COMPOUNDS, PHARMACEUTICAL COMPOSITIONS CONTAINING THEM AND THEIR MEDICAL USE FOR THE TREATMENT OR PREVENTION OF VASCULAR DISEASES

Abstract

The present disclosure provides compounds capable of exhibiting effects similar to those of 2-Cys-peroxyredoxin (2-Cys-Prx) in the body with excellent pharmacological effects and reduced side effects such as reduced cytotoxicity, and pharmaceutical uses thereof. The compounds of the present disclosure and their pharmaceutically acceptable salts are useful for the treatment or prevention of vascular diseases, particularly ischemic coronary artery disease, arteriosclerosis, vascular restenosis, or pulmonary arterial hypertension. The compounds of the present invention and their pharmaceutically acceptable salts are particularly useful for the treatment or prevention of pulmonary arterial hypertension. The invention also provides methods of preparing the compounds of the present disclosure.

Claims

1. A compound of Chemical Formula 1 or Chemical Formula 2: ##STR00036## or a pharmaceutically acceptable salt thereof, in the Chemical Formula 1 and 2, n is an integer of from 1 to 3, R.sub.1 and R.sub.2 are each independently C.sub.1-3alkyl, C.sub.1-3alkoxy-C.sub.1-3alkyl, (CH.sub.2).sub.1-3C(R)(R)OH, (CH.sub.2).sub.1-3N(R)(R), (CH.sub.2).sub.0-3-alkenyl, (CH.sub.2).sub.0-3-alkynyl, (CH.sub.2).sub.0-3C(R)(R)CO.sub.2H, (CH.sub.2).sub.0-5-heterocycloalkyl, (CH.sub.2).sub.0-5-cycloalkyl, (CH.sub.2).sub.0-5-aryl, or (CH.sub.2).sub.0-5-heteroaryl, wherein the alkyl, heterocycloalkyl, cycloalkyl, aryl and heteroaryl are unsubstituted or optionally substituted with one or more substituents selected from the group consisting of C.sub.1-3alkyl, CF.sub.3, C.sub.1-3alkoxy, OCF.sub.3, halogen, CN, amino, N(R)(R), OH, COOH, COOC.sub.1-3alkyl, and =O, wherein R and R are each independently H or C.sub.1-3alkyl, R.sub.3 is C.sub.1-3alkyl, (CH.sub.2)O.sub.3-aryl, or (CH.sub.2).sub.0-3-heteroaryl, wherein the aryl or heteroaryl is unsubstituted or optionally substituted with one or more substituents selected from the group consisting of C.sub.1-3alkyl, CF.sub.3, C.sub.1-3 alkoxy, OCF.sub.3, halogen, CN, amino, OH, and COOH; or R.sub.2 and R.sub.3 are linked together and fused with piperazinedione present in Chemical Formula 1 to form one of the following structures: ##STR00037## wherein, X is S, SO.sub.2, CH.sub.2, O or NR.sub.6, wherein R.sub.6 is H or C.sub.1-3alkyl, R.sub.4 is H or C.sub.1-3alkyl, and R.sub.5 is H, C.sub.1-3alkyl, (CH.sub.2).sub.1-2-aryl, or (CH.sub.2).sub.1-2-heteroaryl.

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, n is an integer of from 1 to 3, R.sub.1 and R.sub.2 are each independently C.sub.1-3alkyl, (CH.sub.2).sub.1-2-heterocycloalkyl, (CH.sub.2).sub.1-2-aryl, or (CH.sub.2).sub.1-2-heteroaryl, wherein the alkyl, heterocycloalkyl, aryl, and heteroaryl are unsubstituted or optionally substituted with one or more substituents selected from the group consisting of C.sub.1-3 alkyl, CF.sub.3, C.sub.1-3alkoxy, CN, halogen, OH, COOH, and COOC.sub.1-3 alkyl, R.sub.3 is C.sub.1-3alkyl, CH.sub.2-aryl, or CH.sub.2-heteroaryl, wherein the aryl or heteroaryl is unsubstituted or optionally substituted with one or more substituents selected from the group consisting of methyl, methoxy, halogen, CN, amino, OH, and COOH; or R.sub.2 and R.sub.3 are linked together and fused with piperazinedione present in Chemical Formula 1 to form one of the following structures: ##STR00038## wherein, X is S, SO.sub.2, CH.sub.2, O or NR.sub.6, wherein R.sub.6 is H or C.sub.1-3alkyl, R.sub.4 is H or C.sub.1-3alkyl, and R.sub.5 is H, C.sub.1-3alkyl, or (CH.sub.2).sub.1-2-aryl.

3. The compound of claim 2 or a pharmaceutically acceptable salt thereof, n is 1, R.sub.1 and R.sub.2 are each independently C.sub.1-3alkyl, CH.sub.2-piperidyl, CH.sub.2 morpholinyl, CH.sub.2-piperazinyl, CH.sub.2-phenyl, CH.sub.2-naphthyl, CH.sub.2 pyridyl, CH.sub.2-quinolinyl, CH.sub.2-pyrazolyl, CH.sub.2-thiophen-2-yl, CH.sub.2-benzo[d]thiazol-2-yl, CH.sub.2-pyrimidyl, CH.sub.2-1H-imidazol-4-yl, CH.sub.2-1H-imidazol-2-yl, CH.sub.2-thiazol-4-yl, CH.sub.2-thiazol-5-yl, CH.sub.2 isoxazolyl, CH.sub.2-indol-2-yl, CH.sub.2-indol-3-yl, CH.sub.2-benzimidazol-5-yl, CH.sub.2-quinolin-4-yl, CH.sub.2-quinazol-2-yl, or CH.sub.2-quinazol-4-yl, wherein the the piperidyl, morpholinyl, piperazinyl, phenyl, naphthyl, pyridyl, quinolinyl, pyrazolyl, thiophene, benzo[d]thiazole, pyrimidyl, imidazole, thiazole, isoxazolyl, indole, benzimidazole, quinoline and quinazole are unsubstituted or optionally substituted with one or more substituents selected from the group consisting of C.sub.1-3alkyl, CF.sub.3, C.sub.1-3 alkoxy, CN, halogen, and COOC.sub.1-3alkyl, R.sub.3 is C.sub.1-3alkyl or CH.sub.2-aryl, or R.sub.2 and R.sub.3 are linked together and fused with piperazinedione present in Chemical Formula 1 to form one of the following structures: ##STR00039## wherein, X is O or NR.sub.6, wherein R.sub.6 is methyl, R.sub.4 is H, and R.sub.5 is H.

4. The compound of 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]nonan-7,9-dione (Compound 1), 6-benzyl-1,8-dimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonan-7,9-dio ne (Compound 2), 1,8-dimethyl-6-(3,4,5-trimethoxybenzyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonan-7,9-dione (Compound 3), 6-(3,5-difluorobenzyl)-1,8-dimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]n onan-7,9-dione (Compound 4), 1,8-dimethyl-6-(quinolin-2-ylmethyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonan-7,9-dione (Compound 5), 1,8-dimethyl-6-(pyridin-2-ylmethyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonan-7,9-dione (Compound 6), 11-benzyltetrahydro-5H,7H-4,9a-(epiminomethano)pyrrolo[2,1-c][1,2,4]dithiazepin-5,10-dione (Compound 7), 12-benzyltetrahydro-5H,10H-4,10a-(epiminomethano)[1,4]oxazino[3,4-c][1,2,4]dithiazepin-5,11-dione (Compound 8), 12-(pyridin-4-ylmethyl)tetrahydro-5H,10H-4,10a-(epiminomethano)[1,4]oxazino[3,4-c][1,2,4]dithiazepin-5,11-dione (Compound 9), 12-ethyltetrahydro-5H,10H-4,10a-(epiminomethano)[1,4]oxazino[3,4-c][1,2,4]dithiazepin-5,11-dione (Compound 10), 11-(pyridin-4-ylmethyl)tetrahydro-5H,7H-4,9a-(epiminomethano)pyrro lo[2,1-c][1,2,4]dithiazepin-5,10-dione (Compound 11), 1,8-dimethyl-6-((6-methylpyridin-2-yl)methyl)-2,3-dithia-6,8-diazabicy clo[3.2.2]nonan-7,9-dione (Compound 12), 1,8-dimethyl-6-((1-methyl-1H-pyrazol-4-yl)methyl)-2,3-dithia-6,8-diaz abicyclo[3.2.2]nonan-7,9-dione (Compound 13), 1,8-dimethyl-6-(thiophen-2-ylmethyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonan-7,9-dione (Compound 14), 6-(benzo[d]thiazol-2-ylmethyl)-1,8-dimethyl-2,3-dithia-6,8-diazabicycl o[3.2.2]nonan-7,9-dione (Compound 15), 1,6-dimethyl-8-(pyrimidin-2-ylmethyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonan-7,9-dione (Compound 16), 1,6-dimethyl-8-((1-methyl-1H-imidazol-4-yl)methyl)-2,3-dithia-6,8-dia zabicyclo[3.2.2]nonan-7,9-dione (Compound 17), methyl 4-((1,6-dimethyl-7,9-dioxo-2,3-dithia-6,8-diazabicyclo[3.2.2]nonan-8-y l)methyl)benzoate (Compound 18), 1-benzyl-6,8-dimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonan-7,9-dio ne (Compound 19), 6,8-diethyl-1-methyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonan-7,9-dione (Compound 20), 12-benzyl-9-methylhexahydro-5H-4,10a-(epiminomethano)pyrazino[2, 1-c][1,2,4]dithiazepin-5,11-dione (Compound 21), 12-benzyl-9-methylhexahydro-5H-4,10a-(epiminomethano)pyrazino[2, 1-c][1,2,4]dithiazepin-5,11-dione (Compound 22), 12-benzyl-9-methylhexahydro-5H-4,10a-(epiminomethano)pyrazino[2, 1-c][1,2,4]dithiazepin-5,11-dione (Compound 23), 6-benzyl-1,4,8-trimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonan-7,9-di one (Compound 24), or 6-benzyl-1,4,8-trimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonan-7,9-di one (Compound 25).

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

6. The method claim 5, 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 lesion, venous ulcer, deep vein thrombosis, vasospasm, arteritis and vascular restenosis.

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

8. Use of a compound of any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment or prevention of a vascular disease.

9. The use of 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 lesion, venous ulcer, deep vein thrombosis, vasospasm, arteritis and vascular restenosis.

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

11. A method for preparing the compound represented by Chemical Formula 3, comprising reacting a 6-(1-hydroxyalkyl)piperazin-2,5-dione derivative represented by Chemical Formula 4 with (a) sulfur (Ss) and (b) lithium bis(trimethylsilyl)amide (LiHMDS) or sodium bis(trimethylsilyl)amide (NaHMDS). ##STR00040## In the Chemical Formula 4, R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are the same as in Chemical Formula 1 of claim 1; R.sub.5 is H; and R is a protecting group, In the Chemical Formula 3, R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are the same as in Chemical Formula 1 of claim 1; R.sub.5 is H; and n is 2 or 3.

12. A method for preparing a compound represented by the following Chemical Formula 1, comprising (S1) reacting a compound represented by Chemical Formula 4 with (a) sulfur (S.sub.8) and (b) LiHMDS (lithium bis(trimethylsilyl)amide) or NaHMDS (sodium bis(trimethylsilyl)amide) to obtain a compound represented by Chemical Formula 3; (S2) reducing the compound of Chemical Formula 3 to obtain a compound represented by Chemical Formula 2; and (S3) forming an intramolecular disulfide crosslink from the compound represented by Chemical Formula 2. ##STR00041## In the Chemical Formula 4, 3, 2 and 1, R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are the same as in Chemical Formula 1 of claim 1; R.sub.5 is H; R is a protecting group; and n is 2 or 3.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0130] FIGS. 1-3 are the results of immunoblot analysis about the effect of the compounds according to the present disclosure on PDGF-induced tyrosine phosphorylation in HASMC and PASMC depleted of PrxII. FIG. 1 is the results of evaluating the con-centration-dependent effect of of Compound 1 on the degree of Tyr 857 phosphorylation of the PDGF receptor- (PDGFR) augmented by PrxII knock-down in human aortic smooth muscle cells (HASMC). FIG. 2 is the results of evaluating the effect of Compound 1, 2, 5, 6, 10, 15, 19, 20, 21, 22, and 23 on the degree of Tyr 857 phosphorylation of PDGF receptor- augmented by PrxII knock-down in human aortic smooth muscle cells. FIG. 3 is the results of evaluating the effect of Compound 8 on the degree of the PDGF-induced intracellular tyrosine phosphorylation augmented by PrxII knock-down and the degree of phosphorylation of signaling proteins such as PLC-1 at downstream of the PDGF receptor- in human pulmonary artery smooth muscle cells (PASMC).

[0131] FIGS. 4-6 are the results of immunoblot analysis of the effect of the compound according to the present disclosure on VEGF-induced tyrosine phosphorylation in HAEC and PAEC depleted of PrxII. FIG. 4 is the results of evaluating the effect of each concentration of Compound 1 on the degree of Tyr 1175 phosphorylation of the VEGF receptor-2 (VEGFR2) augmented by PrxII knock-down in HAECs. FIG. 5 is the results of evaluating the effect of Compound 1, 2, 5, 6, 10, 20, 21, and 23 on the degree of VEGF receptor phosphorylation augmented by PrxII knock-down in HAECs. FIG. 6 is the results of evaluating the effect of Compound 8 on the degree of VEGF-induced intracellular tyrosine phosphorylation augmented by PrxII knock-down and the degree of phosphorylation of signaling proteins such as ERK2 at downstream of the VEGF receptor-2 in PAECs.

[0132] FIGS. 7-9 are the results of preclinical efficacy tests using a Sugen/hypoxia rat model of pulmonary arterial hypertension. FIG. 7 shows significant reduction of right ventricular systolic pressure and inhibition of right ventricular hypertrophy compared to vehicle control group. FIG. 8 is a result showing that occluded blood vessels in the vehicle control group were reversed. FIG. 9 is the immunofluorescence staining results showing the proliferation of pulmonary arterial endothelial cells and the inhibition of smooth muscle cell hyperplasia.

MODE FOR THE INVENTION

[0133] Hereinafter, the present invention is described in considerable detail with examples to help those skilled in the art understand the present invention. However, the following examples are offered by way of illustration and are not intended to limit the scope of the invention. It is apparent that various changes may be made without departing from the spirit and scope of the invention or sacrificing all of its material ad-vantages.

[0134] Amino acids used as starting materials in the following preparations were L-form, which was easy to obtain. However, it would be okay to proceed with synthesis using D-form or racemic form.

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

##STR00010##

Step 1. Methyl N(N-benzyloxycarbonyl)-O-t-butyl-L-seryl)-N-methyl-L-alanine (intermediate 3)

[0135] 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 h. 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 Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure to give a crude residue. This residue was purified by chromatography on silica to give intermediate 3 (20.0 g, 48.2 mmol, 88.9% yield) as yellow oil. MS (ESI) m/z=395.1 [M+1].sup.+

Step 2. (3S,6S)-3-(t-butoxymethyl)-1,6-dimethylpiperazine-2,5-dione (intermediate 4)

[0136] To a mixture of intermediate 3 (35.0 g, 88.7 mmol, 1.00 eq) in MeOH (450 mL) was added Pd/C (3.00 g, 10.0% purity). The mixture was stirred at 50 C. under H.sub.2 (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 light-yellow oil.

Step 3. (3S,6S)-3-(t-butoxymethyl)-1,4,6-trimethylpiperazine-2,5-dione (intermediate 5)

[0137] To a stirred solution of intermediate 4 (19.0 g, 83.2 mmol, 1.00 eq) in DMF (500 mL) at 0 C. was added NaH (3.99 g, 99.9 mmol, 60% purity, 1.20 eq) and Mel (186 g, 1.31 mmol, 81.4 mL, 15.7 eq). The resulting solution was stirred for 12 hours under a nitrogen atmosphere. Water (2 L) was added to the reaction and the mixture was extracted with CH.sub.2Cl.sub.2 (300 mL*3). The combined organic phases were washed with brine (500 mL*2), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated in vacuo. This product was purified on silica to give intermediate 5 (19.0 g, 78.4 mmol, 94.2% yield) as light-yellow oil.

Step 4. (3S,6S)-3-(((t-butyldimethylsilyl)oxy)methyl)-1,4,6-trimethylpiperazine-2,5-dione (intermediate 6)

[0138] To a mixture of intermediate 5 (19.0 g, 78.4 mmol, 1.00 eq) in CH.sub.2Cl.sub.2 (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, then the concentrate was dissolved in DMF (200 mL). To this mixture was added TBSCl (14.6 g, 96.7 mmol, 11.9 mL, 1.20 eq) and imidazole (16.5 g, 242 mmol, 3.00 eq) and stirred at 25 C. for 1 hour. Water (1000 mL) was added to the reaction mixture and the mixture was extracted with CH.sub.2Cl.sub.2 (300 mL*3). The combined organic phases were washed with brine (200 mL*2), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated in vacuo. The residue was purified on silica gel to give intermediate 6 (20.0 g, 66.6 mmol, 82.6% yield) as light-yellow solid.

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

[0139] Sulfur (853 mg, 26.63 mmol, 8 eq) was added to anhydrous THF (53 mL) at room temperature, then NaHMDS (10 mL, 1M in THF, 10 mmol) was slowly added dropwise, followed by 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, and then additional NaHMDS (8.3 mL, 1M in THF, 8.3 mmol) was slowly added dropwise. After the reaction was stirred for 75 minutes, ammonium chloride solution (200 mL) was added to the reaction mixture and extracted with CH.sub.2Cl.sub.2 (200 mL*2). The combined organic phases were washed with brine (100 mL*2), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated in vacuo. Intermediate 7 (trisulfide ring) obtained by purifying the residue on silica gel was used in the next step.

[0140] Intermediate 7 (200 mg, 0.64 mmol) was dissolved in THF (10 mL) and MeOH (10 mL) at room temperature then cooled to 0 C. After NaBH.sub.4 (0.12 g, 3.2 mmol, 5 eq) was slowly added 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 aqueous NaHCO.sub.3 solution (40 mL) were added to the reaction mixture and stirred at 0 C. The mixture was washed with CH.sub.2Cl.sub.2 (100 mL*3) to remove impurities. The aqueous layer was acidified with 1N aqueous hy-drochloric acid and then extracted with CH.sub.2Cl.sub.2 (200 mL*3). The combined organic phases were washed with brine (100 mL*2), dried over anhydrous Na.sub.2SO.sub.4, filtered, and partially concentrated in vacuo to 100 mL to give intermediate 8 which was used directly in the next step without purification.

[0141] In another reaction vessel, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 120 mg, 0.8 eq) was dissolved in THF (10 mL) and CH.sub.2Cl.sub.2 (100 mL). The solution of intermediate 8 obtained above was slowly added dropwise thereto and reacted at room temperature for 30 minutes. To the reaction mixture was added aqueous NaHCO.sub.3 (200 mL) and the mixture was extracted with CH.sub.2Cl.sub.2 (200 mL*3). The combined organic phases were washed with brine (200 mL*2), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated in vacuo. The residue was purified on silica gel to give 101 mg of compound 1 as pale-yellow solid.

[0142] .sup.1H NMR (400 MHz, CDCl.sub.3) 4.29 (s, 1H), 3.09 (s, 3H), 3.02 (s, 3H), 2.82 (dd, J=12.8, 7.5 Hz, 2H), 1.86 (s, 3H).

[0143] .sup.13C NMR (101 MHz, CDCl.sub.3) 168.79 (s), 166.79 (s), 70.19 (s), 63.49 (s), 33.52 (s), 28.56 (s), 28.13 (s), 21.43 (s).

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)

##STR00011##

Step 1. (3S,6S)-1-Benzyl-6-(t-butoxymethyl)-3,4-dimethylpiperazine-2,5-dione (intermediate 2)

[0144] It was synthesized from intermediate 1 (the same material as intermediate 4 in preparation example 1) in a similar manner to the method for preparing intermediate 5 of preparation example 1. However, benzyl bromide was used instead of iodomethane.

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

[0145] It was prepared by a similar synthesis method to compound 1 of preparation example 1. That is, 1 g of intermediate 3 was obtained using intermediate 2 (1.9 g, 5.97 mmol), sulfur (1.53 g, 8 eq), and NaHMDS (29.8 mL, 1M in THF, 29.8 mmol). Then, intermediate 4 was obtained by using intermediate 3 (0.8 g, 2.35 mmol) and NaBH4 (0.44 g, 5 eq), and then used in the next reaction without purification. Thus, compound 2 (0.44 g) was obtained using intermediate 4 and DDQ (0.426 g, 0.8 eq).

[0146] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.37-7.29 (m, 9H), 7.26 (t, J=7.8 Hz, 8H), 5.20 (d, J=14.9 Hz, 1H), 4.75 (d, J=14.7 Hz, 3H), 4.47 (d, J=14.8 Hz, 3H), 4.30 (d, J=4.8 Hz, 3H), 4.01 (d, J=14.8 Hz, 1H), 3.88 (d, J=14.3 Hz, 1H), 3.32 (dd, J=14.4, 6.4 Hz, 1H), 3.09 (s, 8H), 2.98 (s, 2H), 2.59 (dt, J=23.7, 9.9 Hz, 6H), 1.93 (s, 2H), 1.91 (s, 8H)

[0147] .sup.13C NMR (101 MHz, CDCl.sub.3) 168.80 (s), 166.95 (s), 135.17 (s), 129.24 (s), 128.69 (s), 128.48 (s), 69.81 (s), 61.02 (s), 49.67 (s), 28.55 (d, J=7.5 Hz), 21.61 (s)

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)

##STR00012##

Step 1. Methyl-(3,4,5-trimethoxybenzyl)-L-serine (intermediate 1)

[0148] 3,4,5-trimethoxybenzaldehyde (3.25 g, 1 eq) was added a to a solution of serine methyl ester (5 g, 32.1 mmol) in MeOH (50 mL), and cooled to 0 C. After adding Et.sub.3N (4.48 mL, 1 eq) and stirring for 2 hours, NaBH.sub.4 (2.41 g, 2 eq) was added dropwise at 0 C. and stirred for 2 hours. The solvent was removed under reduced pressure after cooling the reactant to room temperature, then quenched with brine. The reaction mixture was extracted with CH.sub.2Cl.sub.2, the organic layer was washed with brine and dried over anhydrous Na.sub.2SO.sub.4. After removing the solvent under reduced pressure, the obtained residue was purified on silica to give intermediate 1 (2.41 g).

Step 2. (3S,6S)-3-(hydroxymethyl)-1,6-dimethyl-4-(3,4,5-trimethoxybenzyl)piperazine-2,5-di one (intermediate 2)

[0149] To intermediate 1 (2.41 g, 8.1 mmol) in CH.sub.2Cl.sub.2 (150 mL) was added NaHCO.sub.3 (1.0 g, 1.5 eq) dissolved in a small amount of water at room temperature. Fmoc-N-Me-alanine chloride (2.77 g, 1 eq) dissolved in CH.sub.2Cl.sub.2 (40 mL) was added to the intermediate 1 solution, and stirred at room temperature for 3 hours. After adding water (100 mL) to the reaction vessel, the organic layer was washed with 1N HCl and brine, and then the organic layer was concentrated under reduced pressure. EtOH (150 mL) and piperidine (0.8 mL, 8.1 mmol, 1 eq) were added to the obtained product, and the mixture was stirred for 4 hours while refluxing. After cooling the reaction mixture to room temperature, the organic solvent was concentrated under reduced pressure, and the residue was purified on silica to obtain intermediate 2 (0.92 g).

Step 3. (3S,6S)-3-(((t-butyldimethylsilyl)oxy)methyl)-1,6-dimethyl-4-(3,4,5-trimethoxybenzyl)piperazine-2, 5-dione (intermediate 3)

[0150] To a stirred solution of intermediate 2 (0.91 g, 2.58 mmol) in anhydrous CH.sub.2Cl.sub.2 (20 mL) was added tert-butyldimethylsilyl chloride (TBSCl, 0.78 g, 2 eq) at 0 C. under nitrogen atmosphere. To this was slowly added a solution of imidazole (0.35 g, 2 eq) in anhydrous CH.sub.2Cl.sub.2 (5 mL). The mixture was stirred at 20 C. for 12 h then quenched with water (20 mL). The organic layer was washed twice with water and brine (100 mL) and dried over anhydrous Na.sub.2SO.sub.4. The organic layer was concentrated, the solvent was completely removed under reduced pressure, and the resulting material was purified on silica to obtain intermediate 3 (1.21 g).

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)

[0151] It was prepared by a similar synthesis method to compound 1 of preparation example 1. That is, 0.53 g of intermediate 4 was obtained using intermediate 3 (1.2 g, 2.57 mmol), sulfur (0.66 g, 8 eq), and NaHMDS (14.1 mL, 1M in THF, 14.1 mmol). Intermediate 5 was obtained by using intermediate 4 (0.25 g, 0.58 mmol) and NaBH.sub.4 (0.1 g, 5 eq), and then used in the next reaction without purification. Thus, compound 3 (0.125 g) was obtained using intermediate 5 and DDQ (0.094 g, 0.42 mmol, 0.8 eq).

[0152] .sup.1H NMR (400 MHz, CDCl.sub.3) 6.48 (s, 2H), 4.65 (d, J=14.7 Hz, 1H), 4.43 (d, J=14.7 Hz, 1H), 4.35 (dd, J=5.7, 2.0 Hz, 1H), 3.84 (s, 6H), 3.83 (s, 3H), 3.10 (s, 3H), 2.57 (qd, J=14.0, 3.9 Hz, 2H), 1.91 (s, 3H)

[0153] .sup.13C NMR (101 MHz, CDCl.sub.3) 168.85, 166.98, 153.82, 138.07, 130.98, 105.72, 69.85, 61.53, 61.05, 56.40, 50.22, 28.62, 28.39, 21.60

Preparation example 4: synthesis of 6-(3,5-difluorobenzyl)-1,8-dimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dion e (Compound 4)

##STR00013##

Step 1. Methyl-(3,5-difluorobenzyl)-L-serine (intermediate 1)

[0154] It was prepared by a similar synthesis method to intermediate 1 of preparation example 3. However, 2,4-difluorobenzaldehyde was used instead of 3,4,5-trimethoxybenzaldehyde.

Step 2. (3S,6S)-1-(3,5-difluorobenzyl)-6-(hydroxymethyl)-3,4-dimethylpiperazine-2,5-dione (intermediate 2)

[0155] It was prepared by a similar synthesis method to intermediate 2 of preparation example 3. However, Fmoc-N-Me-alanine was used instead of Fmoc-N-Me-alanine chloride.

Step 3. (3S,6S)-3-(((t-butyldimethylsilyl)oxy)methyl)-4-(3,5-difluorobenzyl)-1,6-dimethylpipe razine-2,5-dione (intermediate 3)

[0156] It was prepared by a similar synthesis method to intermediate 3 of preparation example 3.

Step 4. 6-(3,5-Difluorobenzyl)-1,8-dimethyl-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-dio ne (compound 4)

[0157] It was prepared by a similar synthesis method to compound 1 of preparation example 1. That is, 0.26 g of intermediate 4 was obtained using intermediate 3 (0.86 g, 2.08 mmol), sulfur (0.54 g, 8 eq), and NaHMDS (11.5 mL, 1M in THF, 11.5 mmol). Intermediate 5 was obtained by using intermediate 4 (0.26 g, 0.69 mmol) and NaBH.sub.4 (0.13 g, 5 eq) and then used in the next reaction without purification. Thus, compound 4 (0.015 g) was obtained using the intermediate 5 and DDQ (0.13 g, 0.8 eq).

[0158] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.37 (dd, J=15.0, 8.1 Hz, 1H), 6.86 (dt, J=20.7, 9.2 Hz, 2H), 4.77 (d, J=14.9 Hz, 1H), 4.44 (d, J=14.9 Hz, 1H), 4.37 (d, J=5.7 Hz, 1H), 3.08 (s, 3H), 2.75 (dd, J=14.1, 5.8 Hz, 1H), 2.67 (d, J=14.0 Hz, 1H), 1.88 (s, 3H)

[0159] .sup.13C NMR (101 MHz, CDCl.sub.3) 168.62 (s), 167.03 (s), 164.21 (d, J=12.3 Hz), 162.46 (d, J=11.9 Hz), 161.71 (d, J=12.3 Hz), 159.99 (d, J=12.0 Hz), 132.44 (dd, J=9.7, 5.3 Hz), 118.46 (d, J=15.1 Hz), 112.38 (dd, J=21.3, 3.7 Hz), 104.52 (s), 104.27 (s), 104.01 (s), 69.8, 61.6, 42.7 (d, J=2.9 Hz), 28.8, 28.6, 21.5

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

##STR00014##

Step 1. (3S,6S)-3-(t-butoxymethyl)-1,6-dimethyl-4-(quinolin-2-ylmethyl)piperazine-2,5-dione (intermediate 2)

[0160] It was synthesized from intermediate 1 (the same material as intermediate 4 in preparation example 1), in a similar manner to the method for preparing intermediate 2 of preparation example 2. However, 2-quinolinemethyl chloride was used instead of iodomethane.

Step 2. 1,8-Dimethyl-6-(quinolin-2-ylmethyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-di one (compound 5)

[0161] It was prepared by a similar synthesis method to compound 1 of preparation example 1. That is, intermediate 2 (1.15 g, 3.11 mmol), sulfur (0.79 g, 8 eq), and NaHMDS (15.6 mL, 1M in THF, 15.6 mmol) was used to obtain 0.72 g (59% yield) of intermediate 3. Intermediate 4 was obtained by using intermediate 3 (0.55 g, 1.44 mmol) and NaBH.sub.4 (0.26 g, 5 eq), and then used in the next reaction without purification. Thus, compound 5 (0.25 g) was obtained using intermediate 4 and DDQ (0.255 g, 0.8 eq).

[0162] .sup.1H NMR (400 MHz, CDCl.sub.3) 8.16 (d, J=8.4 Hz, 1H), 8.02 (dd, J=8.4, 0.7 Hz, 1H), 7.81 (dd, J=8.1, 1.3 Hz, 1H), 7.72 (ddd, J=8.4, 6.9, 1.4 Hz, 1H), 7.55 (ddd, J=8.1, 6.9, 1.2 Hz, 1H), 7.45 (d, J=8.4 Hz, 1H), 5.06 (d, J=14.9 Hz, 1H), 4.71 (d, J=14.9H, 1H), 4.61 (dd, J=6.1, 1.8 Hz, 1H), 3.11 (s, 3H), 2.81 (dd, J=14.0, 1.8 Hz, 1H), 2.71 (dd, J=14.0, 6.1 Hz, 1H), 1.91 (s, 3H)

[0163] .sup.13C NMR (101 MHz, CDCl.sub.3) 168.9, 167.1, 155.8, 147.7, 137.6, 130.1, 129.3, 127.8, 127.7, 127.0, 120.7, 69.8, 62.0, 52.2, 28.8, 28.6, 21.6

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

##STR00015##

Step 1. (3S,6S)-3-(t-butoxymethyl)-1,6-dimethyl-4-(pyridin-2-ylmethyl)piperazine-2,5-dione (intermediate 2)

[0164] It was synthesized from intermediate 1 (the same material as intermediate 4 in preparation example 1), in a similar manner to the method for preparing intermediate 2 of preparation example 2. However, 2-pyridinemethyl chloride was used instead of benzyl bromide.

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

[0165] It was prepared by a similar synthesis method to compound 1 of preparation example 1. That is, 0.68 g of intermediate 3 was obtained using intermediate 2 (1.05 g, 3.29 mmol), sulfur (0.84 g, 8 eq), and NaHMDS (16.4 mL, 1M in THF, 16.4 mmol). Intermediate 4 was obtained by using intermediate 3 (0.58 g, 1.70 mmol) and NaBH4 (0.32 g, 5 eq), and then used in the next reaction without purification. Thus, compound 6 (0.23 g) was obtained using intermediate 4 and DDQ (0.34 g, 0.8 eq).

[0166] .sup.1H NMR (400 MHz, CDCl.sub.3) 8.52 (ddd, J=4.9, 1.7, 0.9 Hz, 1H), 7.67 (td, J=7.7, 1.8 Hz, 1H), 7.33 (d, J=7.8 Hz, 1H), 7.22 (ddd, J=7.5, 4.9, 1.1 Hz, 1H), 4.92 (d, J=14.9 Hz, 1H), 4.56 (dd, J=6.0, 1.9 Hz, 1H), 4.47 (d, J=14.9 Hz, 1H), 3.09 (s, 3H), 2.81 (dd, J=14.0, 1.9 Hz, 1H), 2.72 (dd, J=14.0, 6.0 Hz, 1H), 1.87 (s, 3H)

[0167] .sup.13C NMR (101 MHz, CDCl.sub.3) 168.88, 166.95, 155.48, 149.68, 137.33, 123.24, 123.22, 69.81, 61.94, 51.56, 28.70, 28.56, 21.52

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

##STR00016##

Step 1. Methyl N-((benzyloxycarbonyl)-O-(t-butyl)-L-seryl-L-proline (intermediate 3)

[0168] It was prepared similarly to the synthesis of intermediate 3 in preparation example 1. However, proline methyl ester was used instead of N-methylalanine methyl ester.

Step 2. (3S,8aR)-3-(t-butoxymethyl)hexahydropyrrolo[1,2-a]pyrazine-1,4-dione (intermediate 4)

[0169] To a mixture of intermediate 3 (98.0 g, 1.00 eq) in MeOH (980 mL) was added Pd/C (9.80 g, 10% purity) and the mixture was stirred under H.sub.2 (50 psi) at 70 C. for 12 h. 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 white solid.

Step 3. (3S,8aR)-2-benzyl-3-(t-butoxymethyl)hexahydropyrrolo[1,2-a]pyrazine-1,4-dione (intermediate 5)

[0170] It was synthesized similarly to the method for preparing intermediate 2 of preparation example 2.

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

[0171] It was prepared by a similar synthesis method to compound 1 of preparation example 1. That is, 0.43 g of intermediate 6 was obtained using intermediate 5 (1.5 g, 4.54 mmol), sulfur (1.16 g, 8 eq), and NaHMDS (22.7 mL, 1M in THF, 22.7 mmol). Intermediate 7 was obtained by using intermediate 6 (0.41 g, 1.16 mmol) and NaBH4 (0.22 g, 5 eq) and then used in the next reaction without purification. Thus, compound 7 (0.085 g) was obtained using intermediate 7 and DDQ (0.24 g, 0.8 eq).

[0172] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.40-7.26 (m, 3H), 7.25 (d, J=8.6 Hz, 2H), 4.64 (d, J=14.8 Hz, 1H), 4.51 (d, J=14.8 Hz, 1H), 4.23 (d, J=6.0 Hz, 1H), 3.87-3.79 (m, 1H), 3.73 (dd, J=19.0, 9.8 Hz, 1H), 2.69 (ddd, J=13.8, 11.7, 8.0 Hz, 1H), 2.60 (dd, J=14.1, 6.1 Hz, 1H), 2.52 (d, J=14.1 Hz, 1H), 2.33 (dd, J=14.0, 6.7 Hz, 1H), 2.17 (ddd, J=19.9, 11.7, 4.1 Hz, 2H)

[0173] .sup.13C NMR (101 MHz, CDCl.sub.3) 167.98, 165.57, 135.31, 129.25, 128.71, 128.48, 72.95, 62.96, 49.29, 45.98, 35.16, 27.94, 21.19

Preparation example 8: Synthesis of 12-benzyltetrahydro-5H,10H-4,10a-(epiminomethano)[1,4]oxazino[3,4-c][1,2,4]dithia zepine-5,11-dione (Compound 8)

##STR00017##

Step 1. Methyl-4-(N-((benzyloxy)carbonyl)-O-(t-butyl)-L-seryl)morpholine-3-carboxylate (intermediate 3)

[0174] It was prepared similarly to the synthesis of intermediate 3 in preparation example 7. However, morpholine-3-carboxylic acid methyl ester was used instead of proline methyl ester.

Step 2. (7S)-7-(t-butoxymethyl)hexahydropyrazino[2,1-c][1.4]oxazin-6,9-dione (intermediate 4)

[0175] It was prepared similarly to the synthesis of intermediate 4 in preparation example 7.

Step 3. (7S)-8-Benzyl-7-(t-butoxymethyl)hexahydropyrazino[2,1-c][1.4]oxazine-6,9-dione (intermediate 5)

[0176] It was prepared similarly to the synthesis of intermediate 5 in preparation example 7. Two isomers (diastereomer) were obtained, with one of two isomers (5.20 g, 22.4% yield, 99.3% purity) being white solid while another isomer (5.10 g, 21.8% yield, 98.6% purity) being yellow oil.

[0177] HPLC: 99.3% purity (isomer 1) & 98.6% purity (isomer 2)

[0178] H NMR (isomer 1): 400 MHz, CDCl.sub.3 7.19-7.44 (m, 5H), 5.33 (d, J=15.2 Hz, 1H), 4.50 (dd, J=13.6, 2.0 Hz, 1H), 4.25-4.37 (m, 2H), 3.87-4.00 (m, 3H), 3.71-3.84 (m, 2H), 3.61-3.69 (m, 1H), 3.47 (td, J=11.6, 2.4 Hz, 1H), 2.88 (td, J=12.8, 3.6 Hz, 1H), 1.17 ppm (s, 9H).

[0179] H NMR (isomer 2): 400 MHz, CDCl.sub.3 7.17-7.43 (m, 5H), 5.21 (d, J=15.2 Hz, 1H), 4.48 (dd, J=12.0, 4.2 Hz, 1H), 4.20-4.32 (m, 2H), 3.83-4.08 (m, 3H), 3.71 (dd, J=9.6, 1.6 Hz, 1H), 3.60 (dd, J=9.2, 2.4 Hz, 1H), 3.31-3.55 (m, 2H), 2.87 (td, J=12.8, 4.0 Hz, 1H), 1.11 ppm (s, 9H).

Step 4. 12-benzyltetrahydro-5H,10H-4,10a-(epiminomethano[1,4]oxazino[3,4-c][1,2,4]dithiaz epine-5, 11-dione (Compound 8)

[0180] It was prepared by similar synthesis method to compound 1 of preparation example 1. That is, 1.24 g (58.3% yield) of intermediate 6 was prepared using intermediate 5 (any of the isomers, 2.0 g, 5.77 mmol), sulfur (1.48 g, 8 eq), and NaHMDS (28.9 mL, 1M in THF, 28.9 mmol). Intermediate 7 was obtained by using intermediate 6 (1.2 g, 3.26 mmol) and NaBH4 (0.62 g, 5 eq) and then used in the next reaction without purification. Thus, 0.45 g (41% yield) of compound 8 was obtained by using intermediate 7 and DDQ (0.67 g, 2.93 mmol, 0.8 eq).

[0181] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.34 (t, J=7.9 Hz, 3H), 7.28-7.21 (m, 2H), 4.71 (d, J=14.8 Hz, 1H), 4.46 (d, J=14.8 Hz, 1H), 4.26 (d, J=3.5 Hz, 1H), 4.17 (d, J=13.7 Hz, 1H), 4.13-4.05 (m, 2H), 3.96 (d, J=13.5 Hz, 1H), 3.59 (td, J=12.3, 2.5 Hz, 1H), 3.21 (td, J=13.1, 4.0 Hz, 1H), 2.70-2.58 (m, 2H)

[0182] .sup.13C NMR (101 MHz, CDCl.sub.3) 169.23, 165.90, 134.80, 129.33, 128.74, 128.74, 68.82, 65.81, 65.45, 60.51, 49.19, 38.20, 29.42

[0183] LCMS: RT=0.342 min, m/z=337.0 [M+H].sup.+

Preparation example 9: synthesis of 12-(pyridin-4-ylmethyl)tetrahydro-5H,10H-4,10a-(epiminomethano) [1,4]oxazino[3,4-c][1,2,4]dithiazepin-5,11-dione (Compound 9)

##STR00018##

Step 1. (7S)-7-(tert-butoxymethyl)-8-(pyridin-4-ylmethyl)hexahydropyrazino[2,1-c][1,4]oxazi ne-6,9-dione (intermediate 2)

[0184] It was synthesized from intermediate 1 (the same material as intermediate 4 in preparation example 8) in a similar manner to the method for preparing intermediate 5 of preparation example 8. However, 4-pyridylmethylchloride was used instead of benzyl bromide to give brown oil.

Step 2. 12-(pyridin-4-ylmethyl)tetrahydro-5H,10H-4,10a-(epiminomethano)[1,4]oxazino[3,4-c][1,2,4]Dithiazepine-5,11-dione (Compound 9)

[0185] It was prepared by a similar synthesis method to compound 1 of preparation example 1. That is, 0.68 g of the trisulfide intermediate was obtained as a yellow solid from 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 reduction of trisulfide intermediate (0.68 g, 1.84 mmol) with NaBH.sub.4 (0.2 g, 2.9 eq), it was used in the next reaction without purification. Thus, compound 9 (41 mg) was obtained as white solid using the reduced intermediate and iodine (0.47 g, 1.0 eq).

[0186] .sup.1H NMR (400 MHz, CDCl.sub.3) 2.66-2.73 (m, 2H) 3.17 (td, J=13.20, 4.38 Hz, 1H) 3.55 (td, J=12.26, 3.00 Hz, 1H) 3.89 (d, J=13.63 Hz, 1H) 4.00-4.13 (m, 3H) 4.16-4.37 (m, 2H) 4.75 (d, J=15.63 Hz, 1H) 7.11 (d, J=5.75 Hz, 2H) 8.43-8.68 (m, 2H)

[0187] LCMS: RT=0.342 min, m/z=337.9 [M+H].sup.+

Preparation example 10: Synthesis of 12-ethyltetrahydro-5H,10H-4,10a-(epiminomethano)[1,4]oxazino[3,4-c][1,2,4]dithiaze pine-5,11-dione (Compound 10)

##STR00019##

Step 1. (7S)-7-(tert-butoxymethyl)-8-ethylhexahydropyrazino[2,1-c][1,4]oxazine-6,9-dione (intermediate 2)

[0188] It was synthesized from intermediate 1 (the same material as intermediate 4 in preparation example 8) in a similar manner to the method for preparing intermediate 5 of preparation example 8. However, ethyl bromide was used instead of benzyl bromide.

Step 2. 12-ethyltetrahydro-5H,10H-4,10a-(epiminomethano)[1,4]oxazino[3,4-c][1,2,4]dithiaze pine-5, 11-dione (Compound 10)

[0189] It was prepared by a similar synthesis method to compound 1 of preparation example 1. That is, 0.35 g of trisulfide intermediate was obtained from intermediate 2 (0.57 g, 2.0 mmol), sulfur (0.51 g, 8 eq), and NaHMDS (5.0 mL, 2M in THF, 5 eq). After reduction of trisulfide intermediate (0.35 g, 1.2 mmol) with NaBH.sub.4 (0.23 g, 5 eq), it was used in the next reaction without purification. Thus, compound 10 (130 mg) was obtained as white solid using the reduced intermediate and DDQ (0.24 g, 0.9 eq).

[0190] .sup.1H NMR (400 MHz, DMSO-.sub.d6) 1.07 (t, 3H), 2.95 (m, 1H), 3.00 (t, 2H), 3.13 (m, 1H), 3.55 (m, 2H), 3.85 (d, 2H), 3.98 (m, 2H), 4.60 (dd, 1H)

Preparation example 11: synthesis of 11-(pyridin-4-ylmethyl)tetrahydro-5H,7H-4,9a-(epiminomethano)pyrrolo[2,1-c][1,2,4]dithiazepine-5,10-dione (Compound 11)

##STR00020##

Step 1. (7S)-7-(tert-butoxymethyl)-8-ethylhexahydropyrazino[2,1-c][1,4]oxazine-6,9-dione (intermediate 2)

[0191] It was synthesized from intermediate 1 (the same material as intermediate 4 in preparation example 7) in a similar manner to the method for preparing intermediate 5 of preparation example 7. However, 4-pyridylmethyl chloride was used instead of benzyl bromide.

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

[0192] It was prepared by a similar synthesis method to compound 1 of preparation example 1. That is, 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), 0.6 g of trisulfide intermediate was obtained as a yellow solid. After reduction of trisulfide intermediate (1.0 g, 2.83 mmol) 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).

[0193] .sup.1H NMR (400 MHz, CDCl.sub.3) 2.09-2.28 (m, 2H) 2.35 (ddd, J=14.04, 6.60, 1.38 Hz, 1H) 2.56-2.83 (m, 3H) 3.66-3.98 (m, 2H) 4.22 (dd, J=5.44, 2.06 Hz, 1H) 4.41 (d, J=15.64 Hz, 1H) 4.75 (d, J=15.64 Hz, 1H) 7.19 (d, J=5.64 Hz, 2H) 8.61 (br d, J=5.40 Hz, 2H)

[0194] LCMS: RT=0.343 min, m/z=321.9 [M+H].sup.+

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

##STR00021##

Step 1. (3S,6S)-3-(tert-butoxymethyl)-1,6-dimethyl-4-((6-methylpyridin-2-yl)methyl)piperazin e-2,5-dione (intermediate 2)

[0195] It was synthesized from intermediate 1 (the same material as intermediate 4 in preparation example 1) in a similar manner to the method for preparing intermediate 2 of preparation example 5. However, 6-methyl-2-pyridylmethyl chloride was used instead of quinoline-2-methyl chloride.

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

[0196] It was prepared by a similar synthesis method to compound 1 of preparation example 1. That is, using intermediate 2 (1.0 g, 3 mmol), sulfur (0.77 g, 8 eq), and NaHMDS (7.5 mL, 2M in THF, 5 eq), 0.47 g of trisulfide intermediate was obtained as a yellow oil. After reduction of trisulfide intermediate (0.47 g, 1.32 mmol) with NaBH.sub.4 (0.15 g, 3.5 eq), it was used in the next reaction without purification. Thus, using the reduced intermediate and iodine (0.67 g, 2.0 eq), 140 mg of compound 12 was obtained as pale-yellow solid.

[0197] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.56 (t, J=7.60 Hz, 1H), 7.10 (dd, J=16.0, 7.60 Hz, 2H), 4.90 (d, J=14.8 Hz, 1H), 4.58 (dd, J=6.00, 1.60 Hz, 1H), 4.43 (d, J=14.8 Hz, 1H), 3.10 (s, 3H), 2.82-2.90 (m, 1H), 2.68-2.77 (m, 1H), 2.52 (s, 3H), 1.89 ppm (s, 3H).

[0198] LCMS: RT=0.376 min, m/z=324.0 [M+H].sup.+

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)

##STR00022##

Step 1. (3S,6S)-3-(tert-butoxymethyl)-1,6-dimethyl-4-((1-methyl-1H-pyrazol-4-yl)methyl)pipe razine-2,5-dione (intermediate 2)

[0199] It was synthesized from intermediate 1 (the same material as intermediate 4 in preparation example 1) in a similar manner to the method for preparing intermediate 2 of preparation example 5. However, 1-methyl-4-pyrazolemethyl chloride was used instead of quinoline-2-methyl chloride.

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)

[0200] It was prepared by a similar synthesis method to compound 1 of preparation example 1. That is, using intermediate 2 (1.0 g, 3.1 mmol), sulfur (0.80 g, 8 eq), and NaHMDS (7.75 mL, 2M in THF, 5 eq), 0.5 g of trisulfide intermediate was obtained as a yellow oil. After reduction of trisulfide intermediate (0.5 g, 1.45 mmol) with NaBH.sub.4 (0.16 g, 3 eq), it was used in the next reaction without purification. Thus, using the reduced intermediate and iodine (1.1 g, 3.0 eq), 201 mg of Compound 13 was obtained as pale white solid.

[0201] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.35-7.46 (m, 2H), 4.54-4.66 (m, 1H), 4.29-4.44 (m, 2H), 3.90 (d, J=1.60 Hz, 3H), 3.10 (d, J=1.60 Hz, 3H), 2.63-2.76 (m, 2H), 1.89 ppm (d, J=1.60 Hz, 3H).

[0202] LCMS: RT=0.409 min, m/z=313.0 [M+H].sup.+

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

##STR00023##

Step 1. (3S,6S)-3-(tert-butoxymethyl)-1,6-dimethyl-4-(thiophen-2-ylmethyl)piperazine-2,5-dio ne (intermediate 2)

[0203] It was synthesized from intermediate 1 (the same material as intermediate 4 in preparation example 1) in a similar manner to the method for preparing intermediate 2 of preparation example 5. However, 2-thiopine methyl chloride was used instead of quinoline-2-methyl chloride.

Step 2. 1,8-Dimethyl-6-(thiophen-2-ylmethyl)-2,3-dithia-6,8-diazabicyclo[3.2.2]nonane-7,9-di one (compound 14)

[0204] It was prepared by a similar synthesis method to compound 1 of preparation example 1. That is, using intermediate 2 (1.0 g, 3.1 mmol), sulfur (0.79 g, 8 eq), and NaHMDS (7.7 mL, 2M in THF, 5 eq), 0.5 g of trisulfide intermediate was obtained as a yellow oil. After reduction of trisulfide intermediate (0.5 g, 1.45 mmol) with NaBH.sub.4 (0.16 g, 3 eq), it was used in the next reaction without purification. Thus, using the reduced intermediate and iodine (1.1 g, 3.0 eq), 201 mg of Compound 14 was obtained as pale white solid.

[0205] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.29 (d, J=5.20 Hz, 1H), 7.01 (d, J=3.40 Hz, 1H), 6.94-6.99 (m, 1H), 4.79-4.89 (m, 1H), 4.67-4.77 (m, 1H), 4.41 (d, J=6.00 Hz, 1H), 3.09 (s, 3H), 2.64-2.72 (m, 1H), 2.52-2.60 (m, 1H), 1.90 ppm (s, 3H)

[0206] LCMS: RT=0.496 min, m/z=315.1 [M+H].sup.+

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)

##STR00024##

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

[0207] It was synthesized from intermediate 1 (the same material as intermediate 4 in preparation example 1) in a similar manner to the method for preparing intermediate 2 of preparation example 5. However, 2-benzothiazole methyl chloride was used instead of quinoline-2-methyl chloride.

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)

[0208] It was prepared by a similar synthesis method to compound 1 of preparation example 1. That is, using intermediate 2 (0.5 g, 1.33 mmol), sulfur (0.34 g, 8 eq), and NaHMDS (3.3 mL, 2M in THF, 5 eq), 0.47 g of trisulfide intermediate was obtained as a brown oil. After reduction of trisulfide intermediate (0.47 g, 1.18 mmol) with NaBH.sub.4 (0.13 g, 3 eq), it was used in the next reaction without purification. Thus, Compound 15 (152 mg) was obtained as yellow solid using the reduced intermediate and iodine (0.6 g, 2.0 eq).

[0209] .sup.1H NMR (400 MHz, CDCl.sub.3) 8.00 (d, J=8.00 Hz, 1H), 7.88 (d, J=8.00 Hz, 1H), 7.46-7.55 (m, 1H), 7.38-7.46 (m, 1H), 5.15 (d, J=15.6 Hz, 1H), 4.85 (d, J=15.6 Hz, 1H), 4.62 (dd, J=5.60, 2.40 Hz, 1H), 3.12 (s, 3H), 2.71-2.87 (m, 2H), 1.92 ppm (s, 3H).

[0210] LCMS: RT=0.409 min, m/z=313.0 [M+H].sup.+

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

##STR00025##

Step 1. Methyl O-(tert-butyl)-N-(2-chloropropanoyl)-L-serinate (intermediate 1)

[0211] 2-Chloropropanoyl chloride (26.1 g, 205 mmol) was added to a solution of t-butoxyserine methyl ester (39.5 g, 186 mmol, 1 eq) and TEA (37.8 g, 51.9 mL, 2 eq) in dichloromethane (400 mL). The reaction mixture was stirred at 0 C. for 0.5 h and at 25 C. for 16 h, then diluted with water (100 mL) and extracted with DCM (50 mL). The combined organic layers were washed with brine (100 mL), dried over Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure to give intermediate 1 (48.6 g, 183 mmol, 97.9% yield) as yellow liquid. It was used in the next step without further purification.

Step 2. 3-(tert-butoxymethyl)-6-methyl-1-(pyrimidin-2-ylmethyl)piperazine-2,5-dione (intermediate 2)

[0212] Pyrimidine-2-ylmethanamine (4.44 g, 40.64 mmol, 1 eq) was added to a mixture of intermediate 1 (10.8 g, 40.6 mmol, 1.00 eq), K.sub.2CO.sub.3 (11.2 g, 81.3 mmol, 2.00 eq) and KI (3.37 g, 20.3 mmol, 0.50 eq) in DMF (120 mL) and stirred for 12 hours. The reaction solution was filtered, and the filtrate was concentrated. The product obtained after purification by prep-HPLC was dissolved in toluene, into which DMAP (433 mg, 0.2 eq) was added, 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 prep-HPLC to obtain intermediate 2 (6.04 g, 17.7 mmol, 43.5% yield, 99.0% purity) as yellow oil.

Step 3. 3-(tert-butoxymethyl)-4,6-dimethyl-1l-(pyrimidin-2-ylmethyl)piperazine-2,5-dione temet(intermediate 3)

[0213] NaH (258 mg, 6.46 mmol, 60% pure, 1.10 eq) was added to a solution of intermediate 2 (1.80 m 5.88 mmol. 1.00 ea in DMF (18 mL and the mixture was stirred at 0 C. for 0.5 h, then Mel (834 mg, 5.88 mmol, 365 L, 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 the addition of MeOH (3 mL), then concentrated under reduced pressure. The residue was purified by prep-HPLC to obtain intermediate 3 as brown oil (1.6 g, 4.99 mmol, 85.0% yield).

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

[0214] It was prepared by a similar synthesis method to compound 1 of preparation example 1. That is, using intermediate 3 (1.2 g, 3.75 mmol), sulfur (0.85 g, 8 eq), and NaHMDS (14.2 mL, 2M in THF, 5 eq), 0.64 g of trisulfide intermediate was obtained as a white solid. After reduction of trisulfide intermediate (0.25 g, 0.78 mmol) with NaBH.sub.4 (0.11 g, 4 eq), it was used in the next reaction without purification. Thus, using the reduced intermediate and iodine (0.28 g, 1.5 eq), 110 mg of Compound 16 was obtained as pale white solid.

[0215] .sup.1H NMR (400 MHz, CDCl.sub.3) 8.70 (d, J=4.88 Hz, 1H) 7.20 (t, J=4.88 Hz, 1H) 5.65 (d, J=17.26 Hz, 1H) 4.38-4.48 (m, 2H) 3.05 (s, 3H) 2.81-2.94 (m, 2H) 1.70 (s, 3H)

[0216] LCMS: RT=0.384 min, m/z=311.0 [M+H].sup.+

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)

##STR00026##

Step 1. 3-(tert-butoxymethyl)-6-methyl-1-(pyrimidin-2-ylmethyl)piperazine-2,5-dione (intermediate 2)

[0217] It was synthesized similarly to the method for preparing intermediate 2 of preparation example 16. However, 1-methylimidazolemethanamine was used instead of pyrimidin-2-ylmethanamine.

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

[0218] It was synthesized similarly to the method for preparing intermediate 2 of preparation example 16.

Step 3. 1,6-Dimethyl-8-((1-methyl-1H-imidazol-4-yl)methyl)-2,3-dithia-6,8-diazabicyclo[3.2]nonane-7,9-dione (Compound 17)

[0219] It was prepared by a similar synthesis method to compound 1 of preparation example 1. That is, using intermediate 3 (0.5 g, 1.55 mmol), sulfur (0.4 g, 8 eq), and NaHMDS (5.9 mL, 2M in THF, 5 eq), 0.36 g of trisulfide intermediate was obtained as a yellow oil. After reduction of trisulfide intermediate (0.25 g, 0.73 mmol) with NaBH.sub.4 (0.13 g, 5 eq), it was used in the next reaction without purification. Thus, using the reduced intermediate and iodine (0.18 g, 1 eq), 106 mg of Compound 17 was obtained as pale white solid.

[0220] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.41 (s, 1H) 6.96 (s, 1H) 4.92 (d, J=15.51 Hz, 1H) 4.55 (d, J=15.63 Hz, 1H) 4.31 (t, J=4.00 Hz, 1H) 3.67 (s, 3H) 3.01 (s, 3H) 2.84-2.88 (m, 2H) 2.04 (s, 3H)

[0221] LCMS: RT=0.417 min, m/z=313.0 [M+H].sup.+

Preparation example 18: Synthesis of Methyl 4-((1,6-dimethyl-7,9-dioxo-2,3-dithia-6,8-diazabicyclo[3.2.2]nonan-8-yl)methyl)benzo ate (Compound 18)

##STR00027##

Step 1. Methyl 4-((3-(tert-butoxymethyl)-6-methyl-2,5-dioxopiperazin-1-yl)methyl)benzoate (intermediate 2)

[0222] It was synthesized similarly to the method for preparing intermediate 2 of preparation example 16. However, 4-methoxyphenylmethanamine was used instead of pyrimidin-2-ylmethanamine.

Step 2. Methyl 4-((3-(tert-butoxymethyl)-4,6-dimethyl-2,5-dioxopiperazin-1-yl)methyl)benzoate (intermediate 3)

[0223] It was synthesized similarly to the method for preparing intermediate 3 of preparation example 16.

Step 3. Methyl 4-((1,6-dimethyl-7,9-dioxo-2,3-dithia-6,8-diazabicyclo[3.2.2]nonan-8-yl)methyl)benzo ate (compound 18)

[0224] It was prepared by a similar synthesis method to compound 1 of preparation example 1. That is, using intermediate 3 (1.57 g, 4.16 mmol), sulfur (1.1 g, 8 eq), and NaHMDS (15.7 mL, 1M in THF, 5 eq), 1.0 g of trisulfide intermediate was obtained as a yellow oil. After reduction of trisulfide intermediate (0.13 g, 0.32 mmol) with NaBH.sub.4 (0.015 g, 1.2eq), it was used in the next reaction without purification. Thus, Compound 18 (113 mg) was obtained as pale-yellow solid using the reduced intermediate and iodine (0.08 mg, 1 eq).

[0225] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.99-8.01 (m, 2H) 7.37 (d, J=8.25 Hz, 2H) 5.44 (d, J=16.26 Hz, 1H) 4.43 (dd, J=6.38, 0.88 Hz, 1H) 4.29-4.33 (m, 2H) 3.92 (s, 3H) 3.07 (s, 3H) 2.85 (dd, J=14.88, 6.38 Hz, 1H) 1.70 (s, 3H)

[0226] LCMS: RT=0.503 min, m/z=367.1 [M+H].sup.+

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)

##STR00028##

Step 1. 3-Benzyl-6-(tert-butoxymethyl)-1,4-dimethylpiperazine-2,5-dione (intermediate 2)

[0227] To a solution of compound 1 (15.0 g, 33.6 mmol, 1.00 eq) in DMF (100 mL) was added tBuOK (1M, 134 mL, 4.00 eq) and Mel (29.2 g, 205 mmol, 12.8 mL, 6.12 eq). The mixture was stirred at 25 C. for 2 h, then quenched at 0 C. by adding 300 mL of water and extracted with ethyl acetate (100 mL*2). The combined organic layers were washed with brine (50 mL*6), dried over Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by reverse phase HPLC (column: Kromasil Eternity XT 250*80 mm*10 um; mobile phase: [water (NH.sub.4HCO.sub.3)-ACN]; B %: 29%-59%, 20 min) to obtain intermediate 2 (3.20 g, 9.86 mmol, 14.7% yield, 98.1% purity) as white solid.

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

[0228] It was prepared by the same synthetic method to compound 1 of preparation example 1. That is, by using intermediate 2 (1.0 g, 3.14 mmol), sulfur (0.9 g, 8 eq), and NaHMDS (15.6 mL, 1M in THF, 5 eq), 0.5 g of trisulfide intermediate was obtained as a yellow oil. After reduction of trisulfide intermediate (0.5 g, 1.47 mmol) with NaBH.sub.4 (0.3 g, 5 eq), it was used in the next reaction without purification. Thus, Compound 19 (330 mg) was obtained as a white solid using the reduced intermediate and iodine (0.6 g, 1 eq).

[0229] .sup.1H NMR (400 MHz, CDCl.sub.3) 2.85 (d, J=5.60 Hz, 1H) 2.88-2.96 (m, 1H) 3.02 (s, 3H) 3.10 (s, 3H) 3.24 (d, J=16.4 Hz, 1H) 4.16 (d, J=16.4 Hz, 1H) 4.37 (dd, J=5.6, 2.00 Hz, 1H) 7.14 (d, J=7.2 Hz, 2H) 7.19-7.25 (m, 1H) 7.27-7.32 (m, 2H).

[0230] LCMS: RT=0.498 min, m/z=309.0 [M+H].sup.+

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)

##STR00029##

Step 1. 3-(tert-Butoxymethyl)-1,4-diethyl-6-methylpiperazine-2,5-dione (intermediate 2)

[0231] It was prepared similarly to the synthesis of intermediate 2 in preparation example 19 using starting material 1. However, iodoethane was used instead of iodomethane.

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

[0232] It was prepared by the same synthetic method as compound 1 of preparation example 1. That is, using intermediate 2 (0.65 g, 2.4 mmol), sulfur (0.8 g, 10 eq), and NaHMDS (13.2 mL, 1M in THF, 5 eq), 0.55 g of trisulfide intermediate was obtained as a yellow oil. After reduction of trisulfide intermediate (0.55 g, 1.47 mmol) with NaBH.sub.4 (0.47 g, 6.6 eq), it was used in the next reaction without purification. Thus, Compound 20 (216 mg) was obtained as a white solid using the reduced intermediate and iodine (0.9 g, 2 eq).

[0233] .sup.1H NMR (400 MHz, CDCl.sub.3) 4.40 (t, J=3.80 Hz, 1H), 3.68-3.83 (m, 2H), 3.58-3.67 (m, 1H), 3.20 (dd, J=14.0, 7.20 Hz, 1H), 2.80 (d, J=3.60 Hz, 2H), 1.80 (s, 3H), 1.32 (t, J=7.20 Hz, 3H), 1.16 ppm (t, J=7.20 Hz, 3H).

[0234] LCMS: RT=0.451 min, m/z=261.0 [M+H].sup.+

Preparation example 21: 12-benzyl-9-methylhexahydro-5H-4,10a-(epiminomethano)pyrazino[2,1-c][1,2,4]dithi azepine-5,11-dione (Compound 21)

##STR00030##

Step 1. tert-Butyl 8-benzyl-7-(tert-butoxymethyl)-6,9-dioxooctahydro-2H-pyrazino[1,2-a]pyrazine-2-car boxylate (intermediate 2)

[0235] To a solution of compound 1 (20.0 g, 56.3 mmol, 1.00 eq) in THF (500 mL) was added NaH (6.75 g, 169 mmol, 60% pure, 3.00 eq) at 0 C. over 30 min. Benzyl bromide (10.6 g, 61.9 mmol, 7.35 mL, 1.10 eq) was added dropwise at 0 C. and stirred at 25 C. for 12 hours. The reaction mixture was quenched at 0 C. by adding 100 mL of water and extracted with ethyl acetate (100 mL*2). The combined organic layers were washed with brine (100 mL), dried over Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO.sub.2, petroleum ether:ethyl acetate=2:1 to 1:3) to give intermediate 2 (15.0 g, 17.2 mmol, 76.5% purity) as white solid.

Step 2. 8-Benzyl-7-(tert-butoxymethyl)-2-methylhexahydro-2H-pyrazino[1,2-a]pyrazine-6,9-d ione (intermediate 3)

[0236] To a solution of intermediate 2 (10.0 g, 22.4 mmol, 1.00 eq) in EtOAc (70.0 mL) was added HCV/EtOAc (4M, 30.0 mL, 5.35 eq) and stirred at 25 C. for 2 hours. The reaction mixture was concentrated under reduced pressure and the residue was used in the next step without further purification. After dissolving this compound in MeOH (150 mL), formaldehyde (17.6 g, 217 mmol, 16.2 mL, 37.0% purity, 5.00 eq) was added at 25 C. for 30 minutes, followed by NaBH(OAc).sub.3 (13.8 g, 65.1 mmol, 1.50 eq) addition. The resulting mixture was stirred at 25 C. for 1 hour and the reaction mixture was concentrated under reduced pressure to give a residue. Intermediate 3 (5.50 g, 15.2 mmol, 34.9% yield, 100% purity) was obtained after purification by reverse phase HPLC (column: Kromasil Eternity XT 250*80 mm*Oum; mobile phase: [water (NH.sub.4HCO.sub.3)-ACN]; B %: 23%-53%, 20 min) as yellow oil. Two isomers (diastereomers) were formed, and two were isolated from each other.

[0237] Isomer 1: .sup.1H NMR (400 MHz, DMSO-.sub.d6) ppm 1.05 (s, 9H) 1.76-1.90 (m, 1H) 2.17-2.37 (m, 4H) 2.65-2.80 (m, 2H) 3.06-3.20 (m, 2H) 3.58 (s, 2H) 3.94-4.15 (m, 2H) 4.24-4.39 (m, 2H) 4.84 (d, J=15.2 Hz, 1H) 7.10-7.45 (m, 5H).

[0238] Isomer 2: .sup.1H NMR (400 MHz, DMSO-.sub.d6) 0.99-1.10 (m, 9H) 1.73-1.92 (m, 1H) 2.04-2.33 (m, 4H) 2.61-2.82 (m, 2H) 3.09 (dd, J=10.4, 2.69 Hz, 1H) 3.58 (d, J=2.00 Hz, 2H) 3.88-4.03 (m, 1H) 4.09-4.17 (m, 1H) 4.18-4.39 (m, 2H) 4.85 (d, J=15.2 Hz, 1H) 7.24-7.35 (m, 5H)

[0239] MS: m/z=360.2

Step 3. 12-Benzyl-9-methylhexahydro-5H-4,10a-(epiminomethano)pyrazino[2,1-c][1,2,4]dithi azepine-5,11-dione (Compound 21)

[0240] It was prepared by a similar synthesis method to compound 1 of preparation example 1. That is, 0.48 g of the trisulfide intermediate was obtained as a yellow solid using intermediate 3 (isomer 1 or 2, 1.2 g, 3.3 mmol), sulfur (0.86 g, 8 eq), and NaHMDS (16.7 mL, 1 M in THF, 5 eq). After reduction of trisulfide intermediate (0.78 g, 1.47 mmol) with NaBH.sub.4 (0.14 g, 1.9 eq), it was used in the next reaction without purification. Thus, using the reduced intermediate and iodine (0.68 g, 1 eq), 101 mg of Compound 21 was obtained as white solid.

[0241] .sup.1H NMR (400 MHz, CDCl.sub.3) 2.14 (td, J=12.0, 3.63 Hz, 1H) 2.43 (s, 3H) 2.56-2.80 (m, 3H) 2.87-3.03 (m, 1H) 3.16-3.37 (m, 2H) 4.13-4.29 (m, 2H) 4.44 (d, J=14.8 Hz, 1H) 4.82 (d, J=14.8 Hz, 1H) 7.26-7.31 (m, 3H) 7.35-7.39 (m, 2H).

[0242] LCMS: RT=0.520 min, m/z=350.1 [M+H].sup.+

Preparation example 22: Synthesis of 12-benzyl-9-methylhexahydro-5H-4,10a-(epiminomethano)pyrazino[2,1-c][1,2,4]dithi azepine-5,11-dione (Compound 22)

##STR00031##

Step 1. tert-Butyl 7-(tert-butoxymethyl)-6,9-dioxo-8-(pyridin-4-ylmethyl)octahydro-2H-pyrazino[1,2-a]p yrazin-2-carboxylate (intermediate 2)

[0243] It was prepared similarly to the synthesis of intermediate 2 in preparation example 21. However, 4-pyridylmethyl bromide was used instead of benzyl bromide.

Step 2. 7-(tert-butoxymethyl)-2-methyl-8-(pyridin-4-ylmethyl)hexahydro-2H-pyrazino[1,2-a]p yrazine-6,9-dione (intermediate 3)

[0244] It was prepared similarly to the synthesis of intermediate 3 in preparation example 21. Two isomers (diastereomers) were formed, and each isomer was isolated.

[0245] Isomer 1: .sup.1H NMR (400 MHz, DMSO-.sub.d6) 1.05 (d, J=2.80 Hz, 9H) 1.75-1.98 (m, 2H) 2.19-2.37 (m, 3H) 2.59-2.90 (m, 2H) 3.03-3.29 (m, 3H) 3.44-3.66 (m, 2H) 3.97-4.14 (m, 2H) 4.17-4.59 (m, 2H) 4.60-4.95 (m, 1H) 7.28 (dd, J=18.4, 5.76 Hz, 2H) 8.51 (t, J=6.00 Hz, 2H).

[0246] Isomer 2: .sup.1H NMR (400 MHz, DMSO-.sub.d6) 1.04 (d, J=2.80 Hz, 9H) 1.75-2.00 (m, 2H) 2.25 (d, J=9.20 Hz, 3H) 2.75 (br s, 2H) 3.03-3.39 (m, 2H) 3.47 (br dd, J=9.20, 2.38 Hz, 1H) 3.60 (d, J=2.00 Hz, 1H) 3.98-4.07 (m, 1H) 4.09-4.17 (m, 1H) 4.18-4.57 (m, 2H) 4.59-4.92 (m, 1H) 7.28 (dd, J=18.0, 5.88 Hz, 2H) 8.37-8.60 (m, 2H)

Step 3. 12-Benzyl-9-methylhexahydro-5H-4,10a-(epiminomethano)pyrazino[2,1-c][1,2,4]dithi azepine-5,11-dione (Compound 22)

[0247] It was prepared by a similar synthesis method to 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 2, 1.2 g, 3.3 mmol), sulfur (0.85 g, 8 eq), and NaHMDS (16.6 mL, 1 M in THF, 5 eq). After reduction of trisulfide intermediate (0.55 g, 1.44 mmol) with NaBH.sub.4 (0.18 g, 3.2 eq), it was used in the next reaction without purification. Thus, using the reduced intermediate and iodine (0.53 g, 1 eq), Compound 22 (102 mg) was obtained as yellow solid.

[0248] .sup.1H NMR (400 MHz, CDCl.sub.3) 2.16 (br d, J=3.60 Hz, 1H) 2.43 (s, 3H) 2.62 (d, J=13.6 Hz, 1H) 2.69-2.86 (m, 2H) 2.89-3.04 (m, 1H) 3.16-3.35 (m, 2H) 4.14-4.38 (m, 3H) 4.92 (d, J=15.6 Hz, 1H) 7.19 (d, J=5.60 Hz, 2H) 8.61 (d, J=5.60 Hz, 2H).

[0249] LCMS: RT=0.531 min, m/z=351.3 [M+H].sup.+

Preparation example 23: 12-benzyl-9-methylhexahydro-5H-4,10a-(epiminomethano)pyrazino[2,1-c][1,2,4]dithi azepine-5,11-dione (Compound 23)

##STR00032##

Step 1. tert-Butyl 7-(tert-butoxymethyl)-8-methyl-6,9-dioxooctahydro-2H-pyrazino[1,2-a]pyrazine-2-car boxylate (intermediate 2)

[0250] It was prepared similarly to the synthesis of intermediate 2 in preparation example 21. However, methyl iodide was used instead of benzyl bromide.

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

[0251] It was prepared similarly to the synthesis of intermediate 3 in preparation example 21.

Step 3. 12-Benzyl-9-methylhexahydro-5H-4,10a-(epiminomethano)pyrazino[2,1-c][1,2,4]dithi azepine-5,11-dione (Compound 23)

[0252] It was prepared by a similar synthesis method to compound 1 of preparation example 1. That is, using intermediate 3 (4.0 g, 14.1 mmol), sulfur (3.6 g, 8 eq), and NaHMDS (70.6 mL, 1M in THF, 5 eq), 2.1 g of trisulfide intermediate was obtained as a white solid. After reduction of trisulfide intermediate (0.50 g, 1.44 mmol) with NaBH.sub.4 (0.26 g, 5 eq), it was used in the next reaction without purification. Thus, using the reduced intermediate and DDQ (0.33 g, 0.9 eq), Compound 23 (125 mg) was obtained as pale yellow solid.

[0253] .sup.1H NMR (400 MHz, CDCl.sub.3) 4.24 (dd, J=4.9, 3.1 Hz, 1H), 4.19 (dd, J=13.4, 2.0 Hz, 1H), 3.30-3.22 (m, 2H), 3.03 (s, 3H), 2.97-2.87 (m, 3H), 2.53 (d, J=13.6 Hz, 1H), 2.40 (s, 3H), 2.10 (td, J=12.2, 3.6 Hz, 1H).

[0254] .sup.13C NMR (126 MHz, CDCl3) 169.49, 166.15, 68.29, 62.86, 59.28, 52.72, 46.14, 40.01, 33.07, 29.37.

[0255] MS: m/z=274.0715 [M+H].sup.+

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)

##STR00033##

Step 1. (6S)-3-((R)-1-(tert-butoxy)ethyl)-1,6-dimethylpiperazine-2,5-dione (intermediate 1)

[0256] It was similarly prepared to the synthesis of intermediate 4 in preparation example 7.

Step 2. (3S)-1-Benzyl-6-((S)-1-(tert-butoxy)ethyl)-3,4-dimethylpiperazine-2,5-dione (intermediate 2)

[0257] It was prepared in a similar manner to the synthesis of intermediate 2 in preparation example 2.

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

[0258] It was prepared by a similar synthesis method to compound 1 of preparation example 1. That is, using intermediate 2 (0.9 g, 2.71 mmol), sulfur (0.7 g, 8 eq), and NaHMDS (10.9 mL, 1M in THF, 5 eq), the trisulfide intermediate was obtained as a yellow solid. After reduction of trisulfide intermediate (0.49 g, 1.38 mmol) with NaBH.sub.4 (0.16 g, 3 eq), it was used in the next reaction without purification. Thus, Compound 24 was obtained using the reduced intermediate and iodine (0.7 g, 2 eq), where two isomers (diastereomers) were formed, with isomer 1 being yellow solid (157 mg) and isomer 2 being white solid (13 mg).

[0259] Isomer 1: .sup.1H NMR (400 MHz, CDCl.sub.3) 7.22-7.33 (m, 3H), 7.15-7.22 (m, 2H), 4.41-4.68 (m, 2H), 3.88 (s, 1H), 3.02 (s, 3H), 2.67 (q, J=7.20 Hz, 1H), 1.83 (s, 3H), 1.11 (d, J=7.20 Hz, 3H)

[0260] Isomer 2: .sup.1H NMR (400 MHz, CDCl.sub.3) 7.28-7.38 (m, 3H), 7.10-7.18 (m, 2H), 5.45 (d, J=15.2 Hz, 1H), 4.19 (d, J=6.00 Hz, 1H), 3.84 (d, J=15.2 Hz, 1H), 3.08 (s, 3H), 2.96-3.04 (m, 1H), 1.91 (s, 3H), 1.57-1.60 (m, 3H)

[0261] LCMS (isomer 1): RT=1.925 min, m/z=323 [M+H].sup.+

[0262] LCMS (isomer 2): RT=1.894 min, m/z=323 [M+H].sup.+

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)

##STR00034##

Step 1. (6S)-3-((S)-1-(tert-butoxy)ethyl)-1,6-dimethyl-4-(pyridin-2-ylmethyl)piperazine-2,5-di one (intermediate 2)

[0263] It was prepared in a similar manner to the synthesis of intermediate 2 in preparation example 2. However, 2-pyridylmethyl bromide was used instead of benzyl bromide.

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

[0264] It was prepared by a similar synthesis method to compound 1 of preparation example 1. That is, by using intermediate 2 (3.1 g, 9.3 mmol), sulfur (2.4 g, 8 eq), and NaHMDS (37 mL, 1M in THF, 5 eq), 1.67 g of trisulfide intermediate was obtained as a yellow oil. After reduction of trisulfide intermediate (1.67 g, 4.7 mmol) with NaBH.sub.4 (0.53 g, 3 eq), it was used in the next reaction without purification. Thus, using the reduced intermediate and iodine (1 g, 1 eq), Compound 25 (373 mg) was obtained as white solid.

[0265] .sup.1H NMR (400 MHz, CDCl.sub.3) 8.52 (d, J=4.60 Hz, 1H) 7.62 (td, J=7.60, 1.60 Hz, 1H) 7.12-7.23 (m, 2H) 5.44 (d, J=15.6 Hz, 1H) 4.49 (d, J=15.6 Hz, 1H) 3.95-4.09 (m, 3H) 2.96 (s, 3H) 1.68 (d, J=7.20 Hz, 3H) 1.33 (d, J=6.00 Hz, 3H) 1.21 (s, 9H)

[0266] LCMS: RT=0.684 min, m/z=323.9 [M+H].sup.+

Experimental Example 1: Materials

[0267] A rabbit polyclonal antibody specific to peroxiredoxin type 2 (Prx II or Prdx2) was purchased from AbFrontier (Seoul, Korea). The sequences of siRNA specific to human Prx II used in the present invention is as follows:

TABLE-US-00001 5-CGCUUGUCUGAGGAUUACGUU-3 (PrxII-1) 5-AGGAAUAUUUCUCCAAACAUU-3 (PrxII-2).

[0268] Phospho-Tyrosine antibody (4G10) and PDGF-BB were purchased from Upstate. Prx II, Prx-SO.sub.2/3, and phospho-PDGFR-(pY857) rabbit polyclonal antibodies were prepared as described in literature (M. H. Choi et al., Nature 2005 May 19; 435(7040):347-53). Phospho-VEGFR2 (pY1175), VEGFR2, phospho-PLC1 (pY783), PLC1, pTpY-Erk, Erk2 antibodies were purchased from Cell Signaling Technology (CST). Alpha-Tubulin antibody was purchased from Sigma.VEGF-A was purchased from R&D.

Experimental Example 2: Cell Culture

[0269] Human aortic endothelial cells (HAEC), human pulmonary artery endothelial cells (PAEC), human aortic smooth muscle cells (HASMC), human pulmonary artery smooth muscle cells (PASMC) were purchased from BioWhittaker (Belgium). They were seeded on a 0.1% gelatin-coated culture plate. They were grown in Endothelial basal medium (EBM-2) and Smooth Muscle Cell Basal Medium (SmBM) containing 10% fetal bovine serum (FBS) and full medium supplement (Clonetics-BioWhittaker Cat. no. cc-4176 for HAEC and PAEC; Clonetics-BioWhittaker Cat. no. cc-4149 for HASMC and PASMC). Cell culture was carried out in a humidified CO.sub.2 incubator under 5% carbon dioxide at 37 C. Cells at 5 to 7 passages were mainly used in the invention.

Experimental Example 3: Peroxidase Activity Assay

[0270] Hydrogen peroxide (H.sub.2O.sub.2)-reducing peroxidase activity of compounds in present invention was performed as follows. Peroxyredoxin activity was measured in reaction mixture (200 L volume) containing 50 mM HEPES buffer (pH 7.0), 1 mM EDTA, 250 M NADPH, 3 M yeast thioredoxin (Trx), 1.5 M yeast Trx reductase (TR), 50 M test compound, and 1.2 mM H.sub.2O.sub.2.

[0271] Reaction was initiated by adding H.sub.2O.sub.2 and the decrease in absorbance at 340 nm was measured using Agilent UV8453 spectrophotometry (Hewlett Packard, USA) at 30 C. for 12 min. Initial reaction rate is the amount of NADPH oxidized per minute calculated from slope of the linear part of curve.

Experimental Example 4: Measurement of Intracellular H.SUB.2.O.SUB.2.-Eliminating Activity

[0272] The activity of test compounds in present invention to eliminate intracellular H.sub.2O.sub.2 was measured as follows. NIH-3T3 cells (ATCC) were seeded in a 35-mm culture dish at 210.sup.5 cells/well. After culture for 24 hours, cells were serum-starved with phenol red free-basal DMEM containing 0.5% FBS for 6 h. Starved NIH 3T3 cells were pretreated with compounds diluted with phenol red free-basal DMEM at concentrations (0, 12.5, 25, 50, 100 mM) for 30 min, followed by treatment with 20 mU of glucose oxidase (Gox) for 30 min. A reactive oxygen species-sensitive fluorescence dye, carboxymethyl-dichlorofluorescein-diacetate (CM-H.sub.2DCF-DA), which was prepared in 1 mM DMSO stock and diluted 500 times in phenol red free-basal DMEM, was added to the cells and incubated for 5 min. Then, cells were quickly rinsed with basal medium. Images were taken at excitation 497 nm/emission 518 nm settings using a fluorescence microscope. Three images at 10 magnification per sample were obtained, and fluorescence intensities from 30 or more individual cells per image were averaged.

Experimental Example 5: Cytotoxicity Assay

[0273] For cytotoxicity analysis, human aortic endothelial cells (HAEC) and smooth muscle cells (HASMC) were seeded in a 96-well culture plate at density of 4000 cells/well in a final volume of 100 L. Cells were cultured for 24 h and serum-starved for an additional 18 h. Test compounds were serially diluted with EBM-2 culture medium, added to the plate at 100 L/well, and further incubated for 24 h. The number of viable cells was measured using the WST-1 cell viability assay kit (Roche Diagnostics, USA). Cell number was expressed as absorbance values at 450 nm after subtracting the turbidity value at 600 nm. The absorbance values from three replicate wells were averaged.

Experimental Example 6: Immunoblot Analysis

[0274] Cells were quickly washed in ice-cold phosphate-buffered saline (PBS) and lysed in 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 Na.sub.3VO.sub.4, 5 mM NaF, 1 mM AEBSF, aprotinin (5 g/mL), and leupeptin (5 g/mL). After cen-trifugation at 12,000g, the clarified cell extracts were used for immunoblotting. For re-blotting, the immunoblots were retrieved in a solution of 67 mM Tris (pH 6.7), 2% SDS, and 100 mM 2-mercaptoethanol at 60 C. for 30 min to remove antibodies and rinsed three times with with a Tris-buffered saline (TBS) solution containing 1% Triton X-100. The retrieved membranes were subjected to new immunoblotting.

Experimental Example 7: Measurement of Redox Potential

[0275] As shown in Table 1 below, 10 kinds of redox buffer solutions (0.5 mL) are made by mixing with 50 mM solutions of reduced dithiothreitol (DTT) and its oxidized DTT (trans-1,2-dithiane-4,5-diol) at indicated ratios just before use. A test compound (10 mM) was diluted 20-fold with each redox buffer solution and incubated for 3 h in a 30 C. water bath. Then, compounds were analyzed by HPLC. The area of peaks corresponding to the oxidized and reduced forms of test compound based on specific retention time was calculated and plotted as percent of total peak area against the Nernst equation value (mV) at x-axis. The Nernst equation value corresponding to 50% reduction of test compound was set as the midpoint redox potential value for test compound.

TABLE-US-00002 TABLE 1 Ratio DTT(Red) DTT(Ox) Nernst DTT(Red)/DTT(Ox) (L) (L) equation(mV) 4 400 100 341 1 250 250 323 0.25 100 400 305 0.11 50 450 294 0.05 25 475 285 0.02 10 490 272 0.01 5 495 263 0.005 2.5 497.5 254 0.002 1 499 242 0.0004 0.2 499.8 221

[0276] Specific methods and conditions other than Experimental Examples 1-7 were performed similarly to those disclosed in International Patent Application Publication WO2013-077709 or WO2018-008984. All contents disclosed in International Patent Application Publications WO2013-077709 and WO2018-008984 are hereby incorporated by reference.

Experimental Example 8: Growth Factor-Induced Proliferation Assay in Vascular Smooth Muscle Cell and Vascular Endothelial Cell

[0277] The ability of test compounds to control cell proliferation was measured in vascular smooth muscle cells and vascular endothelial cells.

[0278] HASMCs and PASMCs were transfected with a PrxII-specific siRNA using RNAi MAX transfection reagent according to manufacturer's protocol. After 24 hours, transfected cells (3,000 cells/well) were seeded in a 96-well culture plate. At 12 h after seeding, cells were serum-starved for additional 18 h in SmBM basal medium containing 0.5% FBS. Test compounds were serially diluted with the same basal medium containing 0.5% FBS, added to the cells (100 mL/well), and incubated for 2 h. After treatment, cells were placed in fresh SmBM medium containing 0.5% FBS and PDGF-BB (25 ng/mL) for growth factor stimulation and further cultured for 24 h.

[0279] HAECs and PAECs were transfected with a PrxII-specific siRNA using RNAi MAX transfection reagent according to manufacturer's protocol. After 24 hours, transfected cells (3,000 cells/well) were seeded in a 96-well culture plate. At 12 h after seeding, cells were serum-starved for additional 18 h in EBM basal medium containing 0.5% FBS. Test compounds were serially diluted with the same basal medium containing 0.5% FBS, added to the cells (100 mL/well), and incubated for 2 h. After treatment, cells were placed in fresh EBM medium containing 0.5% FBS and VEGF-A (25 ng/mL) for growth factor stimulation and further cultured for 24 h.

[0280] The degree of cell proliferation was measured using the Cell Titer-GLO kit (Roche Diagnostics, USA). Data are the percent of luminescence intensity averaged from three replicate wells versus that of untreated control group.

Experimental Example 9: Efficacy Evaluation in Preclinical Rat Model of Pulmonary Arterial Hypertension

[0281] The rat study protocol was approved by the Institutional Animal Care and Use Committee of the Ewha Womans University, Republic of Korea, and conforms to the ARRIVE guidelines. Six-week-old rats were acclimatized for one week in the laboratory. Then, Sugen 5416 (Sigma Aldrich) was subcutaneously injected at a dose of 20 mg/kg. The administered subjects were maintained in a normobaric hypoxic (10% O.sub.2) chamber (A chamber, Biosphenix) for 3 weeks. Rats were transferred to normoxia and orally administered with either control vehicle or test compounds for additional 5 weeks (P.O., once daily, 0.1 mg per kg weight).

[0282] Right ventricular systolic pressure (RVSP) was measured by echocardiography using a curved catheter equipped with PowerLab2/26 device (AD Instruments, UK). Rats were anesthetized with 2% isoflurane inhalation. A catheter was inserted through the right jugular vein, the pressure was measured for 10 seconds with stabilized pattern. After echocardiography, rats were perfused with saline, and the heart was carefully removed. The removed heart was dissected into the right ventricle (RV), the rest of the septum (S; septum), and the left ventricle (LV). Each piece of heart was weighed using a microbalance. The RV/(LV+S) value was calculated and represented as a right ventricular hypertrophy index (Fulton's index).

Experimental Example 10: Histological Analysis

[0283] Rats were anesthetized with 2% isoflurane inhalation and subjected to transcardiac perfusion-fixation with heparinized saline solution containing 37% formaldehyde. Then, left lung lobes were incised and fixed in 10% NBF for 3 days. The fixed tissues were paraffin-embedded and sectioned using a rotary microtome (Leica HistoCore MULTICUT). Two serial tissue sections (4 m thick) were placed a slide glass and stained with hematoxylin and eosin (HE). For analysis, 100 pulmonary arteries (diameter 20-100 m) per tissue section were chosen for measurement. The luminal, internal elastic laminal, and external elastic laminal areas were quantified using NIH ImageJ v1.62. The intimal and medial areas were determined by subtraction of the luminal area from the internal elastic area and by subtracting the internal elastic area from the external elastic area. The values were averaged and used for calculating pulmonary arterial vessel thickness.

Experimental Example 11: Immunofluorescence Staining

[0284] Paraffin sections were blocked with 5% normal donkey serum (Vector Laboratories) in PBST (PBS solution of 0.3% Triton X-100) for 1 h at room temperature. Thereafter, lung tissue sections were incubated with antigens for Alexa Fluor 568-conjugated smooth muscle actin (-SMA; 1:300 dilution) and Alexa Fluor 488-conjugated von Willebrand Factor (vWF; 1:200 dilution) at 4 C. for 12 h. Subsequently, nuclei were counterstained with DAPI. Fluorescence images were recorded in random regions of at least 10 pulmonary artery vessels (diameter 20-100 um) per tissue section at a screen magnification of 60 using an LSM 880 confocal microscope equipped with argon and helium-neon lasers.

Result 1: Peroxidase Activity for H.sub.2O.sub.2 Reduction

[0285] The H.sub.2O.sub.2-reducing peroxidase activity of the test compounds in present invention was measured in a reaction mixture containing thioredoxin (Trx)/thioredoxin reductase (TR), respectively. Data are summarized in the table below.

TABLE-US-00003 TABLE 2 Compound no. Peroxidase activity for H.sub.2O.sub.2 reduction(nmole/min) Compound 1 4.92 Compound 2 5.05 Compound 3 4.11 Compound 4 3.82 Compound 5 4.80 Compound 6 4.57 Compound 7 3.83 Compound 8 4.91 Compound 9 5.19 Compound 10 5.06 Compound 11 4.57 Compound 12 4.94 Compound 13 4.78 Compound 14 4.78 Compound 15 4.79 Compound 16 4.93 Compound 17 4.80 Compound 18 4.91 Compound 19 5.69 Compound 20 4.78 Compound 21 8.38 Compound 22 7.97 Compound 23 8.81 Compound 24 4.26 Compound 25 4.51 Control compound (A5) 4.25 [00035]

[0286] As shown in Table 2, most of the test compounds exhibit excellent H.sub.2O.sub.2-reducing peroxidase activity in the presence of the Trx/TR redox system. This indicates that the new compounds have peroxidase activity that specifically mimics 2-Cys peroxiredoxin.

[0287] Compared to a control compound belonging to epidithiodioxopiperazine class (A5), new compounds in the present invention have similar or better peroxidase activity.

Result 2: Assay of Intracellular H.sub.2O.sub.2-Eliminating Activity

[0288] The intracellular H.sub.2O.sub.2-eliminating activity of test compounds in present invention was measured using a live-cell imaging fluorescence probe (CM-HaDCF-DA). H.sub.2O.sub.2-eliminating activity was measured depending on ascending concentrations of the test compound and expressed as the percent of activity compared to the untreated control group. EC.sub.50 represents the concentration of test compound at 50% reduction of intracellular H.sub.2O.sub.2.

TABLE-US-00004 TABLE 3 Compound EC.sub.50 (nM) 1 8.25 2 6.47 5 7.77 6 5.99 8 6.57 A5 15.21

[0289] As shown in Table 3, test compounds show excellent H.sub.2O.sub.2-eliminating ability compared to a control compound belonging to epidithiodioxopiperazine class (A5).

Result 3: Measurement of Oxidation-Reduction Potential

[0290] The redox potential values of the test compounds were analyzed based on the potential values established in redox buffer solutions where reduced and oxidized DTT are mixed at various ratios. Data are shown in the table below.

TABLE-US-00005 TABLE 4 Midpoint redox Compound potential (E m; mV) Compound 1 285.25 Compound 2 293.19 Compound 3 296 Compound 4 292.74 Compound 5 290.3 Compound 6 290.39 Compound 7 300.98 Compound 8 272.6 Compound 10 268.53 Compound 20 296.79 Compound 21 272.86 Trx 270 GSH 240

[0291] As shown in Table 3, all new compounds in present invention show low redox potential values. When compared with the potential value of Trx or GSH, Data confirm that new compounds are real 2-Cys-Prx mimetics capable of undergoing a peroxidase reaction coupled with Trx/TR redox system.

Result 4: Cytotoxicity Test

[0292] Cytotoxicity tests were performed against vascular endothelial cells, vascular smooth muscle cells and hepatocytes. After treatment with ascending concentrations of each compound, the concentration of test compound corresponding to 50% cell viability (CC.sub.50) was measured. Data are shown in the table below.

TABLE-US-00006 TABLE 5 Cell cytotoxicity (CC.sub.50) Smooth Endothelial Liver muscle cell cell cell Compound (M) (M) (M) 1 >500 >500 230.1 2 333.4 >500 >500 3 >500 322.5 82.56 4 128.1 316.7 98 5 148.8 289 >500 6 75.65 34.74 341.3 7 >500 >500 >500 8 340.9 >500 312.4 12 >500 >500 13 >500 >500 14 >500 15 98.45 113.6 156.9 19 >500 >500 >500 23 >500 123.3 A5 20.3 81.9 62.3

[0293] As a result of the measurement, except for Compound 6, most of new compounds have a higher CC.sub.50 above 100 M, indicating very low cytotoxicity. In particular, some of test compounds show no cytotoxicity in the range of concentrations used in the test (10 M to 1 mM). This indicates that new compounds have very excellent safety margin compared to a control compound belonging to epidithiodioxopiperazine class (A5).

Result 5: Analysis of PDGF-Mediated Signaling Pathway in Vascular Smooth Muscle Cells

[0294] In human aortic smooth muscle cell (HASMC) and human pulmonary artery smooth muscle cell (PASMC) in which PrxII was knocked out by transfecting the PrxII siRNA, the ability of test compounds to replace the cellular function of PrxII was evaluated. That is, the efficacy of the new compounds was verified in terms of signaling pathways induced by PDGF that regulates the growth and migration of vascular smooth muscle cells in the aorta and pulmonary artery. Activation of PDGF-induced signaling pathway was analyzed by an immunoblot method using an antibody that specifically recognizes phosphorylation of Tyr 857, which is a major phosphorylation site on PDGF receptor (PDGFR). The results are shown in FIGS. 1 to 3.

[0295] FIG. 1 shows the effect of compound 1 on the degree of phosphorylation of Tyr 857 on PDGFR, which had been augmented by PrxII knock-down in HASMCs, in a concentration-dependent manner. It shows that compound 1 significantly reduces phosphorylation at concentrations above 2.5 nM.

[0296] FIG. 2 shows the effect of Compound 1, 2, 5, 6, 10, 15, 19, 20, 21, 22, and 23 on the degree of phosphorylation of Tyr 857 on PDGFR, which had been augmented by PrxII knock-down in HASMCs. As shown in figure, all compounds tested strongly abolish the phosphorylation of PDGFR in HASMC depleted of PrxII expression.

[0297] FIG. 3 shows the effect of compound 8 on the degree of total tyrosine phosphorylation and the phosphorylation of signaling proteins (e.g. PLCyl) at downstream of PDGFR, all of which was induced by PDGF stimulation, in PASMCs. As shown in figure, compound 8 also significantly inhibits the protein phosphorylation triggered by PDGF.

[0298] As a result of the evaluation, the ability of new compounds to regulate the PDGF signaling pathway is improved ten times better than that of control compound belonging to epidithiodioxopiperazine class (A5). New compounds in present invention also reverse abnormal PDGF signaling augmented by depletion of PrxII expression to a normal level.

Result 6: Analysis of VEGF-Mediated Signaling Pathway in Vascular Endothelial Cells

[0299] In human aortic endothelial cell (HAEC) and human pulmonary artery endothelial cell (PAEC) in which PrxII was knocked out by transfecting the PrxII siRNA, the ability of test compounds to replace the cellular function of PrxII was evaluated. That is, the efficacy of the new compounds was verified in terms of signaling pathways induced by VEGF that regulates the growth and migration of vascular endothelial cells in the aorta and pulmonary artery. Activation of VEGF-induced signaling pathway was analyzed by an immunoblot method using an antibody that specifically recognizes phosphorylation of Tyr 1175, which is a major phosphorylation site on VEGF receptor-2 (VEGFR2). The results are shown in FIGS. 4 to 6.

[0300] FIG. 4 shows the effect of Compound 1 on the degree of phosphorylation of Tyr 1175 on VEGFR2, which had been augmented by PrxII knock-down in HAECs, in a concentration-dependent manner. It shows that compound 1 significantly reduces phosphorylation at concentrations above 2.5 nM.

[0301] FIG. 5 shows the effect of Compound 1, 2, 5, 6, 10, 20, 21, and 23 on the degree of phosphorylation of Tyr 1175 on VEGFR2, which had been augmented by PrxII knock-down in HAECs. As shown in figure, all compounds tested strongly abolish the phosphorylation of VEGFR2 in HAECs depleted of PrxII expression.

[0302] FIG. 6 shows the effect of compound 8 on the degree of total tyrosine phosphorylation and the phosphorylation of signaling proteins (e.g. ERK2) at downstream of VEGFR2, all of which was induced by VEGF stimulation, in PAECs. As shown in figure, Compound 8 also significantly inhibits the protein phosphorylation triggered by VEGF.

[0303] As a result of the evaluation, the ability of new compounds to regulate the VEGF signaling pathway is improved ten times better than that of control compound belonging to epidithiodioxopiperazine class (A5). New compounds in present invention also reverse abnormal VEGF signaling augmented by depletion of PrxII expression to a normal level.

Result 7: Proliferation Analysis in the PDGF-Stimulated Smooth Muscle Cells

[0304] In human aortic smooth muscle cell (HASMC) and human pulmonary artery smooth muscle cell (PASMC) in which PrxII was knocked out by transfecting the PrxII siRNA, the ability of test compounds to inhibit the PDGF-induced proliferation of vascular smooth muscle cells was evaluated. The results are shown in Table 6 below. In Table 6, the degree of proliferation in the compound-treated groups is expressed as percent of the degree of proliferation in untreated control group. The concentrations of test compounds corresponding to 50% growth inhibition (IC.sub.50) is also listed.

TABLE-US-00007 TABLE 6 Conc. (nM) 0 2.5 5 10 25 50 100 IC.sub.50 (nM) Degree of HASMC proliferation (%) Compound 1 100.0 70.0 43.9 17.1 1.5 4.18 Compound 5 100.0 62.3 52.4 36.9 9.7 4.87 Compound 8 100.0 75.1 40.1 46.9 30.2 6.46 Compound 21 100.0 88.1 63.9 27.2 4.0 6.41 Compound 23 100.0 89.8 54.3 34.4 20.4 6.85 Compound 20 100.0 85.8 58.7 34.9 12.3 6.69 Compound 10 100.0 75.5 53.8 31.1 4.6 5.48 Degree of PASMC proliferation (%) Compound 1 100.0 70.8 49.8 29.4 14.3 0.0 0.0 5.01 Compound 5 100.0 71.3 48.3 37.5 26.8 1.9 2.5 5.64 Compound 8 100.0 57.7 41.7 24.8 5.7 0.0 0.0 3.52 Compound 21 100.0 69.9 49.6 18.7 6.0 7.1 0.0 4.51 Compound 23 100.0 70.8 43.3 32.9 14.6 3.2 0.0 4.76 Compound 20 100.0 82.2 51.5 31.6 21.1 7.3 5.2 6.17 Compound 10 100.0 90.5 54.1 44.1 21.9 17.5 7.0 8.28

[0305] As shown in the table above, new compounds in the present invention inhibit the proliferation of HASMCs and PASMCs in a concentration-dependent manner, and show a growth-inhibiting activity started from concentration as low as 2.5 nM.

Result 8: Proliferation Analysis in the VEGF-Stimulated Endothelial Cells

[0306] In human aortic endothelial cell (HAEC) and human pulmonary artery endothelial cell (PAEC) in which PrxII was knocked out by transfecting the PrxII siRNA, the ability of test compounds to augment the VEGF-induced proliferation of vascular endothelial cells was evaluated. The results are shown in Table 7 below. In Table 7, the degree of proliferation in the compound-treated groups is expressed as percent of the degree of proliferation in untreated control group. The effective concentrations of test compounds increasing cell proliferation by 50% (EC.sub.50) is also listed.

TABLE-US-00008 TABLE 7 Conc. (nM) 0 2.5 5 10 25 50 100 EC.sub.50 (nM) Degree of HAEC proliferation (%) Compound 1 100.0 113.8 143.7 162.9 187.8 164.7 170.2 4.93 Compound 5 100.0 112.2 129.7 165.6 191.6 172.7 180.1 5.31 Compound 8 100.0 107.3 147.3 186.3 204.8 157.0 161.6 5.04 Compound 21 100.0 105.0 122.7 154.6 190.1 202.0 141.2 9.96 Compound 23 100.0 109.4 122.6 161.3 187.6 177.1 133.0 9.72 Compound 20 100.0 116.4 120.3 136.3 155.9 169.5 187.6 10.16 Compound 10 100.0 111.6 122.8 127.6 140.7 175.1 159.0 19.32 Degree of PAEC proliferation (%) Compound 1 100.0 104.7 120.0 143.8 164.3 158.8 145.5 9.54 Compound 5 100.0 106.1 129.7 152.3 165.6 167.7 147.4 5.03 Compound 8 100.0 108.5 124.9 143.4 164.8 158.1 144.0 5.12 Compound 21 Compound 23 100.0 109.2 122.9 148.3 173.3 155.3 133.9 5.20 Compound 20 100.0 109.2 124.3 133.5 166.5 180.9 180.4 10.28 Compound 10

[0307] As shown in the table above, new compounds in the present invention promote the proliferation of HAECs and PAECs in a concentration-dependent manner, and show a growth-promoting activity started from concentration as low as 2.5 nM.

Result 9: Preclinical Animal Study for Pulmonary Arterial Hypertension

[0308] The therapeutic effect of new compounds on pulmonary arterial hypertension was evaluated in a preclinical SuHx rat model. For establishing PAH model, rats were administered with Sugen (VEGFR2 inhibitor) and placed in a normobaric hypoxic condition (10% O.sub.2) for 3 weeks. Rats were moved to normoxic condition and orally administered with control vehicle or compound 1 or 8 (P.O., once daily, 0.1 mg/kg) for 5 weeks. Thereafter, right ventricular systolic pressure (RVSP) and right ventricular hypertrophy (Fulton's index) were measured at the end of treatment. The results are shown in FIGS. 7-9.

[0309] FIG. 7 shows that Compound 1 and 8 significantly reduces RVSP and RV hypertrophy compared to vehicle control group.

[0310] FIG. 8 shows that Compound 1 widens the pulmonary arterial vessels which remains to be severely occluded in the vehicle control group. Data indicate that treatment of Ccompound 1 induces normal blood flow in the lumen of the blood vessel, which results in the reduction of RVSP and RV hypertrophy.

[0311] FIG. 9 shows an immunostaining images of endothelium and medial SMC layer in pulmonary arterial vessels. Specifically, the lung tissue sections were immunostained with an endothelial cell-specific vWF antibody and a smooth muscle cell-specific SMA antibody. The results demonstrate that the endothelial damage and medial thickness due to SMC hyperplasia occur in vehicle control group. However, treatment of Compound 1 induces the recovery of endothelial layer (vWF-labeled ECs) as well as the reduction of medial thickness (SMA-labeled SMCs).