that not only causes the death of smooth muscle cells but also causes the death of endothelial cells. Therefore, there is an urgent need to develop drugs with therapeutic potential that can inhibit the growth of vascular smooth muscle cells while selectively promoting the repair of the damaged endothelial cell layer.

[0004] On the other hand, the pulmonary artery is a blood vessel that transports blood from the right ventricle of the heart to the lungs to renew oxygen. Pulmonary hypertension is defined as a mean pulmonary artery pressure of 25 mmHg or higher at rest and a mean pulmonary artery pressure of 30 mmHg or higher during exercise. Pulmonary hypertension is divided into "idiopathic pulmonary hypertension" for which no specific cause has been identified and which is hereditary, and "associated pulmonary hypertension" associated with other diseases. The latter is associated with collagen vascular diseases (systemic sclerosis, lupus erythematosus, etc.), portal hypertension, HIV infection, congenital heart disease, or diseases caused by drugs or toxins such as appetite suppressants or cocaine. Pulmonary hypertension associated with these specific diseases is not significantly different from idiopathic pulmonary hypertension in terms of natural progression, histopathological findings, and response to treatment.

[0005] Currently, there are methods for treating pulmonary hypertension using common vasodilators. Most vasodilators are calcium channel blockers. However, calcium channel blockers often have no significant effect on pulmonary hypertension and have many side effects. Therefore, it is necessary to find an effective therapeutic agent for pulmonary hypertension.

[0006] On the other hand, "PrxII (also known as peroxide reductase II or Prx2)" is one of the 2-Cys-Prx peroxidases that reduce intracellular hydrogen peroxide (H2O2). PrxII removes hydrogen peroxide produced by platelet-derived growth factor (PDGF) in vascular smooth muscle cells, inhibits site-specific phosphorylation of PDGFRβ and PCL (phospholipase C) γ1, and thus inhibits signal amplification. Through this mechanism, PrxII inhibits the proliferation and migration of smooth muscle cells and reverses the intimal thickening of damaged blood vessels (MH Choi et al., Nature 2005 May 19; 435(7040): 347-53). However, it has been reported that PrxII protects VEGFR2 from oxidative inactivation in vascular endothelial cells and activates VEGF-induced signal transduction (DH Kang et al., Mol Cell. 2011 Nov 18; 44(4): 545-58).

[0007] PCT Publication WO2013-077709 confirms that natural compounds with epidithiodioxapiperazine structures can exhibit similar activities to 2-Cys-Prx in cells. By mimicking the activity of 2-Cys-Prx, natural compounds inhibit PDGF-induced vascular smooth muscle cell proliferation and migration, and promote VEGF-induced vascular endothelial cell proliferation and migration. In addition, in experimental animal models, it was shown that natural compounds inhibit intimal thickening caused by excessive proliferation of vascular smooth muscle cells and promote the repair of the vascular endothelium, and ultimately indicate that such compounds are suitable for preventing or treating vascular diseases.

[0008] In addition, PCT Publication WO2018-008984 demonstrates that drugs capable of mimicking the intracellular activity of 2-Cys-Prx peroxidase are suitable for treating or preventing pulmonary hypertension.

[0009] However, with respect to the compounds disclosed in WO2013-077709 and WO2018-008984, the above-mentioned toxicity problems have not been completely solved. Therefore, there is a continuous need for active agents with advanced pharmacological effects and improved adverse cytotoxicity. Summary of the invention

[0010] Technical issues

[0011] An object of the present disclosure is to provide a compound that exhibits an effect similar to that of 2-Cys-peroxide reductase (2-Cys-Prx) in vivo, and a medical use thereof, the compound having excellent pharmacological effects and reduced side effects, such as reduced cytotoxicity.

[0012] Another object to be solved by the present disclosure is to provide one or more pharmaceutical compositions for treating or preventing one or more vascular diseases, the pharmaceutical compositions comprising a compound as an active ingredient, the compound having excellent pharmacological effects and reduced side effects, such as reduced cytotoxicity. That is, the object to be solved by the present disclosure is to provide a method for treating or preventing one or more vascular diseases, the method comprising administering a therapeutically effective amount of a compound of the present disclosure or a salt thereof to a subject in need of treatment or prevention of one or more vascular diseases.

[0013] Another problem to be solved by the present invention is to provide a process for preparing the specific compounds according to the present disclosure.

[0014] Technical solutions to technical problems

[0015] In order to solve the above problems, one embodiment of the present invention provides a compound represented by the following Chemical Formula 1 or Chemical Formula 2, or a pharmaceutically acceptable salt thereof.

[0016] [Chemical formula 1]

[0017]

[0018] [Chemical formula 2]

[0019]

[0020] In Chemical Formula 1 and Chemical Formula 2,

[0021] n is an integer from 1 to 3,

[0022] R1 and R2 are each independently C 1-3Alkyl (preferably methyl or ethyl), C 1-3 Alkoxy-C 1-3 Alkyl, -(CH2) 1-3 -C(R')(R'')OH, -(CH2) 1-3 -N(R')(R''), -(CH2) 0-3 -Alkenyl, -(CH2) 0-3 -Alkynyl, -(CH2) 0-3 -C(R')(R'')CO2H, -(CH2) 0-5 -heterocycloalkyl, -(CH2) 0-5 -cycloalkyl, -(CH2) 0-5 -aryl (preferably -CH2-phenyl) or -(CH2) 0-5 -heteroaryl (preferably -CH2-pyridyl, -CH2-quinolyl, -CH2-pyrazolyl, -CH2-thiophen-2-yl, -CH2-benzo[d]thiazol-2-yl, -CH2-pyrimidinyl or -CH2-1H-imidazol-4-yl), wherein alkyl, heterocycloalkyl, cycloalkyl, aryl and heteroaryl are unsubstituted or optionally substituted with one or more than one substituent selected from C 1-3 Alkyl, -CF3, C 1-3 Alkoxy, -OCF3, halogen (preferably F), CN, amino, -N(R')(R''), -OH, -COOH, -COO-C 1-3 alkyl and =O, wherein R' and R'' are each independently H or C 1-3 alkyl,

[0023] R3 is C 1-3 Alkyl (preferably methyl), -(CH2) 0-3 -Aryl or -(CH2) 0-3 -heteroaryl, wherein the aryl or heteroaryl group is unsubstituted or optionally substituted with one or more than one substituent selected from C 1-3 Alkyl, -CF3, C 1-3 Alkoxy, -OCF3, halogen, -CN, amino, -OH and -COOH; or

[0024] R2 and R3 are connected together and fused with the piperazine dione represented by Chemical Formula 1 to form one of the following structures:

[0025] ,

[0026] in,

[0027] X is S, SO2, CH2, O or NR6, wherein R6 is H or C 1-3 alkyl,

[0028] R4 is H or C 1-3 Alkyl, and

[0029] R5 is H, C 1-3 Alkyl, -(CH2) 1-2 -Aryl or -(CH2) 1-2 -heteroaryl.

[0030] Another embodiment of the present invention provides a compound represented by the above Chemical Formula 1 or Chemical Formula 2, or a pharmaceutically acceptable salt thereof, wherein

[0031] n is an integer from 1 to 3,

[0032] R1 and R2 are each independently C 1-3 Alkyl, -(CH2) 1-2 -heterocycloalkyl, -(CH2) 1-2 -Aryl or -(CH2) 1-2 -heteroaryl, wherein alkyl, heterocycloalkyl, aryl and heteroaryl are unsubstituted or optionally substituted with one or more than one substituent selected from C 1-3 Alkyl, -CF3, C 1-3 Alkoxy, CN, halogen, -OH, -COOH, and -COO-C 1-3 alkyl,

[0033] R3 is C 1-3 alkyl, -CH2-aryl or -CH2-heteroaryl, wherein the aryl or heteroaryl is unsubstituted or optionally substituted with one or more than one substituent selected from methyl, methoxy, halogen, -CN, amino, -OH and -COOH; or

[0034] R2 and R3 are connected together and fused with the piperazine dione represented by Chemical Formula 1 to form one of the following structures:

[0035] ,

[0036] in,

[0037] X is S, SO2, CH2, O or NR6, wherein R6 is H or C 1-3 alkyl,

[0038] R4 is H or C 1-3 Alkyl, and

[0039] R5 is H, C 1-3 Alkyl or -(CH2) 1-2 -Aryl.

[0040] The bridged epidithiadioxapiperazine-based compound according to the present invention is a novel structure, which is different from the common ETP (epidithiadioxapiperazine) derivatives in natural products and does not exist in natural products. As a result confirmed by the present inventors, the compound of the present invention having such a structure has high chemical stability and biological stability, and has better pharmacological activity and reduced toxicity compared to the existing ETP derivative compounds disclosed in WO2013-077709.

[0041] Compared with the existing ETP derivatives disclosed in WO2013-077709, a methylene group (i.e., bridged ETP) is additionally introduced into the compound of the present invention, thereby giving the disulfide structure a relatively high stability. In other words, when comparing the dihedral angles of the disulfide bonds, the existing ETP derivatives have a dihedral angle of about 10° due to the disulfide bonds on the ring. ° The ring generates considerable stress, thus having high reactivity. As shown by the results of X-ray analysis, the dihedral angle of the disulfide bond on the ring of the compound of the present invention is about 50 ° Up to 60 ° , which means that the stress on the ring is relatively low. This relief of ring stress is expected to reduce toxicity by reducing the indiscriminate reactivity of the compound, while it is expected to increase redox reactivity with thioredoxin (Trx) due to increased flexibility. However, the present invention is not limited to these theoretical mechanisms.

[0042] In addition, the compounds of the present invention have structural features that are conducive to interacting with the sulfhydryl group of the cysteine ​​residue of the thioredoxin CXXC motif, wherein the sulfhydryl group is an electron donor for hydrogen peroxide reduction, and thus the compounds are expected to exhibit higher specificity and high activity. For example, for the conventional ETP derivatives disclosed in WO2013-077709, the pharmacological activity of bulky substituents is poor, but in the compounds of the present invention, even bulky substituents such as aryl and heteroaryl groups exhibit good activity. However, the present invention is not limited to these theoretical speculations.

[0043] For the trisulfide compounds with three sulfurs or the tetrasulfide compounds with four sulfurs in the present disclosure, the connection between sulfurs in the main body can be loosened and then connected to the disulfide compound. In this case, the trisulfide compound with three sulfurs or the tetrasulfide compound with four sulfurs is expected to act as a prodrug of the disulfide compound with two sulfurs. However, the present invention is not limited to these theoretical speculations.

[0044] If a substituent is described as "optionally substituted," the substituent may be (1) unsubstituted or (2) substituted with one or more than one of the substituents specified. If a substitutable position is unsubstituted, the default substituent is hydrogen.

[0045] As used herein, the term "alkyl" refers to a saturated straight or branched chain acyclic hydrocarbon having 1 to 10 carbon atoms, unless the context clearly states otherwise. "Lower alkyl" refers to an alkyl having 1 to 4 carbon atoms. Representative saturated straight chain alkyl groups include -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl, -n-octyl, -n-nonyl, and -n-decyl, while saturated branched chain alkyl groups include -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2-methylbutyl, 3-methylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylbutyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, 2-methyl-4-ethylpentyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2-methyl-4-ethylhexyl, 2,2-diethylpentyl, 3,3-diethylhexyl, 2,2-diethylhexyl, 3,3-diethylhexyl, etc. In a preferred embodiment of the present invention, the alkyl group is methyl.

[0046] As used herein, the term "alkenyl" refers to a saturated straight chain or branched non-cyclic hydrocarbon containing 2 to 10 carbon atoms and at least one carbon-carbon double bond. Representative straight chain and branched (C2 to C 10 )Alkenyl includes -vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, -1-hexenyl, -2-hexenyl, -3-hexenyl, -1-heptenyl, -2-heptenyl, -3-heptenyl, -1-octenyl, -2-octenyl, -3-octenyl, -1-nonenyl, -2-nonenyl, -3-nonenyl, -1-decenyl, -2-decenyl and -3-decenyl. In one embodiment of the invention, alkenyl is vinyl or allyl.

[0047] As used herein, the term "alkynyl" refers to a straight or branched non-cyclic hydrocarbon having 2 to 10 carbon atoms and containing at least one carbon-carbon triple bond, unless the context specifically states otherwise. 10) alkyl includes -ethynyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, -3-methyl-1-butynyl, -4-pentynyl, -1-hexynyl, -2-hexynyl, -5-hexynyl, -1-heptynyl, -2-heptynyl, -6-heptynyl, -1-octynyl, -2-octynyl, -7-octynyl, -1-nonynyl, -2-nonynyl, -8-nonynyl, -1-decynyl, -2-decynyl and -9-decynyl. In one embodiment of the invention, alkynyl is -ethynyl or -propynyl.

[0048] As used herein, if the term "C 1-6 ”, “C1 to 6”, “C1 to C6” or “C1 to C6” means that the number of carbon atoms is 1 to 6. For example, C 1-6 The alkyl group refers to an alkyl group having any integer of 1 to 6 carbon atoms.

[0049] As used herein, the term “C 1-6 "Alkoxy" refers to -O-(alkyl) wherein alkyl is as defined above. For example, methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), butoxy, 1-methylpropoxy (sec-butoxy), 2-methylpropoxy (isobutoxy), 1,1-dimethylethoxy (tert-butoxy), pentyloxy, 1-methylbutoxy, 2-methylbutoxy, 3-methylbutoxy, 1,1-dimethylpropoxy, 1,2-dimethylpropoxy, 2,2-dimethylpropoxy, 1-ethylpropoxy, hexyloxy, Oxy, 1-methylpentyloxy, 2-methylpentyloxy, 3-methylpentyloxy, 4-methylpentyloxy, 1,1-dimethylbutoxy, 1,2-dimethylbutoxy, 1,3-dimethylbutoxy, 2,2-dimethylbutoxy, 2,3-dimethylbutoxy, 3,3-dimethylbutoxy, 1-ethylbutoxy, 2-ethylbutoxy, 1,1,2-trimethylpropoxy, 1,2,2-trimethylpropoxy, 1-ethyl-1-methylpropoxy or 1-ethyl-2-methylpropoxy can be given as examples.

[0050] As used herein, the terms "halogen" and "halogen atom" refer to fluorine, chlorine, bromine or iodine.

[0051] As used herein, the term "cycloalkyl" refers to a monocyclic or polycyclic saturated ring having carbon atoms and hydrogen atoms and having no carbon-carbon multiple bonds, and unless otherwise specified herein, it is a C cycloalkyl having 3 to 7 carbon atoms. 3-7Cycloalkyl. Examples of monocyclic rings include, but are not limited to, (C3 to C7) cycloalkyl, including cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. Examples of polycyclic rings include, but are not limited to, fused bicyclic rings, such as octahydropentalene and decahydronaphthalene; spirocyclic rings, such as spiro[3.3]heptane, spiro[3.4]octane, spiro[3.5]nonane, spiro[4.4]nonane, spiro[4.5]decane, and spiro[5.5]undecane; and bridged bicyclic rings, such as bicyclo[2.1.1]hexane, bicyclo[2.2.1]heptane, and bicyclo[2.2.2]octane. Cycloalkyl can be unsubstituted or optionally substituted. In an embodiment of the present invention, the cycloalkyl is a monocyclic ring.

[0052] The term "heterocycle" or "heterocycloalkyl" refers to a 4- to 7-membered monocyclic saturated ring or a 7- to 12-membered bicyclic saturated ring containing 1 to 4 heteroatoms, the heteroatoms being independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen heteroatom and the sulfur heteroatom may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized. Unless otherwise expressly provided herein, "heterocycloalkyl" refers to "hetero(C 3-7 )cycloalkyl". Representative heterocycles include oxirane, oxetane, tetrahydrofuran, tetrahydropyran, 1,4-dioxane, aziridine, azetidine, pyrrolidine, piperidine, piperazine, pyrrolidone, hydantoin, pentyl propionamide, thiirane, thietane, tetrahydrothiophene, tetrahydrothiopyran, morpholine, tetrahydropyridine, tetrahydropyrimidine, and the like. Heterocycles include bicyclic rings in which a portion of the heterocycle is fused to a benzene ring or a cyclopenta-1,3-diene ring . The heterocyclic ring may be connected via any heteroatom or carbon atom. In addition, the heterocyclic ring also includes fused bicyclic rings, spirocyclic rings and bridged bicyclic rings, in which one or more carbon atoms of the above polycyclic rings are replaced by nitrogen atoms, oxygen atoms or sulfur atoms. For example, when the heteroatom is nitrogen, the heterocyclic ring includes but is not limited to fused heterobicyclic rings, such as octahydrocyclopenta[c]pyrrole, octahydropyrrolo[3,4-c]pyrrole, decahydroisoquinoline and decahydro-2,6-naphthyridine; spirocyclic rings, such as 2-Azaspiro[3.3]heptane, 2,6-diazaspiro[3.3]heptane, 2-azaspiro[3.4]octane, 2,6-diazaspiro[3.4]octane, 2-azaspiro[3.5]nonane, 2,7-diazaspiro[3.5]nonane, 2-azaspiro[4.4]nonane, 2,7-diazaspiro[4.4]nonane, 8-azaspiro[4.5]decane, 2,8-diazaspiro[4.5]decane, 3 ...3.3]heptane, 2,6-diazaspiro[3.3]heptane, 2-azaspiro[3.4]octane, 2,6-diazaspiro[3.4]octane, 2-azaspiro[3.5]nonane, 2,7-diazaspiro[3.5]nonane, 2-azaspiro[4.4]nonane, 2,7-diazaspiro[4.4]nonane, 8-azaspiro[4.5]decane, 2,8-diazaspiro[4.5]decane, 2,8-diazaspiro[4.5]decane, 2,8-diazaspiro[4.5]decane, 2,8-diazaspiro[4.5]decane, 2,8-diazaspiro[4.5]decane, heterospiro[5.5]undecane and 3,9-diazaspiro[5.5]undecane; and bridged heterobicycles such as 2-azabicyclo[2.1.1]hexane, 2-azabicyclo[2.2.1]heptane, 2,5-diazabicyclo[2.2.1]heptane, 2-azabicyclo[2.2.2]octane and 2,5-diazabicyclo[2.2.2]octane. In an embodiment of the invention, the heterocycle is piperidinyl, morpholinyl or piperazinyl.

[0053] As used herein, the term "aryl" refers to a carbocyclic aromatic group containing 5 to 10 ring atoms. Representative examples include, but are not limited to, phenyl, tolyl, xylyl, naphthyl, tetrahydronaphthyl, anthracenyl, Indenyl and The carbocyclic aromatic group may be unsubstituted or optionally substituted. In an embodiment of the present invention, the aryl group is phenyl or naphthyl.

[0054] As used herein, the term "heteroaryl" refers to a 5- to 10-membered aromatic heterocyclic ring having at least one heteroatom selected from nitrogen, oxygen and sulfur and containing at least 1 carbon atom, including monocyclic and bicyclic ring systems. Representative heteroaryl groups include furan, 4H-pyran, pyrrole, imidazole, pyrazole, triazole, tetrazole, pyridine, pyrimidine, pyridazine, pyrazine, triazine, thiophene, Azoles, Isopropylamine Azoles, thiazoles, isothiazoles, oxadiazole, benzofuran, benzothiophene, quinoline, indole, benzo In an embodiment of the present invention, heteroaryl is pyridine, quinoline, pyrazole, thiophene, benzo[d]thiazole, pyrimidine, imidazole, thiazole, isocyanate, phthalazine, quinazoline, 1H-azacycloheptatriene, etc. oxazole, indole, quinazoline or benzimidazole.

[0055] A preferred embodiment of the present invention provides a compound represented by the above Chemical Formula 1 or Chemical Formula 2 or a pharmaceutically acceptable salt thereof, wherein

[0056] n is 1,

[0057] R1 and R2 are each independently C 1-3 alkyl, -CH2-piperidinyl, -CH2-morpholinyl, -CH2-piperazinyl, -CH2-phenyl, -CH2-naphthyl, -CH2-pyridinyl, -CH2-quinolinyl, -CH2-pyrazolyl, -CH2-thiophen-2-yl, -CH2-benzo[d]thiazol-2-yl, -CH2-pyrimidinyl, -CH2-1H-imidazol-4-yl, -CH2-1H-imidazol-2-yl, -CH2-thiazol-4-yl, -CH2-thiazol-5-yl, -CH2-isothiazol-6-yl, -CH2- oxazolyl, -CH2-indol-2-yl, -CH2-indol-3-yl, -CH2-benzimidazol-5-yl, -CH2-quinolin-4-yl, -CH2-quinazol-2-yl or -CH2-quinazol-4-yl, wherein piperidinyl, morpholinyl, piperazinyl, phenyl, naphthyl, pyridinyl, quinolinyl, pyrazolyl, thiophene, benzo[d]thiazole, pyrimidinyl, imidazole, thiazole, isothiophene, oxazolyl, indole, benzimidazole, quinoline and quinazole are unsubstituted or optionally substituted with one or more than one substituent selected from C 1-3 Alkyl, -CF3, C 1-3 Alkoxy, CN, halogen and -COO-C 1-3 alkyl,

[0058] R3 is C 1-3 Alkyl or -CH2-aryl, or,

[0059] R2 and R3 are connected together and fused with the piperazine dione represented by Chemical Formula 1 to form one of the following structures:

[0060] ,

[0061] in,

[0062] X is O or NR6, wherein R6 is methyl,

[0063] R4 is H, and

[0064] R5 is H.

[0065] Among the compounds of Chemical Formula 1, 1,6,8-trimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 1), 6-benzyl-1,8-dimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 2), 1,8-dimethyl-6-(3,4,5-trimethoxybenzyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 3), 6-(3,5-difluorobenzyl)-1,8-dimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 4), 6-(3,5-difluorobenzyl)-1,8-dimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 5), 6-(3,5-difluorobenzyl)-1,8-dimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 6), 6-(3,5-difluorobenzyl)-1,8-dimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 7), 6-(3,5-difluorobenzyl)-1,8-dimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 8), 6-(3,5-difluorobenzyl)-1,8-dimethyl-2,3-dithia-6,8-diazabicyclo Cyclo[3.2.2]nonane-7,9-dione (Compound 4), 1,8-dimethyl-6-(quinolin-2-ylmethyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 5), 1,8-dimethyl-6-(pyridin-2-ylmethyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 6), 11-benzyltetrahydro-5H,7H-4,9a-(epi-iminomethoxy)pyrrolo[2,1-c][1,2,4]dithiazepine-5,10-dione (Compound 7), 12-benzyltetrahydro-5H,10H-4,10a-(epi-iminomethoxy)[1,4] Azino[3,4-c][1,2,4]dithiazepine-5,11-dione (Compound 8), 12-(pyridin-4-ylmethyl)tetrahydro-5H,10H-4,10a-(epi-imidomethoxy)[1,4] Azino(3,4-c)(1,2,4)dithiazepine-5,11-dione (Compound 9), 12-ethyltetrahydro-5H,10H-4,10a-(epi-iminomethyl)[1,4] azino[3,4-c][1,2,4]dithiazepine-5,11-dione (Compound 10), 11-(pyridin-4-ylmethyl)tetrahydro-5H,7H-4,9a-(epi-imidomethyl)pyrrolo[2,1-c][1,2,4]dithiazepine-5,10-dione (Compound 11), 1,8-dimethyl-6-((6-methylpyridin-2-yl)methyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 12), 1,8-dimethyl-6-((1-methyl-1H-pyrazol-4-yl)methyl)-2,3-dithia-6,8-diazabicyclo[3. 2.2]nonane-7,9-dione (Compound 13), 1,8-dimethyl-6-(thien-2-ylmethyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 14), 6-(benzo[d]thiazol-2-ylmethyl)-1,8-dimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 15), 1,6-dimethyl-8-(pyrimidin-2-ylmethyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 16), 1,6-dimethyl-8-((1-methyl-1H-imidazol-4-ylmethyl)- )methyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 17), methyl 4-((1,6-dimethyl-7,9-dioxa-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-8-yl)methyl)benzoate (Compound 18), 1-benzyl-6,8-dimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 19), 6,8-diethyl-1-methyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 20), 12-benzyl-9-methylhexahydro-5H- 4,10a-(Iminomethoxy)pyrazino[2,1-c][1,2,4]dithiazepine-5,11-dione (Compound 21), 12-benzyl-9-methylhexahydro-5H-4,10a-(Iminomethoxy)pyrazino[2,1-c][1,2,4]dithiazepine-5,11-dione (Compound 22), 12-benzyl-9-methylhexahydro-5H-4,10a-(Iminomethoxy)pyrazino[2,1-c][1,2,4]dithiazepine-5,11-dione (Compound 23), 6-benzyl-1,4,8-trimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 24) and 6-benzyl-1,4,8-trimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 25).

[0066] The compound of Chemical Formula 1 or Chemical Formula 2 of the present invention may be used in the form of a pharmaceutically acceptable salt.

[0067] As used herein, the term "pharmaceutically acceptable salt" refers to any salt that retains the desired biological activity and / or physiological activity of the compound and exhibits minimal undesirable toxicological effects. In the present invention, any type of salt can be used without restriction as long as the diketopiperazine ring containing the intramolecular disulfide bridge is retained. Acid addition salts formed by pharmaceutically acceptable free acids are useful as the salts. Acid addition salts can be prepared by conventional methods, for example, by dissolving the compound in an excess of aqueous solution and precipitating the salt using a water-soluble organic solvent such as methanol, ethanol, acetone or acetonitrile. Equimolar amounts of the compound and an acid or alcohol (e.g., ethylene glycol monomethyl ether) in water can be heated, and the mixture is then evaporated to dryness, or the precipitated salt is filtered by suction. At this time, inorganic acids and organic acids can be used as free acids. Hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, tartaric acid, etc. can be used as inorganic acids, and methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, etc. can be used as organic acids. However, the acid used in the present disclosure is not limited thereto.

[0068] In addition, a base can also be used to prepare a pharmaceutically acceptable metal salt. For example, by dissolving the compound in an excess of an alkali metal hydroxide solution or an alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and then evaporating and drying the filtrate to obtain an alkali metal salt or an alkaline earth metal salt. In this case, it is particularly suitable but not limited to prepare a sodium salt, a potassium salt or a calcium salt as the metal salt. In addition, the corresponding silver salt can also be obtained by reacting an alkali metal salt or an alkaline earth metal salt with a suitable silver salt (such as silver nitrate).

[0069] Unless otherwise indicated, the pharmaceutically acceptable salts of the compounds according to the present invention include salts of all possible acidic groups or basic groups. For example, pharmaceutically acceptable salts may include sodium salts, calcium salts and potassium salts of hydroxyl groups, and pharmaceutically acceptable salts of other amino groups include hydrobromates, sulfates, bisulfates, phosphates, hydrogen phosphates, hydrogen phosphates, acetates, succinates, citrates, tartrates, lactates, mandelates, mesylates and tosylates etc. These salts may be manufactured by salt-making methods known in the art.

[0070] As used herein, the phrase "the invention / one or more than one compound of the present invention" includes any one or more than one compound of Chemical Formula 1, and its clathrate, its hydrate, its solvate or its polymorph. Moreover, even if the term "one or more than one compound of the present invention" does not mention its pharmaceutically acceptable salt, the term also includes its salt. In one embodiment, the compounds of the present disclosure include stereochemically pure compounds, such as compounds that are substantially free of (e.g., higher than 85% ee, higher than 90% ee, higher than 95% ee, higher than 97% ee or higher than 99% ee) other stereoisomers. That is, if the compound of Chemical Formula 1 according to the present disclosure or its salt is a tautomeric isomer and / or stereoisomer (e.g., geometric isomer and conformational isomer), these individual isomers and mixtures thereof are also included in the scope of the present disclosure. If the compound of the present disclosure or its salt has an asymmetric carbon in its structure, its active optical isomers and its racemic mixture are also included in the scope of the present disclosure.

[0071] As used herein, the term "polymorph" refers to a solid crystalline form of a compound of the present disclosure or its coordination compound. Different polymorphs of the same compound may exhibit different physical properties, chemical properties and / or spectral properties. Different physical properties include, but are not limited to, stability (e.g., stability to heat or light), compressibility and density (important in formulation and product production), and dissolution rate (which may affect bioavailability). Differences in stability may be due to changes in chemical reactivity (e.g., differential oxidation, which causes a dosage form containing one polymorph to fade faster than a formulation containing another polymorph) or changes in mechanical properties (e.g., tablets may break during storage because a kinetically favorable polymorph is converted into a thermodynamically more stable polymorph) or both (e.g., tablets of one polymorph are more easily broken at high humidity). The different physical properties of polymorphs may affect their processing. For example, one polymorph may be more likely to form a solvate than another polymorph, or more difficult to filter or wash to remove impurities due to, for example, the shape or size distribution of its particles.

[0072] As used herein, the term "solvate" refers to a compound according to the present disclosure or a salt thereof that further comprises a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. Preferred solvents are volatile, non-toxic and acceptable for administration to humans in trace amounts.

[0073] As used herein, the term "hydrate" refers to a compound according to the present disclosure or a salt thereof that further comprises a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.

[0074] As used herein, the term "clathrate" refers to a compound or a salt thereof in the form of a lattice containing spaces (eg, channels) in which guest molecules (eg, solvent or water) are trapped.

[0075] As used herein, the term "purified" means that upon isolation, the isolate is greater than 90% pure, in one embodiment greater than 95% pure, in another embodiment greater than 99% pure, and in another embodiment greater than 99.9% pure.

[0076] "2-Cys-Prx" is a thiol-specific antioxidant enzyme that protects cells by reducing the peroxidase activity of hydrogen peroxide, peroxynitrite and other hydroperoxides in cells. Its functional unit is a homodimer, which has a unique intramolecular redox-active disulfide center that plays an important role in enzyme activity. In the reduced state, each cysteine ​​in the dimer exists in the form of a sulfhydryl group. On the other hand, when reacting with peroxide, the peroxidized cysteine ​​of 2-Cys-Prx is oxidized to a sulfinic acid intermediate, which is different from the cysteine ​​of the other subunits of the dimer that retains the sulfhydryl group. The sulfinic acid group and the sulfhydryl group of the intermediate undergo dehydration condensation to form an intramolecular disulfide bridge. For example, in cells, 2-Cys-Trx is oxidized from a reduced form containing two sulfhydryl groups to an oxidized form in which the two sulfhydryl groups form an intramolecular disulfide bond, thereby reducing intracellular hydroperoxides. At this point, the oxidized form of 2-Cys-Prx containing an intramolecular disulfide bond can be converted into an active reduced form with two sulfhydryl groups by coupling with a redox system, including thioredoxin (Trx) and thioredoxin reductase (Trx reductase; TR); AhpF (alkyl hydroperoxide reductase); trypanothione reductase, trypanothione and tryparedoxin or lipoamide dehydrogenase, dihydrolipoyl succinyltransferase (SucB) and AhpD.

[0077] The compounds of the present invention mimic 2-Cys-Prx activity in cells, specifically couple with thioredoxin (Trx) and thioredoxin reductase (Trx reductase; TR) system, and show excellent activity of inhibiting the proliferation and migration of vascular smooth muscle cells induced by PDGF. In addition, on the contrary, the compounds of the present invention promote the proliferation or migration of vascular endothelial cells induced by VEGF.

[0078] Therefore, one embodiment of the present invention provides a pharmaceutical composition for preventing or treating vascular diseases, the composition comprising a compound represented by Chemical Formula 1 or Chemical Formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0079] Another embodiment of the present invention provides a method for treating or preventing vascular diseases, comprising administering a therapeutically effective amount of a compound represented by Chemical Formula 1 or Chemical Formula 2 or a pharmaceutically acceptable salt thereof to a subject in need of prevention or treatment of vascular diseases or a subject suspected of having vascular diseases.

[0080] Other embodiments of the present invention provide use of a compound represented by Chemical Formula 1 or Chemical Formula 2 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating or preventing vascular diseases.

[0081] In the pharmaceutical composition, method or use, the vascular disease can be hypertension (e.g., pulmonary hypertension), ischemic coronary artery disease (e.g., angina pectoris, myocardial infarction, unstable angina pectoris), cerebral artery occlusion (e.g., stroke), atherosclerosis (e.g., coronary atherosclerosis, carotid atherosclerosis), peripheral arterial occlusive disease (e.g., Burger's disease), thromboembolism, diabetic foot disease, venous ulcer, deep vein thrombosis, vasospasm, arteritis, and vascular smooth muscle hyperplasia, such as vascular restenosis and / or vascular endothelial loss caused by vascular disease. That is, the pharmaceutical composition of the present disclosure can be used to treat or prevent any of the above vascular diseases. In one embodiment of the present invention, the vascular disease is ischemic coronary artery disease, arteriosclerosis, vascular restenosis, or pulmonary hypertension. In another embodiment of the present invention, the vascular disease is pulmonary hypertension.

[0082] In one embodiment of the present invention, vascular restenosis can be caused by vascular transplantation, vascular dissection, arteriosclerosis, intravascular fat accumulation, hypertension, vascular inflammation or angioplasty. Although the cause of vascular restenosis is not yet clear, it is known that after vascular damage caused by various reasons or vascular endothelial damage caused by the device inserted during angioplasty, the surrounding cells will secrete growth factors and / or cytokines through a repair mechanism, and these growth factors and / or cytokines will cause abnormal vascular smooth muscle cell migration and proliferation, thereby causing intimal thickening. Blood vessels include but are not limited to the aorta, carotid artery, coronary artery, peripheral artery, renal artery, etc.

[0083] In one embodiment of the invention, pulmonary hypertension is a type of hypertension that affects the pulmonary artery and the right artery of the heart, and refers to a pulmonary artery mean pressure of 25mmHg or higher than 25mmHg at rest, and a pulmonary artery mean pressure of 30mmHg or higher than 30mmHg during exercise. In a form of pulmonary hypertension, the arterioles and capillaries of the lungs, which are called pulmonary arteries, may become narrow, blocked or damaged, which makes it difficult for blood to flow through the lungs and the pressure in the pulmonary artery increases. Due to the increase in pressure, the right lower chamber (right ventricle) is difficult to pump blood through the lungs, and eventually causes right ventricular hypertrophy, which may lead to heart failure.

[0084] Compositions according to the present invention may also include suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. Compositions are sterile or aseptic, and may be water, buffers, isotonic agents, etc., and solutions are sterile or aseptic. Compositions do not cause allergic reactions or other adverse reactions when applied to animals or humans. Compositions may also contain other ingredients known to those skilled in the art.

[0085] As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic agents, and the like. The use of these media and agents for pharmaceutically active carriers is well known in the art. In addition to conventional media or conventional formulations that are incompatible with the active ingredient, their use in therapeutic compositions is also considered. Supplementary active ingredients may also be added to the composition.

[0086] The composition can be formulated into a preparation such as a liquid, emulsion, suspension or cream, and can be administered parenterally. The amount of the composition can be used as a normal amount for preventing or treating vascular diseases, and is preferably used differently depending on the patient's age, sex, condition, absorption of the active ingredient in the body, inactivation rate, and concomitant drugs, etc.

[0087] In the present invention, the term "prevention" refers to any effect of inhibiting or delaying the onset of vascular disease by administering the pharmaceutical composition according to the present invention, and the term "treatment" refers to any effect of improving or beneficially changing the symptoms caused by vascular disease by administering the pharmaceutical composition.

[0088] In the present invention, the term "subject" refers to all animals that suffer from or may develop vascular diseases, including humans, and vascular diseases can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to the subject. In addition, the pharmaceutical composition of the present invention can also be administered in combination with known therapeutic agents for vascular diseases.

[0089] The compound of the present invention or its pharmaceutically acceptable salt is administered in a therapeutically effective amount. The term "therapeutically effective amount" refers to an amount sufficient to treat a disease without causing side effects at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level depends on the patient's gender, age, weight, health status, severity of disease, drug activity, sensitivity to the drug, method of administration, time of administration, route of administration and excretion rate, duration of treatment, factors including drugs used in combination or concomitantly, and other factors well known in the medical field. Those skilled in the art can easily determine.

[0090] For example, the compound according to the present invention or its pharmaceutically acceptable salt can be used by any suitable route, in the form of pharmaceutical composition adapted to this route and in the dosage effective for the expected treatment. The effective dose of single administration or divided administration is generally about 0.0001mg per kg body weight per day to about 100mg per kg body weight per day, preferably about 0.001mg / kg / day to about 50mg / kg / day. More preferably, the effective dose of single administration or divided administration is about 0.005mg / kg / day to about 20mg / kg / day. According to age, species and treated disease or illness, the dosage level below this range lower limit can be suitable. In other cases, larger dosage can be used without harmful side effects. Larger doses can also be divided into several smaller doses for full-day administration.

[0091] As used herein, the term "administer" refers to the introduction of a predetermined substance into a patient's body by a suitable method, and the route of administration of the composition can be administered by any conventional route as long as the target tissue can be reached. As a method of administration, oral administration, intravenous administration, subcutaneous administration, intraperitoneal administration, etc. can be used but are not limited to. In a preferred embodiment of the present invention, the compound of the present invention is administered orally. In another embodiment of the present invention, the compound of the present invention can be applied locally to the lesion. In particular, the compound of the present invention can be administered using a drug eluting stent. That is, the compound of the present invention can be used in or on a stent and directly applied to a stenotic position. Double balloon catheters, dispensers or microporous balloons can be used for the administration of local drugs, and in particular, stents or sustained-release microparticles can be used for long-term drug delivery.

[0092] In addition, one embodiment of the present invention provides a drug delivery device for local administration, which includes a pharmaceutical composition according to the present invention for preventing or treating vascular diseases. The drug delivery device for local administration may include but is not limited to a double balloon catheter, a distributor, a microporous balloon, a stent, etc., preferably a stent.

[0093] As used herein, the term "stent" refers to a conventional device for intraluminal applications, such as intravascular applications. In particular, a stent refers to a cylindrical medical material that is inserted into a narrowed or blocked blood vessel under X-ray fluoroscopy to improve blood flow in an area where blood flow should be smooth but is blocked. In a preferred embodiment of the present invention, the stent is a sustained-release drug-releasing stent.

[0094] As a method for coating the pharmaceutical composition of the present invention on a stent, conventional coating methods known to those skilled in the art, such as dip coating and / or polymer coating methods, can be used. Dip coating is the simplest coating method, and since only the pharmaceutical composition is coated, it is easy to observe the biological effect of only the drug, but it is not limited thereto. Preferably, the stent of the present invention can be prepared by mixing the composition according to the present invention with a polymer material and then coating the stent so that the composition can be slowly released. The polymer materials that can be used for drug eluting stents are well known in the art, and examples thereof include but are not limited to polyurethane, polyethylene terephthalate, polylactic acid-polyglycolic acid copolymer (PLGA), polycaprolactone, poly(hydroxybutyrate / hydroxyvalerate) copolymer, polyvinyl pyrrolidone, polytetrafluoroethylene, poly(2-hydroxyethyl methacrylate), poly(ether urethane urea), polysiloxane, polyacrylic acid, polyepoxide, polyester, carbamate, polyparaxylene, polyphosphazene polymer, fluoropolymer, polyamide, polyolefin and mixtures thereof.

[0095] The stent may be coated with at least one material selected from polysaccharides, heparin, gelatin, collagen, alginate, hyaluronic acid, alginic acid, carrageenan, chondroitin, pectin, chitosan and derivatives thereof and copolymers thereof, or may be coated to form a layer comprising any of these materials or an antithrombotic agent. Suitably, these materials may be added to a biocompatible top layer as described in US 2006 / 0083772.

[0096] One embodiment of the present invention also provides a method for preparing a compound represented by Chemical Formula 1', which comprises the step of forming an intramolecular disulfide bridge from the compound 3-mercapto-6-methylmercaptopiperazinedione represented by Chemical Formula 2 through an oxidation reaction. As described above, the compound represented by Chemical Formula 1' improves intracellular permeability and mimics 2-Cys-Prx activity by being reduced in cells.

[0097] [Chemical Formula 1']

[0098]

[0099] [Chemical formula 2]

[0100]

[0101] (In Chemical Formula 1′ and Chemical Formula 2, R1, R2, R3, R4, and R5 are the same as those in the above Chemical Formula 1.)

[0102] As described above, unless otherwise specified, if there are stereoisomers in the compounds represented by the chemical formula of the present invention, these stereoisomers are also included in the scope of the compounds and production methods according to the present invention.

[0103] For example, the derivative in which R5 in the chemical formula 1' of the present invention is hydrogen can be prepared by oxidizing 3-mercapto-6-methylmercaptopiperazine dione represented by the chemical formula 2. At this time, the oxidation reaction can be carried out using a reaction known in the art, but there is no limitation. It is preferred but not limited to use iodine (I2) or DDQ (2,3-dichloro-5,6-dicyano-1,4-benzoquinone).

[0104] In Formula 1', derivatives wherein R5 is not hydrogen can be prepared by treating the compound of Formula 1' wherein R5 is hydrogen with a suitable electrophilic reagent.

[0105] Another embodiment of the present invention also provides a method for preparing a 3-mercapto-6-methylmercaptopiperazinedione derivative represented by Chemical Formula 2, which comprises reducing an intermediate represented by Chemical Formula 3 having a ring structure containing 3 to 4 sulfur atoms to form two mercapto groups of Chemical Formula 2.

[0106] [Chemical formula 3]

[0107]

[0108] (In Chemical Formula 3, R1, R2, R3, and R4 are the same as in the above Chemical Formula 1; R5 is H; and n is 2 or 3.)

[0109] At this time, the reduction reaction can be carried out using reactions known in the art, but there is no limitation. It is preferred to use a hydride-based reducing agent to react, such as sodium borohydride or lithium borohydride. It is more preferred but not limited to use sodium borohydride to react.

[0110] Another embodiment of the present invention provides a method for preparing a compound represented by Chemical Formula 3, which comprises reacting a 6-(1-hydroxyalkyl)piperazine-2,5-dione derivative represented by Chemical Formula 4 (wherein R5=hydrogen) with (a) sulfur (S8) and (b) lithium bis(trimethylsilyl)amide (LiHMDS) or sodium bis(trimethylsilyl)amide (NaHMDS).

[0111] [Chemical formula 4]

[0112]

[0113] (In Chemical Formula 4, R1, R2, R3 and R4 are the same as those in the above Chemical Formula 1; R5 is H; and R is a protecting group.)

[0114] In Chemical Formula 4, R is a protecting group, and a hydroxy protecting group known in the art can be used as R, but there is no limitation. It is preferred but not limited to use a silicon-based protecting group such as TBDMS (t-Bu(Me)2silyl) and TMS (trimethylsilyl); an ether-based protecting group such as t-Bu; an ester-based protecting group such as acetyl; and a protecting group such as dihydropyran.

[0115] For conventional 3,6-substituted-piperazinediones, when reacting with sulfur under strong alkaline conditions, a cyclic structure in which sulfur atoms are directly connected to the 3,6-position is formed, while for Chemical Formula 4, a hydroxymethyl group is first eliminated under strong alkaline conditions, followed by the generation of a highly reactive α,β-unsaturated ketone intermediate, and the intermediate immediately reacts with sulfur to form a unique cyclic structure having a methylene bridge similar to Chemical Formula 3. At this time, the number of sulfur atoms forming the cyclic structure is 3 to 4, which varies depending on the structure of the starting material or the reaction conditions (see the mechanism below).

[0116] [Mechanism of formation of chemical formula 3 from chemical formula 4]

[0117]

[0118] One embodiment of the present invention also provides a method for preparing a compound represented by the following chemical formula 1', comprising

[0119] (S1) reacting the compound represented by Chemical Formula 4 with (a) sulfur (S8) and (b) LiHMDS (lithium bis(trimethylsilyl)amide) or NaHMDS (sodium bis(trimethylsilyl)amide) to obtain the compound represented by Chemical Formula 3;

[0120] (S2) reducing the compound of Chemical Formula 3 to obtain the compound represented by Chemical Formula 2; and

[0121] (S3) The compound represented by Chemical Formula 2 forms an intramolecular disulfide crosslink.

[0122] [Chemical formula 4]

[0123]

[0124] [Chemical formula 3]

[0125]

[0126] [Chemical formula 2]

[0127]

[0128] [Chemical Formula 1']

[0129]

[0130] In Chemical Formula 4, Chemical Formula 3, Chemical Formula 2 and Chemical Formula 1′, R1, R2, R3 and R4 are the same as in the above Chemical Formula 1; R5 is H; R is a protecting group; and n is 2 or 3.

[0131] The reagents mentioned in the above methods can be used as reaction reagents in this embodiment.

[0132] Advantageous Effects of the Invention

[0133] The present invention provides a -CH2-S-(S) n -bridged piperazine dione compounds and pharmaceutically acceptable salts thereof. The compounds disclosed herein contain intramolecular disulfide crosslinks to improve cell permeability, and simulate the function of 2-Cys-Prx by being rapidly reduced to compounds with two sulfhydryl groups in cells. In particular, the compounds disclosed herein inhibit the migration and proliferation of vascular smooth muscle cells induced by PDGF by simulating the function of PrxII isoforms in arterial vascular cells, thereby inhibiting intimal thickening, while the compounds disclosed herein promote the migration and proliferation of vascular endothelial cells induced by VEGF, thereby enhancing re-endothelialization. Therefore, according to the compounds disclosed herein and pharmaceutically acceptable salts thereof, pharmaceutical compositions that can be used to prevent or treat vascular diseases. In particular, the compounds disclosed herein or their salts are suitable for treating or preventing vascular diseases, such as ischemic coronary artery disease, arteriosclerosis, vascular restenosis and pulmonary hypertension. BRIEF DESCRIPTION OF THE DRAWINGS

[0134] Figures 1 to 3 are the results of immunoblot analysis regarding the effects of compounds according to the present disclosure on PDGF-induced tyrosine phosphorylation in HASMCs and PASMCs deficient in PrxII. Figure 1 The results are the results of evaluating the concentration-dependent effect of compound 1 on the phosphorylation level of Tyr 857 of PDGF receptor-β (PDGFRβ) in human aortic smooth muscle cells (HASMC), wherein the phosphorylation level of Tyr 857 of PDGF receptor-β was enhanced by PrxII knockdown. Figure 2 The results are those evaluating the effects of Compound 1, Compound 2, Compound 5, Compound 6, Compound 10, Compound 15, Compound 19, Compound 20, Compound 21, Compound 22, and Compound 23 on the degree of Tyr 857 phosphorylation of PDGF receptor-β enhanced by PrxII knockdown in human aortic smooth muscle cells. Figure 3The results are the results of evaluating the effect of compound 8 on the level of intracellular tyrosine phosphorylation induced by PDGF enhanced by PrxII knockdown in human pulmonary artery smooth muscle cells (PASMCs), as well as the effect on the phosphorylation level of signal transduction proteins in PASMCs such as PLC-γ1 downstream of PDGF receptor-β.

[0135] Figures 4 to 6 are the results of immunoblot analysis of the effects of compounds according to the present disclosure on VEGF-induced tyrosine phosphorylation in HAEC and PAEC deficient for PrxII. Figure 4 The results are obtained by evaluating the effects of various concentrations of compound 1 on the phosphorylation level of Tyr 1175 of VEGF receptor-2 (VEGFR2) in HAECs, wherein the phosphorylation level of Tyr 1175 of VEGFR2 was enhanced by PrxII knockdown. Figure 5 The results are those evaluating the effects of Compound 1, Compound 2, Compound 5, Compound 6, Compound 10, Compound 20, Compound 21, and Compound 23 on the degree of VEGF receptor phosphorylation enhanced by PrxII knockdown in HAECs. Figure 6 The results are the results of evaluating the effect of compound 8 on the level of intracellular tyrosine phosphorylation induced by VEGF enhanced by PrxII knockdown in PAEC, and the effect on the phosphorylation level of signal transduction proteins such as ERK2 downstream of VEGF receptor-2 in PAEC.

[0136] Figures 7 to 9 are the results of a preclinical efficacy trial using the Sugen / hypoxia rat model of pulmonary hypertension. Figure 7 It was shown that right ventricular systolic pressure was significantly reduced and right ventricular hypertrophy was inhibited compared with the vehicle control group. Figure 8 is a result showing that the occluded blood vessels in the vehicle control group were reversed. Fig. 9 The results of immunofluorescence staining showed that the proliferation of pulmonary artery endothelial cells and smooth muscle cell hyperplasia were inhibited. DETAILED DESCRIPTION

[0137] Hereinafter, the present invention is described in considerable detail by way of example to help those skilled in the art understand the present invention. However, the following examples are provided by way of illustration and are not intended to limit the scope of the present invention. Obviously, various changes may be made without violating the spirit and scope of the present invention or abandoning all substantial advantages of the present invention.

[0138] The amino acids used as starting materials in the following preparations are readily available in the L-form. However, the synthesis can also be carried out using the D-form or the racemic form.

[0139] Preparation Example 1: Synthesis of 1,6,8-trimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 1)

[0140]

[0141] Step 1. Methyl N-(N-benzyloxycarbonyl)-O-tert-butyl-L-seryl)-N-methyl-L-alanine methyl ester (Intermediate 3)

[0142] To a solution of intermediate 2 (8.32 g, 54.2 mmol, 1.00 eq) in DMF (100 mL) was added DIPEA (35.0 g, 271 mmol, 47.2 mL, 5.00 eq), intermediate 1 (16.0 g, 54.2 mmol, 1.00 eq) and HATU (30.9 g, 81.3 mmol, 1.50 eq) and the reaction mixture was stirred at 25 °C for 12 hours. Water (1000 mL) was added to the reaction mixture and extracted with ethyl acetate (100 mL*2). The combined organic layers were washed with brine (200 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a crude residue. The residue was purified by silica gel chromatography to give intermediate 3 (20.0 g, 48.2 mmol, 88.9% yield) as a yellow oil. MS (ESI) m / z = 395.1 [M+1] +

[0143] Step 2. (3S,6S)-3-(tert-Butoxymethyl)-1,6-dimethylpiperazine-2,5-dione (Intermediate 4)

[0144] To a mixture of intermediate 3 (35.0 g, 88.7 mmol, 1.00 equiv) in MeOH (450 mL) was added Pd / C (3.00 g, 10.0% purity). The mixture was stirred at 50° C. under H2 (50 Psi) for 12 hours. After filtering the mixture, the filtrate was concentrated under reduced pressure to give intermediate 4 (15.0 g, 65.7 mmol, 74.1% yield) as a light yellow oil.

[0145] Step 3. (3S,6S)-3-(tert-Butoxymethyl)-1,4,6-trimethylpiperazine-2,5-dione (Intermediate 5)

[0146] To a solution of intermediate 4 (19.0 g, 83.2 mmol, 1.00 eq) in DMF (500 mL) stirred at 0°C, NaH (3.99 g, 99.9 mmol, 60% purity, 1.20 eq) and MeI (186 g, 1.31 mmol, 81.4 mL, 15.7 eq) were added. The resulting solution was stirred for 12 hours under a nitrogen atmosphere. Water (2 L) was added to the reactant, and the mixture was extracted with CH2Cl2 (300 mL*3). The combined organic phases were washed with brine (500 mL*2), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The product was purified on silica gel to obtain intermediate 5 (19.0 g, 78.4 mmol, 94.2% yield) as a light yellow oil.

[0147] Step 4. (3S,6S)-3-(((tert-butyldimethylsilyl)oxy)methyl)-1,4,6-trimethylpiperazine-2,5-dione (Intermediate 6)

[0148] To a mixture of intermediate 5 (19.0 g, 78.4 mmol, 1.00 eq) in CH2Cl2 (50.0 mL) was added TFA (77.0 g, 675 mmol, 50.0 mL, 8.61 eq) and stirred at 25°C for 12 hours. The reaction mixture was concentrated under reduced pressure, and the concentrate was subsequently dissolved in DMF (200 mL). TBSCl (14.6 g, 96.7 mmol, 11.9 mL, 1.20 eq) and imidazole (16.5 g, 242 mmol, 3.00 eq) were added to the mixture and stirred at 25°C for 1 hour. Water (1000 mL) was added to the reaction mixture, and the mixture was extracted with CH2Cl2 (300 mL*3). The combined organic phases were washed with brine (200 mL*2), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified on a silica gel column to give intermediate 6 (20.0 g, 66.6 mmol, 82.6% yield) as a light yellow solid.

[0149] Step 5. 1,6,8-Trimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 1)

[0150] Sulfur (853 mg, 26.63 mmol, 8 eq) was added to anhydrous THF (53 mL) at room temperature, followed by slow dropwise addition of NaHMDS (10 mL, 1 M in THF, 10 mmol) and subsequent stirring for 10 minutes. Intermediate 6 (1 g, 3.33 mmol, 1 eq) dissolved in THF (12 mL) was added dropwise at room temperature for 10 minutes, followed by slow dropwise addition of additional NaHMDS (8.3 mL, 1 M in THF, 8.3 mmol). After stirring the reaction for 75 minutes, ammonium chloride solution (200 mL) was added to the reaction mixture and extracted with CH2Cl2 (200 mL*2). The combined organic phases were washed with brine (100 mL*2), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified on a silica gel column to give intermediate 7 (trisulfide ring) for the next step.

[0151] Intermediate 7 (200 mg, 0.64 mmol) was dissolved in THF (10 mL) and MeOH (10 mL) at room temperature and then cooled to 0°C. After slowly adding NaBH4 (0.12 g, 3.2 mmol, 5 equivalents) dropwise at 0°C, the temperature was raised to room temperature and the mixture was stirred for 30 minutes. The reaction mixture was concentrated under reduced pressure. Water (10 mL) and NaHCO3 aqueous solution (40 mL) were added to the reaction mixture and stirred at 0°C. The mixture was washed with CH2Cl2 (100 mL*3) to remove impurities. The aqueous layer was acidified with 1N hydrochloric acid aqueous solution and then extracted with CH2Cl2 (200 mL*3). The combined organic phase was washed with brine (100 mL*2), dried over anhydrous Na2SO4, filtered, and partially concentrated to 100 mL under vacuum to obtain intermediate 8, which was used directly in the next step without purification.

[0152] In another reaction vessel, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 120 mg, 0.8 equiv) was dissolved in THF (10 mL) and CH2Cl2 (100 mL). The solution of intermediate 8 obtained above was slowly added dropwise thereto and reacted at room temperature for 30 minutes. An aqueous solution of NaHCO3 (200 mL) was added to the reaction mixture, and then the mixture was extracted with CH2Cl2 (200 mL*3). The combined organic phase was washed with brine (200 mL*2), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified on a silica gel column to obtain 101 mg of compound 1 as a light yellow solid.

[0153]

[0154]

[0155] Preparation Example 2: Synthesis of 6-benzyl-1,8-dimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 2)

[0156]

[0157] Step 1. (3S,6S)-1-Benzyl-6-(tert-butoxymethyl)-3,4-dimethylpiperazine-2,5-dione (Intermediate 2)

[0158] It was synthesized from Intermediate 1 (the same material as Intermediate 4 in Preparation Example 1) in a manner similar to the preparation method of Intermediate 5 in Preparation Example 1. However, benzyl bromide was used instead of iodomethane.

[0159] Step 2. 6-Benzyl-1,8-dimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 2)

[0160] It was prepared by a synthetic method similar to that of compound 1 of Preparation Example 1. That is, intermediate 2 (1.9 g, 5.97 mmol), sulfur (1.53 g, 8 eq.) and NaHMDS (29.8 mL, 1 M in THF, 29.8 mmol) were used to obtain 1 g of intermediate 3. Subsequently, intermediate 3 (0.8 g, 2.35 mmol) and NaBH4 (0.44 g, 5 eq.) were used to obtain intermediate 4, which was then used in the next reaction without purification. Therefore, intermediate 4 and DDQ (0.426 g, 0.8 eq.) were used to obtain compound 2 (0.44 g).

[0161]

[0162]

[0163] Preparation Example 3: Synthesis of 1,8-dimethyl-6-(3,4,5-trimethoxybenzyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 3)

[0164]

[0165] Step 1. Methyl-(3,4,5-trimethoxybenzyl)-L-serine (Intermediate 1)

[0166] To a solution of serine methyl ester (5 g, 32.1 mmol) in MeOH (50 mL) was added 3,4,5-trimethoxybenzaldehyde (3.25 g, 1 eq) and cooled to 0 ° C. After adding Et3N (4.48 mL, 1 eq) and stirring for 2 hours, NaBH4 (2.41 g, 2 eq) was added dropwise at 0 ° C and stirred for 2 hours. After the reactant was cooled to room temperature, the solvent was removed under reduced pressure and then quenched with brine. The reaction mixture was extracted with CH2Cl2, the organic layer was washed with brine and dried over anhydrous Na2SO4. After removing the solvent under reduced pressure, the resulting residue was purified on silica gel to obtain intermediate 1 (2.41 g).

[0167] Step 2. (3S,6S)-3-(Hydroxymethyl)-1,6-dimethyl-4-(3,4,5-trimethoxybenzyl)piperazine-2,5-dione (Intermediate 2)

[0168] At room temperature, NaHCO (1.0 g, 1.5 equivalents) dissolved in a small amount of water was added to a solution of intermediate 1 (2.41 g, 8.1 mmoles) in CH2Cl2 (150 mL). Fmoc-N-Me-chloroalanine (2.77 g, 1 equivalent) dissolved in CH2Cl2 (40 mL) was added to the solution of intermediate 1, and stirred at room temperature for 3 hours. After adding water (100 mL) to the reaction vessel, the organic layer was washed with 1NHCl and salt water, and then the organic layer was concentrated under reduced pressure. EtOH (150 mL) and piperidine (0.8 mL, 8.1 mmoles, 1 equivalent) were added to the obtained product, and the mixture was stirred under reflux for 4 hours. After the reaction mixture was cooled to room temperature, the organic solvent was concentrated under reduced pressure, and the residue was purified on silica gel to obtain intermediate 2 (0.92 g).

[0169] Step 3. (3S,6S)-3-(((tert-butyldimethylsilyl)oxy)methyl)-1,6-dimethyl-4-(3,4,5-trimethoxybenzyl)piperazine-2,5-dione (Intermediate 3)

[0170] Under nitrogen atmosphere, tert-butyldimethylsilyl chloride (TBSCl, 0.78 g, 2 eq.) was added to a stirred solution of intermediate 2 (0.91 g, 2.58 mmoles) in anhydrous CH2Cl2 (20 mL) at 0°C. A solution of imidazole (0.35 g, 2 eq.) in anhydrous CH2Cl2 (5 mL) was slowly added thereto. The mixture was stirred at 20°C for 12 hours and then quenched with water (20 mL). The organic layer was washed twice with water and brine (100 mL) and dried over anhydrous Na2SO4. The organic layer was concentrated, the solvent was completely removed under reduced pressure, and the resulting material was purified on silica gel to obtain intermediate 3 (1.21 g).

[0171] Step 4. 1,8-Dimethyl-6-(3,4,5-trimethoxybenzyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 3)

[0172] It was prepared by a synthetic method similar to that of compound 1 of Preparation Example 1. That is, intermediate 3 (1.2 g, 2.57 mmol), sulfur (0.66 g, 8 eq.) and NaHMDS (14.1 mL, 1 M in THF, 14.1 mmol) were used to obtain 0.53 g of intermediate 4. Intermediate 4 (0.25 g, 0.58 mmol) and NaBH4 (0.1 g, 5 eq.) were used to obtain intermediate 5, which was then used in the next reaction without purification. Therefore, intermediate 5 and DDQ (0.094 g, 0.42 mmol, 0.8 eq.) were used to obtain compound 3 (0.125 g).

[0173]

[0174]

[0175] Preparation Example 4: Synthesis of 6-(3,5-difluorobenzyl)-1,8-dimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 4)

[0176]

[0177] Step 1. Methyl-(3,5-difluorobenzyl)-L-serine (Intermediate 1)

[0178] It was prepared by a synthetic method similar to that of Intermediate 1 of Preparation Example 3. However, 2,4-difluorobenzaldehyde was used instead of 3,4,5-trimethoxybenzaldehyde.

[0179] Step 2. (3S,6S)-1-(3,5-difluorobenzyl)-6-(hydroxymethyl)-3,4-dimethylpiperazine-2,5-dione (Intermediate 2)

[0180] It was prepared by a synthetic method similar to that of Intermediate 2 of Preparation Example 3. However, Fmoc-N-Me-alanine was used instead of Fmoc-N-Me-chloroalanine.

[0181] Step 3. (3S,6S)-3-(((tert-butyldimethylsilyl)oxy)methyl)-4-(3,5-difluorobenzyl)-1,6-dimethylpiperazine-2,5-dione (Intermediate 3)

[0182] It was prepared by a synthetic method similar to that of Intermediate 3 in Preparation Example 3.

[0183] Step 4. 6-(3,5-difluorobenzyl)-1,8-dimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 4)

[0184] It was prepared by a synthetic method similar to that of compound 1 of Preparation Example 1. That is, intermediate 3 (0.86 g, 2.08 mmol), sulfur (0.54 g, 8 eq.) and NaHMDS (11.5 mL, 1 M in THF, 11.5 mmol) were used to obtain 0.26 g of intermediate 4. Intermediate 4 (0.26 g, 0.69 mmol) and NaBH4 (0.13 g, 5 eq.) were used to obtain intermediate 5, which was then used in the next reaction without purification. Therefore, intermediate 5 and DDQ (0.13 g, 0.8 eq.) were used to obtain compound 4 (0.015 g).

[0185]

[0186]

[0187] Preparation Example 5: Synthesis of 1,8-dimethyl-6-(quinolin-2-ylmethyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 5)

[0188]

[0189] Step 1. (3S,6S)-3-(tert-Butoxymethyl)-1,6-dimethyl-4-(quinolin-2-ylmethyl)piperazine-2,5-dione (Intermediate 2)

[0190] It was synthesized from Intermediate 1 (the same material as Intermediate 4 of Preparation Example 1) in a manner similar to the preparation method of Intermediate 2 of Preparation Example 2. However, 2-chloromethylquinoline was used instead of iodomethane.

[0191] Step 2. 1,8-Dimethyl-6-(quinolin-2-ylmethyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 5)

[0192] It was prepared by a synthetic method similar to that of compound 1 of Preparation Example 1. That is, intermediate 2 (1.15 g, 3.11 mmoles), sulfur (0.79 g, 8 equivalents) and NaHMDS (15.6 mL, 1 M in THF, 15.6 mmoles) were used to obtain 0.72 g (59% yield) of intermediate 3. Intermediate 4 was obtained using intermediate 3 (0.55 g, 1.44 mmoles) and NaBH4 (0.26 g, 5 equivalents) and was then used for the next reaction without purification. Therefore, intermediate 4 and DDQ (0.255 g, 0.8 equivalents) were used to obtain compound 5 (0.25 g).

[0193]

[0194]

[0195] Preparation Example 6: Synthesis of 1,8-dimethyl-6-(pyridin-2-ylmethyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 6)

[0196]

[0197] Step 1. (3S,6S)-3-(tert-Butoxymethyl)-1,6-dimethyl-4-(pyridin-2-ylmethyl)piperazine-2,5-dione (Intermediate 2)

[0198] It was synthesized from Intermediate 1 (the same material as Intermediate 4 in Preparation Example 1) in a similar manner to the preparation method of Intermediate 2 in Preparation Example 2. However, 2-chloromethylpyridine was used instead of benzyl bromide.

[0199] Step 2. 1,8-Dimethyl-6-(pyridin-2-ylmethyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 6)

[0200] It was prepared by a synthetic method similar to that of compound 1 of Preparation Example 1. That is, 0.68 g of intermediate 3 was obtained using intermediate 2 (1.05 g, 3.29 mmoles), sulfur (0.84 g, 8 equiv.) and NaHMDS (16.4 mL, 1 M in THF, 16.4 mmoles). Intermediate 4 was obtained using intermediate 3 (0.58 g, 1.70 mmoles) and NaBH4 (0.32 g, 5 equiv.), which was then used in the next reaction without purification. Therefore, compound 6 (0.23 g) was obtained using intermediate 4 and DDQ (0.34 g, 0.8 equiv.).

[0201]

[0202]

[0203] Preparation Example 7: Synthesis of 11-benzyltetrahydro-5H,7H-4,9a-(epi-iminomethyl)pyrrolo[2,1-c][1,2,4]dithiazepine-5,10-dione (Compound 7)

[0204]

[0205] Step 1. N-((Benzyloxycarbonyl)-O-(tert-butyl)-L-seryl-L-proline methyl ester (Intermediate 3)

[0206] It was prepared by a synthesis similar to that of Intermediate 3 in Preparation Example 1. However, proline methyl ester was used instead of N-methylalanine methyl ester.

[0207] Step 2. (3S,8aR)-3-(tert-Butoxymethyl)hexahydropyrrolo[1,2-a]pyrazine-1,4-dione (Intermediate 4)

[0208] To a mixture of intermediate 3 (98.0 g, 1.00 equiv.) in MeOH (980 mL) was added Pd / C (9.80 g, 10% purity), and the mixture was stirred at 70 ° C. for 12 hours under H2 (50 psi). After filtration, the filtrate was concentrated. The residue was purified by column chromatography to give intermediate 4 (35.0 g, 59.8% yield, 99.0% purity) as a white solid.

[0209] Step 3. (3S,8aR)-2-Benzyl-3-(tert-butoxymethyl)hexahydropyrrolo[1,2-a]pyrazine-1,4-dione (Intermediate 5)

[0210] It was synthesized by a preparation method similar to that of Intermediate 2 in Preparation Example 2.

[0211] Step 4. 11-Benzyltetrahydro-5H,7H-4,9a-(epi-imidomethoxy)pyrrolo[2,1-c][1,2,4]dithiazepine-5,10-dione (Compound 7)

[0212] It was prepared by a synthetic method similar to that of compound 1 of Preparation Example 1. That is, intermediate 5 (1.5 g, 4.54 mmoles), sulfur (1.16 g, 8 equiv.) and NaHMDS (22.7 mL, 1 M in THF, 22.7 mmoles) were used to obtain 0.43 g of intermediate 6. Intermediate 6 (0.41 g, 1.16 mmoles) and NaBH4 (0.22 g, 5 equiv.) were used to obtain intermediate 7, which was then used in the next reaction without purification. Therefore, intermediate 7 and DDQ (0.24 g, 0.8 equiv.) were used to obtain compound 7 (0.085 g).

[0213]

[0214]

[0215] Preparation Example 8: 12-Benzyltetrahydro-5H,10H-4,10a-(epi-imidomethyl)[1,4] Synthesis of Azino[3,4-c][1,2,4]dithiazepine-5,11-dione (Compound 8)

[0216]

[0217] Step 1. Methyl 4-(N-((Benzyloxy)carbonyl)-O-(tert-butyl)-L-seryl)morpholine-3-carboxylate (Intermediate 3)

[0218] It was prepared by a synthesis similar to that of Intermediate 3 in Preparative Example 7. However, morpholine-3-carboxylic acid methyl ester was used instead of proline methyl ester.

[0219] Step 2. (7S)-7-(tert-Butyloxymethyl)hexahydropyrazino[2,1-c][1.4] Oxazine-6,9-dione (Intermediate 4)

[0220] It was prepared by a synthesis similar to that of Intermediate 4 in Preparative Example 7.

[0221] Step 3. (7S)-8-Benzyl-7-(tert-butoxymethyl)hexahydropyrazino[2,1-c][1.4] Oxazine-6,9-dione (Intermediate 5)

[0222] It was prepared by a synthesis similar to that of Intermediate 5 in Preparation Example 7. Two isomers (diastereomers) were obtained, one of which (5.20 g, 22.4% yield, 99.3% purity) was a white solid, and the other isomer (5.10 g, 21.8% yield, 98.6% purity) was a yellow oil.

[0223]

[0224]

[0225]

[0226] Step 4. 12-Benzyltetrahydro-5H,10H-4,10a-(epi-imidomethoxy)[1,4] Azino[3,4-c][1,2,4]dithiazepine-5,11-dione (Compound 8)

[0227] It was prepared by a synthetic method similar to that of compound 1 of Preparation Example 1. That is, intermediate 5 (any isomer, 2.0 g, 5.77 mmoles), sulfur (1.48 g, 8 equivalents) and NaHMDS (28.9 mL, 1 M in THF, 28.9 mmoles) were used to obtain 1.24 g (58.3% yield) of intermediate 6. Intermediate 6 (1.2 g, 3.26 mmoles) and NaBH4 (0.62 g, 5 equivalents) were used to obtain intermediate 7, which was then used in the next reaction without purification. Therefore, intermediate 7 and DDQ (0.67 g, 2.93 mmoles, 0.8 equivalents) were used to obtain 0.45 g (41% yield) of compound 8.

[0228]

[0229]

[0230]

[0231] Preparation Example 9: 12-(pyridin-4-ylmethyl)tetrahydro-5H,10H-4,10a-(epi-imidomethyl)[1,4] Synthesis of Azino(3,4-c)(1,2,4)dithiazepine-5,11-dione (Compound 9)

[0232]

[0233] Step 1. (7S)-7-(tert-Butyloxymethyl)-8-(pyridin-4-ylmethyl)hexahydropyrazino[2,1-c][1,4] Oxazine-6,9-dione (Intermediate 2)

[0234] It was synthesized from Intermediate 1 (the same material as Intermediate 4 in Preparation Example 8) in a similar manner to the preparation method of Intermediate 5 in Preparation Example 8. However, 4-chloromethylpyridine was used instead of benzyl bromide to give a brown oil.

[0235] Step 2. 12-(Pyridin-4-ylmethyl)tetrahydro-5H,10H-4,10a-(epi-imidomethoxy)[1,4] Azino(3,4-c)(1,2,4)dithiazepine-5,11-dione (Compound 9)

[0236] It was prepared by a synthetic method similar to that of compound 1 of Preparation Example 1. That is, 0.68 g of the trisulfide intermediate was obtained as a yellow solid using intermediate 2 (0.2 g, 0.58 mmol), sulfur (0.14 g, 8 eq.) and NaHMDS (1.14 mL, 2M in THF, 5 eq.). After the trisulfide intermediate (0.68 g, 1.84 mmol) was reduced with NaBH4 (0.2 g, 2.9 eq.), it was used in the next reaction without purification. Therefore, the reduced intermediate and iodine (0.47 g, 1.0 eq.) were used to obtain compound 9 (41 mg) as a white solid.

[0237]

[0238]

[0239] Preparation Example 10: 12-Ethyltetrahydro-5H,10H-4,10a-(epi-iminomethyl)[1,4] Synthesis of Azino[3,4-c][1,2,4]dithiazepine-5,11-dione (Compound 10)

[0240]

[0241] Step 1. (7S)-7-(tert-Butyloxymethyl)-8-ethylhexahydropyrazino[2,1-c][1,4] Oxazine-6,9-dione (Intermediate 2)

[0242] It was synthesized from Intermediate 1 (the same material as Intermediate 4 in Preparation Example 8) in a similar manner to the preparation method of Intermediate 5 in Preparation Example 8. However, ethyl bromide was used instead of benzyl bromide.

[0243] Step 2. 12-Ethyltetrahydro-5H,10H-4,10a-(epi-iminomethoxy)[1,4] Azino[3,4-c][1,2,4]dithiazepine-5,11-dione (Compound 10)

[0244] It was prepared by a synthetic method similar to that of Compound 1 of Preparation Example 1. That is, 0.35 g of the trisulfur intermediate was obtained using Intermediate 2 (0.57 g, 2.0 mmoles), sulfur (0.51 g, 8 eq.) and NaHMDS (5.0 mL, 2M in THF, 5 eq.). After the trisulfur intermediate (0.35 g, 1.2 mmoles) was reduced with NaBH4 (0.23 g, 5 eq.), it was used in the next reaction without purification. Thus, Compound 10 (130 mg) was obtained as a white solid using the reduced intermediate and DDQ (0.24 g, 0.9 eq.).

[0245]

[0246] Preparation Example 11: Synthesis of 11-(pyridin-4-ylmethyl)tetrahydro-5H,7H-4,9a-(epi-iminomethyl)pyrrolo[2,1-c][1,2,4]dithiazepine-5,10-dione (Compound 11)

[0247]

[0248] Step 1. (7S)-7-(tert-Butyloxymethyl)-8-ethylhexahydropyrazino[2,1-c][1,4] Oxazine-6,9-dione (Intermediate 2)

[0249] It was synthesized from Intermediate 1 (the same material as Intermediate 4 in Preparation Example 7) in a similar manner to the preparation method of Intermediate 5 in Preparation Example 7. However, 4-chloromethylpyridine was used instead of benzyl bromide.

[0250] Step 2. 11-(Pyridin-4-ylmethyl)tetrahydro-5H,7H-4,9a-(epi-imidomethoxy)pyrrolo[2,1-c][1,2,4]dithiazepine-5,10-dione (Compound 11)

[0251] It was prepared by a synthetic method similar to that of Compound 1 of Preparation Example 1. That is, 0.6 g of the trisulfide intermediate was obtained as a yellow solid using Intermediate 2 (0.2 g, 0.60 mmol), sulfur (0.15 g, 8 eq.) and NaHMDS (1.2 mL, 2M in THF, 5 eq.). After the trisulfide intermediate (1.0 g, 2.83 mmol) was reduced with NaBH4 (0.37 g, 3.5 eq.), it was used in the next reaction without purification. Thus, Compound 11 (40 mg) was obtained as a white solid using the reduced intermediate and iodine (0.72 g, 1.0 eq.).

[0252]

[0253]

[0254] Preparation Example 12: Synthesis of 1,8-dimethyl-6-((6-methylpyridin-2-yl)methyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 12)

[0255]

[0256] Step 1. (3S,6S)-3-(tert-Butoxymethyl)-1,6-dimethyl-4-((6-methylpyridin-2-yl)methyl)piperazine-2,5-dione (Intermediate 2)

[0257] It was synthesized from Intermediate 1 (the same material as Intermediate 4 in Preparation Example 1) in a manner similar to the preparation method of Intermediate 2 in Preparation Example 5. However, 6-methyl-2-chloromethylpyridine was used instead of 2-chloromethylquinoline.

[0258] Step 2. 1,8-Dimethyl-6-((6-methylpyridin-2-yl)methyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 12)

[0259] It was prepared by a synthetic method similar to that of Compound 1 of Preparation Example 1. That is, 0.47 g of the trisulfide intermediate was obtained as a yellow oil using Intermediate 2 (1.0 g, 3 mmoles), sulfur (0.77 g, 8 equiv.) and NaHMDS (7.5 mL, 2M in THF, 5 equiv.). After reducing the trisulfide intermediate (0.47 g, 1.32 mmoles) with NaBH4 (0.15 g, 3.5 equiv.), it was used in the next reaction without purification. Thus, 140 mg of Compound 12 was obtained as a pale yellow solid using the reduced intermediate and iodine (0.672 g, 2.0 equiv.).

[0260]

[0261]

[0262] Preparation Example 13: Synthesis of 1,8-dimethyl-6-((1-methyl-1H-pyrazol-4-yl)methyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 13)

[0263]

[0264] Step 1. (3S,6S)-3-(tert-Butoxymethyl)-1,6-dimethyl-4-((1-methyl-1H-pyrazol-4-yl)methyl)piperazine-2,5-dione (Intermediate 2)

[0265] It was synthesized from Intermediate 1 (the same material as Intermediate 4 in Preparation Example 1) in a manner similar to the preparation method of Intermediate 2 in Preparation Example 5. However, 1-methyl-4-chloromethylpyrazole was used instead of 2-chloromethylquinoline.

[0266] Step 2. 1,8-Dimethyl-6-((1-methyl-1H-pyrazol-4-yl)methyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 13)

[0267] It was prepared by a synthetic method similar to that of Compound 1 of Preparation Example 1. That is, 0.5 g of the trisulfur intermediate was obtained as a yellow oil using Intermediate 2 (1.0 g, 3.1 mmoles), sulfur (0.80 g, 8 equiv.) and NaHMDS (7.75 mL, 2M in THF, 5 equiv.). After reducing the trisulfur intermediate (0.5 g, 145 mmoles) with NaBH4 (0.16 g, 3 equiv.), it was used in the next reaction without purification. Thus, 201 mg of Compound 13 was obtained as a pale white solid using the reduced intermediate and iodine (1.1 g, 3.0 equiv.).

[0268]

[0269]

[0270] Preparation Example 14: Synthesis of 1,8-dimethyl-6-(thiophen-2-ylmethyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 14)

[0271]

[0272] Step 1. (3S,6S)-3-(tert-Butoxymethyl)-1,6-dimethyl-4-(thiophen-2-ylmethyl)piperazine-2,5-dione (Intermediate 2)

[0273] It was synthesized from Intermediate 1 (the same material as Intermediate 4 in Preparation Example 1) in a manner similar to the preparation method of Intermediate 2 in Preparation Example 5. However, 2-chloromethylthiophene was used instead of 2-chloromethylquinoline.

[0274] Step 2. 1,8-Dimethyl-6-(thiophen-2-ylmethyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 14)

[0275] It was prepared by a synthetic method similar to that of compound 1 of Preparation Example 1. That is, 0.5 g of the trisulfide intermediate was obtained as a yellow oil using intermediate 2 (1.0 g, 3.1 mmoles), sulfur (0.79 g, 8 equiv.) and NaHMDS (7.7 mL, 2M in THF, 5 equiv.). After the trisulfide intermediate (0.5 g, 1.45 mmoles) was reduced with NaBH4 (0.16 g, 3 equiv.), it was used in the next reaction without purification. Thus, 201 mg of compound 14 was obtained as a pale white solid using the reduced intermediate and iodine (1.1 g, 3.0 equiv.).

[0276]

[0277]

[0278] Preparation Example 15: Synthesis of 6-(Benzo[d]thiazol-2-ylmethyl)-1,8-dimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 15)

[0279]

[0280] Step 1. (3S,6S)-1-(Benzo[d]thiazol-2-ylmethyl)-6-(tert-butoxymethyl)-3,4-dimethylpiperazine-2,5-dione (Intermediate 2)

[0281] It was synthesized from Intermediate 1 (the same material as Intermediate 4 in Preparation Example 1) in a manner similar to the preparation method of Intermediate 2 in Preparation Example 5. However, 2-chloromethylbenzothiazole was used instead of 2-chloromethylquinoline.

[0282] Step 2. 6-(Benzo[d]thiazol-2-ylmethyl)-1,8-dimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 15)

[0283] It was prepared by a synthetic method similar to that of compound 1 of Preparation Example 1. That is, 0.47 g of the trisulfide intermediate was obtained as a brown oil using intermediate 2 (0.5 g, 1.33 mmoles), sulfur (0.34 g, 8 equiv.) and NaHMDS (3.3 mL, 2M in THF, 5 equiv.). After the trisulfide intermediate (0.47 g, 1.18 mmoles) was reduced with NaBH4 (0.13 g, 3 equiv.), it was used in the next reaction without purification. Therefore, the reduced intermediate and iodine (0.6 g, 2.0 equiv.) were used to obtain compound 15 (152 mg) as a yellow solid.

[0284]

[0285]

[0286] Preparation Example 16: Synthesis of 1,6-dimethyl-8-(pyrimidin-2-ylmethyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 16)

[0287]

[0288] Step 1. O-(tert-butyl)-N-(2-chloropropionyl)-L-serine methyl ester (Intermediate 1)

[0289] 2-Chloropropionyl chloride (26.1 g, 205 mmol) was added to a solution of tert-butyloxyserine methyl ester (39.5 g, 186 mmol, 1 equivalent) and TEA (37.8 g, 51.9 mL, 2 equivalents) in dichloromethane (400 mL). The reaction mixture was stirred at 0 ° C for 0.5 hours and at 25 ° C for 16 hours, then diluted with water (100 mL) and extracted with DCM (50 mL). The combined organic layer was washed with brine (100 mL), dried with Na2SO4, filtered, and concentrated under reduced pressure to obtain intermediate 1 (48.6 g, 183 mmol, 97.9% yield) as a yellow liquid. It was used for the next step without further purification.

[0290] Step 2. 3-(tert-Butoxymethyl)-6-methyl-1-(pyrimidin-2-ylmethyl)piperazine-2,5-dione (Intermediate 2)

[0291] To a mixture of intermediate 1 (10.8 g, 40.6 mmol, 1.00 eq), K2CO3 (11.2 g, 81.3 mmol, 2.00 eq) and KI (3.37 g, 20.3 mmol, 0.50 eq) in DMF (120 mL) was added pyrimidine-2-ylmethylamine (4.44 g, 40.64 mmol, 1 eq) and stirred for 12 hours. The reaction solution was filtered and the filtrate was concentrated. The product obtained after purification by preparative HPLC was dissolved in toluene, DMAP (433 mg, 0.2 eq) was added thereto, and the mixture was stirred at 130 ° C for 36 hours. The reaction mixture was filtered and the filtrate was concentrated. The residue was purified by preparative HPLC to obtain intermediate 2 (6.04 g, 17.7 mmol, 43.5% yield, 99.0% purity) as a yellow oil.

[0292] Step 3. 3-(tert-Butoxymethyl)-4,6-dimethyl-1-(pyrimidin-2-ylmethyl)piperazine-2,5-dione (Intermediate 3)

[0293] To the solution of intermediate 2 (1.80 g, 5.88 mmol, 1.00 eq) in DMF (18 mL) was added NaH (258 mg, 6.46 mmol, 60% purity, 1.10 eq), and the mixture was stirred at 0 ° C for 0.5 hours, and then MeI (834 mg, 5.88 mmol, 365 uL, 1.00 eq) was added to the reaction mixture at 0 ° C. The reaction was stirred at 25 ° C for 2.5 hours and quenched by adding MeOH (3 mL), and then concentrated under reduced pressure. The residue was purified by preparative HPLC to obtain intermediate 3 (1.6 g, 4.99 mmol, 85.0% yield) as a brown oil.

[0294] Step 4. 1,6-Dimethyl-8-(pyrimidin-2-ylmethyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 16)

[0295] It was prepared by a synthetic method similar to that of compound 1 of Preparation Example 1. That is, 0.64 g of the trisulfide intermediate was obtained as a white solid using intermediate 3 (1.2 g, 3.75 mmoles), sulfur (0.85 g, 8 eq.) and NaHMDS (14.2 mL, 2 M in THF, 5 eq.). After the trisulfide intermediate (0.25 g, 0.78 mmoles) was reduced with NaBH4 (0.11 g, 4 eq.), it was used in the next reaction without purification. Thus, 110 mg of compound 16 was obtained as a pale white solid using the reduced intermediate and iodine (0.28 g, 1.5 eq.).

[0296]

[0297]

[0298] Preparation Example 17: Synthesis of 1,6-dimethyl-8-((1-methyl-1H-imidazol-4-yl)methyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 17)

[0299]

[0300] Step 1. 3-(tert-Butoxymethyl)-6-methyl-1-(pyrimidin-2-ylmethyl)piperazine-2,5-dione (Intermediate 2)

[0301] It was synthesized by a similar preparation method to Intermediate 2 in Preparation Example 16. However, 1-methylimidazolmethylamine was used instead of pyrimidin-2-ylmethylamine.

[0302] Step 2. 3-(tert-Butoxymethyl)-4,6-dimethyl-1-((1-methyl-1H-imidazol-4-yl)methyl)piperazine-2,5-dione (Intermediate 3)

[0303] It was synthesized by a preparation method similar to that of Intermediate 2 in Preparation Example 16.

[0304] Step 3. 1,6-Dimethyl-8-((1-methyl-1H-imidazol-4-yl)methyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 17)

[0305] It was prepared by a synthetic method similar to that of Compound 1 of Preparation Example 1. That is, 0.36 g of the trisulfide intermediate was obtained as a yellow oil using Intermediate 3 (0.5 g, 1.55 mmoles), sulfur (0.4 g, 8 eq.) and NaHMDS (5.9 mL, 2 M in THF, 5 eq.). After the trisulfide intermediate (0.25 g, 0.73 mmoles) was reduced with NaBH4 (0.13 g, 5 eq.), it was used in the next reaction without purification. Thus, 106 mg of Compound 17 was obtained as a pale white solid using the reduced intermediate and iodine (0.18 g, 1 eq.).

[0306]

[0307]

[0308] Preparation Example 18: Synthesis of methyl 4-((1,6-dimethyl-7,9-dioxa-2,3-dithia-6,8-diazabicyclo[3.2.2]nonan-8-yl)methyl)benzoate (Compound 18)

[0309]

[0310] Step 1. Methyl 4-((3-(tert-butoxymethyl)-6-methyl-2,5-dioxapiperazin-1-yl)methyl)benzoate (Intermediate 2)

[0311] It was synthesized by a similar preparation method to Intermediate 2 of Preparation Example 16. However, 4-methoxyphenylmethylamine was used instead of pyrimidin-2-ylmethylamine.

[0312] Step 2. Methyl 4-((3-(tert-butoxymethyl)-4,6-dimethyl-2,5-dioxapiperazin-1-yl)methyl)benzoate (Intermediate 3)

[0313] It was synthesized by a preparation method similar to that of Intermediate 3 in Preparation Example 16.

[0314] Step 3. Methyl 4-((1,6-dimethyl-7,9-dioxa-2,3-dithia-6,8-diazabicyclo[3.2.2]nonan-8-yl)methyl)benzoate (Compound 18)

[0315] It was prepared by a synthetic method similar to that of compound 1 of Preparation Example 1. That is, 1.0 g of the trisulfide intermediate was obtained as a yellow oil using intermediate 3 (1.57 g, 4.16 mmoles), sulfur (1.1 g, 8 eq.) and NaHMDS (15.7 mL, 1 M in THF, 5 eq.). After the trisulfide intermediate (0.13 g, 0.32 mmoles) was reduced with NaBH4 (0.015 g, 1.2 eq.), it was used in the next reaction without purification. Thus, compound 18 (113 mg) was obtained as a pale yellow solid using the reduced intermediate and iodine (0.08 mg, 1 eq.).

[0316]

[0317]

[0318] Preparation Example 19: Synthesis of 1-benzyl-6,8-dimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 19)

[0319]

[0320] Step 1. 3-Benzyl-6-(tert-butoxymethyl)-1,4-dimethylpiperazine-2,5-dione (Intermediate 2)

[0321] To a solution of compound 1 (15.0 g, 33.6 mmol, 1.00 eq) in DMF (100 mL) was added tBuOK (1 M, 134 mL, 4.00 eq) and MeI (29.2 g, 205 mmol, 12.8 mL, 6.12 eq). The mixture was stirred at 25 ° C for 2 hours, then quenched by adding 300 mL of water at 0 ° C, and extracted with ethyl acetate (100 mL * 2). The combined organic layer was washed with brine (50 mL * 6), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by reverse phase HPLC (column: Kromasil Eternity XT 250*80mm*10um; mobile phase: [water(NH4HCO3)-ACN]; B%: 29% to 59%, 20 minutes) to give Intermediate 2 (3.20 g, 9.86 mmol, 14.7% yield, 98.1% purity) as a white solid.

[0322] Step 2. 1-Benzyl-6,8-dimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 19)

[0323] It was prepared by the same synthetic method as compound 1 of Preparation Example 1. That is, 0.5 g of the trisulfide intermediate was obtained as a yellow oil using intermediate 2 (1.0 g, 3.14 mmoles), sulfur (0.9 g, 8 eq.) and NaHMDS (15.6 mL, 1 M in THF, 5 eq.). After the trisulfide intermediate (0.5 g, 1.47 mmoles) was reduced with NaBH4 (0.3 g, 5 eq.), it was used in the next reaction without purification. Therefore, the reduced intermediate and iodine (0.6 g, 1 eq.) were used to obtain compound 19 (330 mg) as a white solid.

[0324]

[0325]

[0326] Preparation Example 20: Synthesis of 6,8-diethyl-1-methyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 20)

[0327]

[0328] Step 1. 3-(tert-Butoxymethyl)-1,4-diethyl-6-methylpiperazine-2,5-dione (Intermediate 2)

[0329] It was prepared by a synthesis similar to Intermediate 2 in Preparative Example 19 using Starting Material 1. However, ethyl iodide was used instead of methyl iodide.

[0330] Step 2. 6,8-Diethyl-1-methyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 20)

[0331] It was prepared by the same synthetic method as compound 1 of Preparation Example 1. That is, 0.55 g of the trisulfide intermediate was obtained as a yellow oil using intermediate 2 (0.65 g, 2.4 mmoles), sulfur (0.8 g, 10 equivalents) and NaHMDS (13.2 mL, 1 M in THF, 5 equivalents). After reducing the trisulfide intermediate (0.55 g, 1.47 mmoles) with NaBH4 (0.47 g, 6.6 equivalents), it was used in the next reaction without purification. Therefore, the reduced intermediate and iodine (0.9 g, 2 equivalents) were used to obtain compound 20 (216 mg) as a white solid.

[0332]

[0333]

[0334] Preparation Example 21: 12-Benzyl-9-methylhexahydro-5H-4,10a-(iminomethyl)pyrazino[2,1-c][1,2,4]dithiazepine-5,11-dione (Compound 21)

[0335]

[0336] Step 1. 8-Benzyl-7-(tert-butoxymethyl)-6,9-dioxaoctahydro-2H-pyrazino[1,2-a]pyrazine-2-carboxylic acid tert-butyl ester (Intermediate 2)

[0337] At 0 ℃, NaH (6.75g, 169 mmoles, 60% purity, 3.00 equivalents) was added to a solution of compound 1 (20.0g, 56.3 mmoles, 1.00 equivalents) in THF (500mL) for 30 minutes. Benzyl bromide (10.6g, 61.9 mmoles, 7.35mL, 1.10 equivalents) was added dropwise at 0 ℃, and stirred for 12 hours at 25 ℃. The reaction mixture was quenched by adding 100mL water at 0 ℃ and extracted with ethyl acetate (100mL*2). The combined organic layer was washed with saline (100mL), dried with Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. By column chromatography (SiO2, petroleum ether: ethyl acetate = 2: 1 to 1: 1) the residue was purified to obtain an intermediate 2 (15.0g, 17.2 mmoles, 76.5% purity) as a white solid.

[0338] Step 2. 8-Benzyl-7-(tert-butoxymethyl)-2-methylhexahydro-2H-pyrazino[1,2-a]pyrazine-6,9-dione (Intermediate 3)

[0339] HCl / EtOAc (4M, 30.0mL, 5.35 equivalents) was added to a solution of intermediate 2 (10.0g, 22.4 mmoles, 1.00 equivalents) in EtOAc (70.0mL) and stirred for 2 hours at 25°C. The reaction mixture was concentrated under reduced pressure, and the residue was used in the next step without further purification. After the compound was dissolved in MeOH (150mL), formaldehyde (17.6g, 217 mmoles, 16.2mL, 37.0% purity, 5.00 equivalents) was added at 25°C for 30 minutes, followed by NaBH (OAc) 3 (13.8g, 65.1 mmoles, 1.50 equivalents). The resulting mixture was stirred for 1 hour at 25°C, and the reaction mixture was concentrated under reduced pressure to obtain a residue. After purification by reverse phase HPLC (column: Kromasil Eternity XT 250*80mm*10um; mobile phase: [water (NH4HCO3)-ACN]; B%: 23% to 53%, 20 minutes), intermediate 3 (5.50 g, 15.2 mmol, 34.9% yield, 100% purity) was obtained as a yellow oil. Two isomers (diastereomers) were formed and the two were separated from each other.

[0340]

[0341]

[0342]

[0343] Step 3. 12-Benzyl-9-methylhexahydro-5H-4,10a-(epi-imidomethoxy)pyrazino[2,1-c][1,2,4]dithiazepine-5,11-dione (Compound 21)

[0344] It was prepared by a synthetic method similar to that of compound 1 of Preparation Example 1. That is, 0.48 g of the trisulfur intermediate was obtained as a yellow solid using intermediate 3 (isomer 1 or isomer 2, 1.2 g, 3.3 mmoles), sulfur (0.86 g, 8 equivalents) and NaHMDS (16.7 mL, 1 M in THF, 5 equivalents). After reducing the trisulfur intermediate (0.78 g, 1.47 mmoles) with NaBH4 (0.14 g, 1.9 equivalents), it was used for the next reaction without purification. Therefore, 101 mg of compound 21 was obtained as a white solid using the reduced intermediate and iodine (0.68 g, 1 equivalent).

[0345]

[0346]

[0347] Preparation Example 22: Synthesis of 12-benzyl-9-methylhexahydro-5H-4,10a-(iminomethyl)pyrazino[2,1-c][1,2,4]dithiazepine-5,11-dione (Compound 22)

[0348]

[0349] Step 1. tert-Butyl 7-(tert-butoxymethyl)-6,9-dioxa-8-(pyridin-4-ylmethyl)octahydro-2H-pyrazino[1,2-a]pyrazine-2-carboxylate (Intermediate 2)

[0350] It was prepared by a synthesis similar to that of Intermediate 2 in Preparative Example 21. However, 4-bromomethylpyridine was used instead of benzyl bromide.

[0351] Step 2. 7-(tert-Butoxymethyl)-2-methyl-8-(pyridin-4-ylmethyl)hexahydro-2H-pyrazino[1,2-a]pyrazine-6,9-dione (Intermediate 3)

[0352] It was prepared by a synthesis similar to Intermediate 3 in Preparative Example 21. Two isomers (diastereomers) were formed and each was isolated.

[0353]

[0354]

[0355] Step 3. 12-Benzyl-9-methylhexahydro-5H-4,10a-(epi-imidomethoxy)pyrazino[2,1-c][1,2,4]dithiazepine-5,11-dione (Compound 22)

[0356] It was prepared by a synthetic method similar to that of compound 1 of Preparation Example 1. That is, 0.40 g of the trisulfide intermediate was obtained as a yellow solid using intermediate 3 (isomer 1 or isomer 2, 1.2 g, 3.3 mmoles), sulfur (0.85 g, 8 eq.) and NaHMDS (16.6 mL, 1 M in THF, 5 eq.). After the trisulfide intermediate (0.55 g, 1.44 mmoles) was reduced with NaBH4 (0.18 g, 3.2 eq.), it was used in the next reaction without purification. Therefore, the reduced intermediate and iodine (0.53 g, 1 eq.) were used to obtain compound 22 (101 mg) as a yellow solid.

[0357]

[0358]

[0359] Preparation Example 23: 12-Benzyl-9-methylhexahydro-5H-4,10a-(iminomethyl)pyrazino[2,1-c][1,2,4]dithiazepine-5,11-dione (Compound 23)

[0360]

[0361] Step 1. 7-(tert-Butyloxymethyl)-8-methyl-6,9-dioxaoctahydro-2H-pyrazino[1,2-a]pyrazine-2-carboxylic acid tert-butyl ester (Intermediate 2)

[0362] It was prepared by a synthesis similar to that of Intermediate 2 in Preparative Example 21. However, iodomethane was used instead of benzyl bromide.

[0363] Step 2. 7-(tert-Butoxymethyl)-2,8-dimethylhexahydro-2H-pyrazino[1,2-a]pyrazine-6,9-dione (Intermediate 3)

[0364] It was prepared by a synthesis similar to that of Intermediate 3 in Preparative Example 21.

[0365] Step 3. 12-Benzyl-9-methylhexahydro-5H-4,10a-(epi-imidomethoxy)pyrazino[2,1-c][1,2,4]dithiazepine-5,11-dione (Compound 23)

[0366] It was prepared by a synthetic method similar to that of Compound 1 of Preparation Example 1. That is, 2.1 g of the trisulfide intermediate was obtained as a white solid using Intermediate 3 (4.0 g, 14.1 mmoles), sulfur (3.6 g, 8 eq.) and NaHMDS (70.6 mL, 1 M in THF, 5 eq.). After the trisulfide intermediate (0.50 g, 1.44 mmoles) was reduced with NaBH4 (0.26 g, 5 eq.), it was used in the next reaction without purification. Thus, Compound 23 (125 mg) was obtained as a pale yellow solid using the reduced intermediate and DDQ (0.33 g, 0.9 eq.).

[0367]

[0368]

[0369]

[0370] Preparation Example 24: Synthesis of 6-benzyl-1,4,8-trimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 24)

[0371]

[0372] Step 1. (6S)-3-((R)-1-(tert-Butoxy)ethyl)-1,6-dimethylpiperazine-2,5-dione (Intermediate 1)

[0373] It was prepared by a synthesis similar to that of Intermediate 4 in Preparative Example 7.

[0374] Step 2. (3S)-1-Benzyl-6-((S)-1-(tert-butoxy)ethyl)-3,4-dimethylpiperazine-2,5-dione (Intermediate 2)

[0375] It was prepared by a similar manner to the synthesis of Intermediate 2 in Preparative Example 2.

[0376] Step 3. 6-Benzyl-1,4,8-trimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 24)

[0377] It was prepared by a synthetic method similar to that of compound 1 of Preparation Example 1. That is, the trisulfur intermediate was obtained as a yellow solid using intermediate 2 (0.9 g, 2.71 mmoles), sulfur (0.7 g, 8 equivalents) and NaHMDS (10.9 mL, 1 M in THF, 5 equivalents). After the trisulfur intermediate (0.49 g, 1.38 mmoles) was reduced with NaBH4 (0.16 g, 3 equivalents), it was used for the next reaction without purification. Therefore, the reduced intermediate and iodine (0.7 g, 2 equivalents) were used to obtain compound 24, in which two isomers (diastereomers) were formed, isomer 1 was a yellow solid (157 mg), and isomer 2 was a white solid (13 mg).

[0378]

[0379]

[0380]

[0381]

[0382] Preparation Example 25: Synthesis of 6-benzyl-1,4,8-trimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 25)

[0383]

[0384] Step 1. (6S)-3-((S)-1-(tert-butoxy)ethyl)-1,6-dimethyl-4-(pyridin-2-ylmethyl)piperazine-2,5-dione (Intermediate 2)

[0385] It was prepared in a similar manner to the synthesis of Intermediate 2 in Preparative Example 2. However, 2-bromomethylpyridine was used instead of benzyl bromide.

[0386] Step 2. 6-Benzyl-1,4,8-trimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 25)

[0387] It was prepared by a synthetic method similar to that of compound 1 of Preparation Example 1. That is, 1.67 g of the trisulfide intermediate was obtained as a yellow oil using intermediate 2 (3.1 g, 9.3 mmoles), sulfur (2.4 g, 8 eq.) and NaHMDS (37 mL, 1 M in THF, 5 eq.). After the trisulfide intermediate (1.67 g, 4.7 mmoles) was reduced with NaBH4 (0.53 g, 3 eq.), it was used in the next reaction without purification. Thus, the reduced intermediate and iodine (1 g, 1 eq.) were used to obtain compound 25 (373 mg) as a white solid.

[0388]

[0389]

[0390] Experimental Example 1: Materials

[0391] Rabbit polyclonal antibody specific for peroxiredoxin type 2 (Prx II or Prdx2) was purchased from AbFrontier (Seoul, Korea). The siRNA sequence specific for human PrxII used in the present invention is as follows:

[0392] .

[0393] Phospho-tyrosine antibody (4G10) and PDGF-BB were purchased from Upstate. Prx II, Prx-SO were prepared as described in the literature (MH Choi et al., Nature 2005 May 19; 435(7040): 347-53). 2 / 3 Rabbit polyclonal antibodies against phosphorylated PDGFR-β (pY857) and phosphorylated PDGFR-β (pY857). Antibodies against phosphorylated VEGFR2 (pY1175), VEGFR2, phosphorylated PLCη1 (pY783), PLCη1, pTpY-Erk, and Erk2 were purchased from Cell Signaling Technology (CST). Antibodies against α-tubulin were purchased from Sigma. VEGF-A was purchased from R&D.

[0394] Experimental Example 2: Cell culture

[0395] Human aortic endothelial cells (HAEC), human pulmonary artery endothelial cells (PAEC), human aortic smooth muscle cells (HASMC), and human pulmonary artery smooth muscle cells (PASMC) were purchased from BioWhittaker (Belgium). They were seeded on 0.1% gelatin-coated culture plates. They were grown in endothelial cell basal medium (EBM-2) and smooth muscle cell basal medium (SmBM) containing 10% fetal bovine serum (FBS) and complete medium supplement (Clonetics-BioWhittaker product number cc-4176 for HAEC and PAEC; Clonetics-BioWhittaker product number cc-4149 for HASMC and PASMC). Cell culture was carried out in a humidified CO2 incubator at 37°C under 5% carbon dioxide. The present invention mainly uses cells with a passage number of 5 to 7 generations.

[0396] Experimental Example 3: Peroxidase activity test

[0397] The peroxidase activity assay of the compounds of the invention for the reduction of hydrogen peroxide (H2O2) was performed as follows. Peroxidase activity was detected in a reaction mixture (200 μL volume) containing 50 mM HEPES buffer (pH 7.0), 1 mM EDTA, 250 μM NADPH, 3 μM yeast thioredoxin (Trx), 1.5 μM yeast Trx reductase (TR), 50 μM test compound and 1.2 mM H2O2.

[0398] The reaction was started by adding H2O2 and the decrease in absorbance at 340 nm was measured for 12 min at 30°C using an Agilent UV8453 spectrophotometer (Hewlett Packard, USA). The initial reaction rate was the amount of NADPH oxidized per minute calculated from the slope of the linear portion of the curve.

[0399] Experimental Example 4: Detection of intracellular H2O2 scavenging activity

[0400] The activity of the test compound of the present invention in removing intracellular H2O2 was detected as follows. NIH-3T3 cells (ATCC) were cultured at 2×10 5Cells / wells were seeded in a 35mm culture dish. After 24 hours of cultivation, cells were serum starved for 6 hours with the basic DMEM containing 0.5% FBS without phenol red. The compound pre-treatment of the hungry NIH 3T3 cells 30 minutes with the different concentrations (0mM, 12.5mM, 25mM, 50mM, 100mM) of the basic DMEM dilution without phenol red was used, and then 20mU glucose oxidase (Gox) was used to process 30 minutes. In the cell, the fluorescent dye carboxymethyl-dichlorofluorescein-diacetate (CM-H2DCF-DA) sensitive to reactive oxygen species was added and incubated for 5 minutes, and the CM-H2DCF-DA was prepared as 1mM DMSO stock solution and diluted 500 times in the basic DMEM without phenol red. Subsequently, cells were quickly rinsed with basic culture medium. Images were taken under exciting 497nm / emission 518nm using a fluorescence microscope. Three images at 10× magnification were obtained for each sample, and the fluorescence intensity of 30 or more individual cells in each image was averaged.

[0401] Experimental Example 5: Cytotoxicity Test

[0402] For cytotoxicity analysis, human aortic endothelial cells (HAEC) and human aortic smooth muscle cells (HASMC) were seeded into 96-well culture plates at a density of 4000 cells / well and a final volume of 100 μL. The cells were cultured for 24 hours and serum starved for 18 hours. The test compounds were serially diluted with EBM-2 culture medium, added to the culture plates at 100 μL / well, and incubated for another 24 hours. The number of viable cells was detected using the WST-1 cell viability assay kit (Roche Diagnostics, USA). The number of cells was expressed as the absorbance value at 450nm minus the turbidity value at 600nm. The absorbance values ​​of three replicate wells were averaged.

[0403] Experimental Example 6: Immunoblotting analysis

[0404] The cells were quickly washed in ice-cold phosphate buffered saline (PBS) and lysed in an extraction buffer containing 20 mM Hepes (pH 7.0), 1% Triton X-100, 150 mM NaCl, 10% glycerol, 1 mM EDTA, 2 mM EGTA and 1 mM DTT, 5 mM Na3VO4, 5 mM NaF, 1 mM AEBSF, aprotinin (5 μg / mL) and leupeptin (5 μg / mL). After centrifugation at 12000 × g, the clarified cell extracts were used for immunoblotting. For reblotting, the antibodies were removed by treatment in a solution of 67 mM Tris (pH 6.7), 2% SDS and 100 mM 2-mercaptoethanol at 60°C for 30 minutes and the immunoblots were recovered by washing three times with Tris buffered saline (TBS) containing 1% Triton X-100. The recovered membranes were used for new immunoblots.

[0405] Experimental Example 7: Measurement of Redox Potential

[0406] As shown in Table 1 below, 10 redox buffer solutions (0.5 mL) were prepared by mixing a 50 mM solution of reduced dithiothreitol (DTT) with its oxidized DTT (trans-1,2-dithiane-4,5-diol) at a specified ratio before use. The test compound (10 mM) was diluted 20 times with each redox buffer solution and incubated in a water bath at 30 ° C for 3 hours. The compound was then analyzed by HPLC. The corresponding peak areas of the oxidized test compound and the reduced test compound were calculated based on a specific retention time and plotted as a graph of the Nernst equation value (mV) of the percentage of the total peak area to the x-axis. The corresponding Nernst equation value of the reduced 50% test compound was set as the midpoint redox potential value of the test compound.

[0407] [Table 1]

[0408]

[0409] The specific methods and conditions other than Experimental Examples 1 to 7 were carried out in a similar manner to those disclosed in International Patent Application Publication No. WO2013-077709 or WO2018-008984. All contents disclosed in International Patent Application Publication No. WO2013-077709 and WO2018-008984 are incorporated herein by reference.

[0410] Experimental Example 8: Growth factor-induced proliferation assay in vascular smooth muscle cells and vascular endothelial cells

[0411] The ability of the test compounds to control cell proliferation was examined in vascular smooth muscle cells and endothelial cells.

[0412] According to the manufacturer's scheme, RNAi MAX transfection reagent was used to transfect HASMC and PASMC with siRNA specific for PrxII. After 24 hours, the transfected cells (3000 cells / well) were inoculated into 96-well culture plates. After 12 hours of inoculation, the cells were serum starved for 18 hours in addition in the SmBM basal medium containing 0.5% FBS. The test compound was serially diluted with the same basal medium containing 0.5% FBS, added to the cells (100mL / well), and incubated for 2 hours. After treatment, the cells were placed in the fresh SmBM medium containing 0.5% FBS and PDGF-BB (25ng / mL) for growth factor stimulation, and continued to be cultured for 24 hours.

[0413] According to the manufacturer's protocol, HAEC and PAEC were transfected with PrxII-specific siRNA using RNAi MAX transfection reagent. After 24 hours, the transfected cells (3000 cells / well) were seeded into 96-well culture plates. After 12 hours of inoculation, the cells were serum starved for an additional 18 hours in EBM basal medium containing 0.5% FBS. The test compound was serially diluted with the same basal medium containing 0.5% FBS, added to the cells (100mL / well), and incubated for 2 hours. After treatment, the cells were placed in fresh EBM medium containing 0.5% FBS and VEGF-A (25ng / mL) for growth factor stimulation and continued to be cultured for 24 hours.

[0414] The degree of cell proliferation was detected using the Cell Titer-GLO kit (Roche Diagnostics, USA). The data are the percentages of the average luminescence intensity of three replicate wells relative to the average luminescence intensity of three replicate wells of the untreated control group.

[0415] Experimental Example 9: Evaluation of therapeutic efficacy in a preclinical pulmonary hypertension rat model

[0416] The rat study protocol was approved by the Laboratory Animal Care and Use Committee of Ewha Womans University, Republic of Korea, and the ARRIVE guidelines were followed. Six-week-old rats were acclimatized in the laboratory for one week. Sugen 5416 (Sigma Aldrich) was then injected subcutaneously at a dose of 20 mg / kg. The subjects of administration were maintained in a hypoxic (10% O2) chamber (A chamber, Biosphenix) at normal pressure for 3 weeks. Rats were transferred to normoxia and additionally orally administered with a control vehicle or test compound for 5 weeks (PO, once a day, 0.1 mg per kg body weight).

[0417] Right ventricular systolic pressure (RVSP) was measured by echocardiography using a curved catheter equipped with a PowerLab2 / 26 device (AD Instruments, UK). Rats were anesthetized by 2% isoflurane inhalation. A catheter was inserted through the right jugular vein and the pressure was measured for 10 seconds in a stable mode. After echocardiography, the rats were perfused with saline and the heart was carefully removed. The removed heart was dissected into the right ventricle (RV), the remainder of the septum (S; septum) and the left ventricle (LV). The weight of each heart was weighed using a microbalance. The RV / (LV+S) value was calculated and expressed as the right ventricular hypertrophy index (Fulton index).

[0418] Experimental Example 10: Histological Analysis

[0419] Rats were anesthetized with 2% isoflurane inhalation and fixed by transcardial perfusion using a heparinized saline solution containing 37% formaldehyde. The left lung lobe was then excised and fixed in 10% NBF for 3 days. The fixed tissue was paraffin-embedded and sliced ​​using a rotary slicer (Leica HistoCore MULTICUT). Two consecutive tissue sections (4 μm thick) were placed on slides and stained with hematoxylin and eosin (HE). During analysis, approximately 100 pulmonary arteries (20 μm to 100 μm in diameter) were selected for measurement per tissue section. The areas of the lumen, internal elastic layer, and external elastic layer were quantified using NIH ImageJ v1.62. The areas of the intima and media were determined by subtracting the lumen area from the area of ​​the internal elastic layer and subtracting the internal elastic layer area from the area of ​​the external elastic layer. These values ​​were averaged and used to calculate the pulmonary artery vascular thickness.

[0420] Experimental Example 11: Immunofluorescence staining

[0421] Paraffin sections were blocked in PBST (PBS containing 0.3% Triton X-100) solution with 5% normal donkey serum (Vector Laboratories) for 1 h at room temperature. Subsequently, lung tissue sections were incubated with antigens of smooth muscle actin (α-SMA; 1:300 dilution) coupled to Alexa Fluor 568 and antigens of von Willebrand factor (vWF; 1:200 dilution) coupled to Alexa Fluor 488 for 12 h at 4°C. Subsequently, cell nuclei were counterstained with DAPI. Fluorescence images of random areas of at least 10 pulmonary artery vessels (20 μm to 100 μm in diameter) per tissue section were recorded at 60x screen magnification using an LSM 880 confocal microscope equipped with an argon laser and a helium-neon laser.

[0422] Result 1: Peroxidase activity in reducing H2O2

[0423] The peroxidase activity of the test compounds of the present invention for reducing H2O2 was measured in reaction mixtures containing thioredoxin (Trx) / thioredoxin reductase (TR), respectively. The data are summarized in the table below.

[0424] [Table 2]

[0425]

[0426] A5:

[0427] As shown in Table 2, in the presence of the Trx / TR redox system, most of the tested compounds exhibited excellent peroxidase activity for reducing H2O2, indicating that the novel compounds have the peroxidase activity of specifically mimicking 2-Cys peroxidase reductase.

[0428] Compared to the reference compound (A5) belonging to the class of epidithiodioxapiperazines, the novel compounds of the present invention have similar or better peroxidase activity.

[0429] Result 2: Determination of intracellular H2O2 scavenging activity

[0430] The intracellular H2O2 scavenging activity of the test compound of the present invention was detected using a live cell imaging fluorescent probe (CM-H2DCF-DA). The H2O2 scavenging activity was detected according to increasing test compound concentrations and expressed as a percentage of activity compared to the untreated control group. EC 50 The concentration of the test compound at which intracellular H2O2 was reduced by 50%.

[0431] [Table 3]

[0432]

[0433] As shown in Table 3, the test compounds showed excellent ability to scavenge H2O2 compared to the control compound (A5) belonging to the epidithiodioxapiperazines.

[0434] Result 3: Measurement of redox potential

[0435] The redox potential values ​​of the test compounds were analyzed based on the potential values ​​established in the redox buffer solution, in which reduced DTT and oxidized DTT were mixed in different ratios. The data are shown in the table below.

[0436] [Table 4]

[0437]

[0438] As shown in Table 3, all novel compounds of the present invention exhibited lower redox potential values. When compared to the potential values ​​of Trx or GSH, the data confirmed that the novel compounds are true 2-Cys-Prx mimetics that are able to couple the Trx / TR redox system to the peroxidase reaction.

[0439] Results 4: Cytotoxicity test

[0440] Cytotoxicity tests were performed on endothelial cells, vascular smooth muscle cells, and hepatocytes. After treatment with increasing concentrations of each compound, the concentration of the test compound corresponding to 50% cell viability (CC 50 ). The data are shown in the following table.

[0441] [Table 5]

[0442]

[0443] The measurement results showed that most of the novel compounds, except compound 6, had high CC values ​​above 100 μM. 50 , indicating that its cytotoxicity is very low. In particular, some of the test compounds showed no cytotoxicity within the concentration range used in the test (10 μM to 1 mM). This shows that the new compounds have a very excellent safety margin compared to the control compound (A5) belonging to the epidithiodioxapiperazine class.

[0444] Results 5: Analysis of PDGF-mediated signal transduction pathways in vascular smooth muscle cells

[0445] The ability of the test compounds to replace the PrxII function of the cells was evaluated in human aortic smooth muscle cells (HASMC) and human pulmonary artery smooth muscle cells (PASMC) in which PrxII was knocked down by transfection with PrxII siRNA. That is, the efficacy of the novel compounds was verified from the perspective of the PDGF-induced signal transduction pathway that regulates the growth and migration of vascular smooth muscle cells in the aorta and pulmonary artery. The activation of the PDGF-induced signal transduction pathway was analyzed by immunoblotting using an antibody that specifically recognizes phosphorylation of Tyr 857, where Tyr 857 is the main phosphorylation site on the PDGF receptor (PDGFRβ). The results are shown in Figure 2. Figures 1 to 3 shown.

[0446] Figure 1 The effect of compound 1 on the phosphorylation level of Tyr 857 on PDGFRβ is shown, which is enhanced by genetic knockdown of PrxII in HASMC in a concentration-dependent manner. This shows that compound 1 significantly reduces the phosphorylation level at concentrations above 2.5 nM.

[0447] Figure 2The effect of compound 1, compound 2, compound 5, compound 6, compound 10, compound 15, compound 19, compound 20, compound 21, compound 22 and compound 23 on the phosphorylation level of Tyr 857 on PDGFRβ is shown, and the effect is enhanced by genetic knockdown of PrxII in HASMC. As shown in the figure, all tested compounds strongly abolished PDGFRβ phosphorylation in HASMC with loss of PrxII expression.

[0448] Figure 3 The effect of compound 8 on the total tyrosine phosphorylation level and the phosphorylation level of PDGFRβ downstream signal transduction proteins (such as PLCγ1) in PASMC is shown, wherein all tyrosine phosphorylation and PLCγ1 phosphorylation are induced by PDGF stimulation. As shown in the figure, compound 8 also significantly inhibited the protein phosphorylation triggered by PDGF.

[0449] As a result of the evaluation, the ability of the novel compound to regulate the PDGF signal transduction pathway was increased tenfold compared to the control compound (A5) belonging to the epidithiodioxapiperazine class. The novel compound of the present invention can also reverse the abnormal PDGF signal transduction enhanced by the loss of PrxII expression to normal levels.

[0450] Results 6: Analysis of VEGF-mediated signal transduction pathways in vascular endothelial cells

[0451] The ability of the test compounds to replace the PrxII function of the cells was evaluated in human aortic endothelial cells (HAEC) and human pulmonary artery endothelial cells (PAEC) in which PrxII was knocked down by transfection with PrxII siRNA. That is, the efficacy of the novel compounds was verified from the perspective of the VEGF-induced signal transduction pathway that regulates the growth and migration of vascular endothelial cells in the aorta and pulmonary artery. The activation of the VEGF-induced signal transduction pathway was analyzed by immunoblotting using an antibody that specifically recognizes the phosphorylation of Tyr 1175, where Tyr 1175 is the main phosphorylation site on VEGF receptor-2 (VEGFR2). The results are shown in Figure 2. Figures 4 to 6 shown.

[0452] Figure 4 The effect of compound 1 on the phosphorylation level of Tyr 1175 on VEGFR2 is shown, which is enhanced by genetic knockdown of PrxII in HAEC in a concentration-dependent manner. This shows that compound 1 significantly reduces phosphorylation at concentrations above 2.5 nM.

[0453] Figure 5The effects of Compound 1, Compound 2, Compound 5, Compound 6, Compound 10, Compound 20, Compound 21 and Compound 23 on the phosphorylation level of Tyr 1175 on VEGFR2 enhanced by PrxII knockdown in HAEC are shown. As shown, all tested compounds strongly abolished VEGFR2 phosphorylation in HAECs lacking PrxII expression.

[0454] Figure 6 The effect of compound 8 on the total tyrosine phosphorylation and phosphorylation of VEGFR2 downstream signal transduction proteins (such as ERK2) in PAEC is shown, wherein all tyrosine phosphorylation and ERK2 phosphorylation are induced by VEGF stimulation. As shown in the figure, compound 8 also significantly inhibits protein phosphorylation triggered by VEGF.

[0455] As a result of the evaluation, the ability of the novel compound to regulate the VEGF signal transduction pathway was increased tenfold compared to the control compound (A5) belonging to the epidithiodioxapiperazine class. The novel compound of the present invention can also reverse the abnormal VEGF signal transduction enhanced by the loss of PrxII expression to normal levels.

[0456] Results 7: Proliferation analysis in PDGF-stimulated smooth muscle cells

[0457] The ability of the test compound to inhibit PDGF-induced vascular smooth muscle cell proliferation was evaluated in human aortic smooth muscle cells (HASMC) and human pulmonary artery smooth muscle cells (PASMC) knocked out PrxII by transfection with PrxII siRNA. The results are shown in Table 6 below. In Table 6, the degree of proliferation of the compound-treated group is expressed as a percentage of the degree of proliferation of the untreated control group. The concentration of the test compound corresponding to 50% growth inhibition (IC 50 ).

[0458] [Table 6]

[0459]

[0460] As shown in the above table, the novel compounds of the present invention inhibited the proliferation of HASMC and PASMC in a concentration-dependent manner and showed growth inhibitory activity starting from a concentration as low as 2.5 nM.

[0461] Results 8: Proliferation analysis in VEGF-stimulated endothelial cells

[0462] The ability of the test compound to enhance VEGF-induced vascular endothelial cell proliferation was evaluated in human aortic endothelial cells (HAEC) and human pulmonary artery endothelial cells (PAEC) in which PrxII was knocked out by transfection with PrxII siRNA. The results are shown in Table 7 below. In Table 7, the degree of proliferation of the compound-treated group is expressed as a percentage of the degree of proliferation of the untreated control group. The effective concentration (EC) of the test compound that increases cell proliferation by 50% is also listed. 50 ).

[0463] [Table 7]

[0464]

[0465] As shown in the above table, the novel compounds of the present invention promoted the proliferation of HAEC and PAEC in a concentration-dependent manner and exhibited growth promoting activity starting from a concentration as low as 2.5 nM.

[0466] Results 9: Preclinical Animal Studies of Pulmonary Hypertension

[0467] The therapeutic effects of novel compounds on pulmonary arterial hypertension were evaluated in the preclinical SuHx rat model. To establish the PAH model, Sugen (VEGFR2 inhibitor) was administered to rats and placed under normobaric hypoxic conditions (10% O2) for 3 weeks. The rats were moved to normoxic conditions and orally administered with control vehicle or compound 1 or compound 8 (PO, once daily, 0.1 mg / kg) for 5 weeks. Thereafter, right ventricular systolic pressure (RVSP) and right ventricular hypertrophy (Fulton index) were measured at the end of treatment. The results are shown in Figure 2. Figures 7 to 9 shown.

[0468] Figure 7 It was shown that Compound 1 and Compound 8 significantly reduced RVSP and RV hypertrophy compared to the vehicle control group.

[0469] Figure 8 It was shown that Compound 1 widened the pulmonary artery vessels, while the pulmonary artery vessels of the vehicle control group remained severely occluded. The data indicate that treatment with Compound 1 induces normal blood flow within the lumen of the vessels, which results in reduced RVSP and RV hypertrophy.

[0470] Fig. 9 Shown are immunostaining images of endothelial and media SMC layers in pulmonary artery vessels. Specifically, lung tissue sections were immunostained with endothelial cell-specific vWF antibodies and smooth muscle cell-specific SMA antibodies. The results showed that in the vehicle control group, endothelial damage and media thickening were caused due to SMC hyperplasia. However, the treatment of compound 1 induced the recovery of the endothelial layer (vWF-labeled EC) and the reduction of media thickness (SMA-labeled SMC).

Claims

1. A compound of Chemical Formula 1 or Chemical Formula 2: ， or a pharmaceutically acceptable salt thereof, In Chemical Formula 1 and Chemical Formula 2, n is an integer from 1 to 3, R1 and R2 are each independently C 1-3 Alkyl, C 1-3 Alkoxy-C 1-3 Alkyl, -(CH2) 1-3 -C(R')(R'')OH, -(CH2) 1-3 -N(R')(R''), -(CH2) 0-3 -Alkenyl, -(CH2) 0-3 -Alkynyl, -(CH2) 0-3 -C(R')(R'')CO2H, -(CH2) 0-5 -heterocycloalkyl, -(CH2) 0-5 -cycloalkyl, -(CH2) 0-5 -Aryl or -(CH2) 0-5 -heteroaryl, wherein alkyl, heterocycloalkyl, cycloalkyl, aryl and heteroaryl are unsubstituted or optionally substituted with one or more than one substituent selected from C 1-3 Alkyl, -CF3, C 1-3 Alkoxy, -OCF3, halogen, CN, amino, -N(R')(R''), -OH, -COOH, -COO-C 1-3 alkyl and =O, wherein R' and R'' are each independently H or C 1-3 alkyl, R3 is C 1-3 Alkyl, -(CH2) 0-3 -Aryl or -(CH2) 0-3 -heteroaryl, wherein the aryl or heteroaryl group is unsubstituted or optionally substituted with one or more than one substituent selected from C 1-3 Alkyl, -CF3, C 1-3 Alkoxy, -OCF3, halogen, -CN, amino, -OH and -COOH; or R2 and R3 are connected together and fused with the piperazine dione represented by Chemical Formula 1 to form one of the following structures: ， in, X is S, SO2, CH2, O or NR6, wherein R6 is H or C 1-3 alkyl, R4 is H or C 1-3 Alkyl, and R5 is H, C 1-3 Alkyl, -(CH2) 1-2 -Aryl or -(CH2) 1-2 -heteroaryl.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, n is an integer from 1 to 3, R1 and R2 are each independently C 1-3 Alkyl, -(CH2) 1-2 -heterocycloalkyl, -(CH2) 1-2 -Aryl or -(CH2) 1-2 -heteroaryl, wherein alkyl, heterocycloalkyl, aryl and heteroaryl are unsubstituted or optionally substituted with one or more than one substituent selected from C 1-3 Alkyl, -CF3, C 1-3 Alkoxy, CN, halogen, -OH, -COOH, and -COO-C 1-3 alkyl, R3 is C 1-3 alkyl, -CH2-aryl or -CH2-heteroaryl, wherein the aryl or heteroaryl is unsubstituted or optionally substituted with one or more than one substituent selected from methyl, methoxy, halogen, -CN, amino, -OH and -COOH; or R2 and R3 are connected together and fused with the piperazine dione represented by Chemical Formula 1 to form one of the following structures: ， in, X is S, SO2, CH2, O or NR6, wherein R6 is H or C 1-3 alkyl, R4 is H or C 1-3 Alkyl, and R5 is H, C 1-3 Alkyl or -(CH2) 1-2 -Aryl.

3. The compound according to claim 2 or a pharmaceutically acceptable salt thereof, n is 1, R1 and R2 are each independently C 1-3 alkyl, -CH2-piperidinyl, -CH2-morpholinyl, -CH2-piperazinyl, -CH2-phenyl, -CH2-naphthyl, -CH2-pyridinyl, -CH2-quinolinyl, -CH2-pyrazolyl, -CH2-thiophen-2-yl, -CH2-benzo[d]thiazol-2-yl, -CH2-pyrimidinyl, -CH2-1H-imidazol-4-yl, -CH2-1H-imidazol-2-yl, -CH2-thiazol-4-yl, -CH2-thiazol-5-yl, -CH2-isothiazol-6-yl, -CH2- oxazolyl, -CH2-indol-2-yl, -CH2-indol-3-yl, -CH2-benzimidazol-5-yl, -CH2-quinolin-4-yl, -CH2-quinazol-2-yl or -CH2-quinazol-4-yl, wherein piperidinyl, morpholinyl, piperazinyl, phenyl, naphthyl, pyridinyl, quinolinyl, pyrazolyl, thiophene, benzo[d]thiazole, pyrimidinyl, imidazole, thiazole, isothiophene, Oxylidene, indole, benzimidazole, quinoline and quinazole are unsubstituted or optionally substituted with one or more than one substituent selected from C 1-3 Alkyl, -CF3, C 1-3 Alkoxy, CN, halogen and -COO-C 1-3 alkyl, R3 is C 1-3 Alkyl or -CH2-aryl, or, R2 and R3 are connected together and fused with the piperazine dione represented by Chemical Formula 1 to form one of the following structures: ， in, X is O or NR6, wherein R6 is methyl, R4 is H, and R5 is H.

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is 1,6,8-trimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 1), 6-benzyl-1,8-dimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 2), 1,8-dimethyl-6-(3,4,5-trimethoxybenzyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 3), 6-(3,5-difluorobenzyl)-1,8-dimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione. 2.2]nonane-7,9-dione (Compound 4), 1,8-dimethyl-6-(quinolin-2-ylmethyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 5), 1,8-dimethyl-6-(pyridin-2-ylmethyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 6), 11-benzyltetrahydro-5H,7H-4,9a-(epi-iminomethoxy)pyrrolo[2,1-c][1,2,4]dithiazepine-5,10-dione (Compound 7), 12-benzyltetrahydro-5H,10H-4,10a-(epi-iminomethoxy)[1,4] Azino[3,4-c][1,2,4]dithiazepine-5,11-dione (Compound 8), 12-(pyridin-4-ylmethyl)tetrahydro-5H,10H-4,10a-(epi-imidomethoxy)[1,4] Azino(3,4-c)(1,2,4)dithiazepine-5,11-dione (Compound 9), 12-ethyltetrahydro-5H,10H-4,10a-(epi-iminomethyl)[1,4] azino[3,4-c][1,2,4]dithiazepine-5,11-dione (Compound 10), 11-(pyridin-4-ylmethyl)tetrahydro-5H,7H-4,9a-(epi-imidomethyl)pyrrolo[2,1-c][1,2,4]dithiazepine-5,10-dione (Compound 11), 1,8-dimethyl-6-((6-methylpyridin-2-yl)methyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 12), 1,8-dimethyl-6-((1-methyl-1H-pyrazol-4-yl)methyl)-2,3-dithia-6,8-diazabicyclo[3. 2.2]nonane-7,9-dione (Compound 13), 1,8-dimethyl-6-(thien-2-ylmethyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 14), 6-(benzo[d]thiazol-2-ylmethyl)-1,8-dimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 15), 1,6-dimethyl-8-(pyrimidin-2-ylmethyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 16), 1,6-dimethyl-8-((1-methyl-1H-imidazol-4-ylmethyl)- )methyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 17), methyl 4-((1,6-dimethyl-7,9-dioxa-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-8-yl)methyl)benzoate (Compound 18), 1-benzyl-6,8-dimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 19), 6,8-diethyl-1-methyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 20), 12-benzyl-9-methylhexahydro-5H- 4,10a-(Iminomethoxy)pyrazino[2,1-c][1,2,4]dithiazepine-5,11-dione (Compound 21), 12-benzyl-9-methylhexahydro-5H-4,10a-(Iminomethoxy)pyrazino[2,1-c][1,2,4]dithiazepine-5,11-dione (Compound 22), 12-benzyl-9-methylhexahydro-5H-4,10a-(Iminomethoxy)pyrazino[2,1-c][1,2,4]dithiazepine-5,11-dione (Compound 23), 6-benzyl-1,4,8-trimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2] nonane-7,9-dione (Compound 24) or 6-benzyl-1,4,8-trimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dione (Compound 25).

5. A method for treating or preventing a vascular disease, the method comprising administering a therapeutically effective amount of a compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof to a subject in need of prevention or treatment of a vascular disease or a subject suspected of having a vascular disease.

6. The method according to claim 5, wherein the vascular disease is any one selected from hypertension, ischemic coronary artery disease, cerebral artery occlusion, arteriosclerosis, peripheral arterial occlusive disease, thromboembolism, diabetic foot lesions, venous ulcers, deep vein thrombosis, vasospasm, arteritis and vascular restenosis.

7. The method according to claim 6, wherein the vascular disease is ischemic coronary artery disease, arteriosclerosis, vascular restenosis or pulmonary hypertension.

8. Use of the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating or preventing vascular diseases.

9. The method according to claim 8, wherein the vascular disease is any one selected from the group consisting of hypertension, ischemic coronary artery disease, cerebral artery occlusion, arteriosclerosis, peripheral arterial occlusive disease, thromboembolism, diabetic foot lesions, venous ulcers, deep vein thrombosis, vasospasm, arteritis and vascular restenosis.

10. The use according to claim 9, wherein the vascular disease is ischemic coronary artery disease, arteriosclerosis, vascular restenosis or pulmonary hypertension.

11. A method for preparing a compound represented by Chemical Formula 3, the method comprising reacting a 6-(1-hydroxyalkyl)piperazine-2,5-dione derivative represented by Chemical Formula 4 with (a) sulfur (S8) and (b) lithium bis(trimethylsilyl)amide (LiHMDS) or sodium bis(trimethylsilyl)amide (NaHMDS), ， In Chemical Formula 4, R1, R2, R3 and R4 are the same as those in Chemical Formula 1 of claim 1; R5 is H; and R is a protecting group, In Chemical Formula 3, R1, R2, R3 and R4 are the same as in Chemical Formula 1 of claim 1; R5 is H; and n is 2 or 3.

12. A method for preparing a compound represented by the following chemical formula 1', the method comprising (S1) reacting the compound represented by Chemical Formula 4 with (a) sulfur (S8) and (b) LiHMDS (lithium bis(trimethylsilyl)amide) or NaHMDS (sodium bis(trimethylsilyl)amide) to obtain the compound represented by Chemical Formula 3; (S2) reducing the compound of Chemical Formula 3 to obtain the compound represented by Chemical Formula 2; and (S3) forming intramolecular disulfide crosslinks with the compound represented by Chemical Formula 2, ， In Chemical Formula 4, Chemical Formula 3, Chemical Formula 2 and Chemical Formula 1′, R1, R2, R3 and R4 are the same as in Chemical Formula 1 of claim 1; R5 is H; R is a protecting group; and n is 2 or 3.

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