EPIDITHIODIOXOPIPERAZINE COMPOUND OR ITS DERIVATIVES, AND THE USE THEREOF
20170239261 · 2017-08-24
Inventors
Cpc classification
A61K31/548
HUMAN NECESSITIES
A61K31/00
HUMAN NECESSITIES
G01N2500/02
PHYSICS
International classification
Abstract
The present invention relates to an epidithiodioxopiperazine derivative represented by the following Chemical Formula 1 or its reduced derivative; a method for preparing a compound represented by Chemical Formula 1 having improved intracellular permeability and mimicking the activity of 2-Cys-Prx in its reduced form in the cells; a pharmaceutical composition for preventing or treating vascular diseases comprising an epidithiodioxopiperazine compound or its derivatives or pharmaceutically acceptable salts thereof as an active ingredient; a drug delivery device for local administration including the pharmaceutical composition; and a pharmaceutical composition for inhibiting melanoma metastasis comprising the epidithiodioxopiperazine compound or its derivatives or pharmaceutically acceptable salts thereof as an active ingredient.
##STR00001##
Claims
1. A method for treating vascular diseases and melanoma, induced by PrxII deficiency or inactivation, comprising administering a pharmaceutical composition comprising an epidithiodioxopiperazine derivative represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient into a subject suspected of having vascular diseases: ##STR00037## wherein, R1 to R4 are each independently hydrogen, a halogen atom, a hydroxyl group, linear or branched C1 to C6 alkyl, alkenyl or alkynyl, linear or branched C1 to C6 alkoxy, linear or branched C1 to C6 hydroxyalkyl, substituted or unsubstituted benzyl, linear or branched C1 to C6 alkylaryl, a linear or branched C1 to C6 perfluoroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted perfluoroaryl group, a substituted or unsubstituted heteroaryl group comprising an oxygen, nitrogen or sulfur atom in a ring as a heteroatom, or a substituted or unsubstituted epidithiodioxopiperazine group, and the alkyl and the aryl group may optionally include a heteroatom of oxygen, nitrogen or sulfur in the middle of the chain, and each of the substituted epidithiodioxopiperazine groups may independently optionally include the substituents defined above and have a structure identical to or different from mother nucleus epidithiodioxopiperazine; R1 and R2, and R3 and R4 each independently form a substituted or unsubstituted C3 to C6 cycloalkyl group with a carbon atom to which these are attached; or form a substituted or unsubstituted heterocyclic ring having 5 to 8 ring atoms with a carbon atom to which these are attached, and additional carbon or heteroatoms, and herein, 1 or 2 ring atoms of the heterocyclic ring are selected from nitrogen (N), oxygen (O) or sulfur (S), and the derivative represented by Chemical Formula 1 does not include a compound represented by the following Chemical Formula 16. ##STR00038##
2. The method of claim 1, wherein the epidithiodioxopiperazine compound is any one of compounds represented by the following Chemical Formulae 7 to 15 and 17 to 31: ##STR00039## ##STR00040## ##STR00041## wherein, A and B are each independently hydrogen; methoxy; or a hydroxyl group. ##STR00042## ##STR00043## ##STR00044##
3. The method of claim 1, wherein the vascular disease is selected from the group consisting of hypertension, an ischemic coronary artery disease such as unstable angina pectoris, angina pectoris and myocardial infarction, cerebral artery occlusion such as stroke, artherosclerosis, a pheripheral arterial occlusive disease such as a bergers disease, thromboembolism, diabetic foot lesion, venous ulcer, deep vein thrombosis, carotidal artherosclerosis, vasospasm, arteritis and vascular restenosis.
4. The method of claim 3, wherein the vascular restenosis is caused by vascular graft, vascular cutting, artherosclerosis, intravascular lipid accumulation, hypertension, arteritis or angioplasty.
5. The method of claim 1, wherein the composition promotes proliferation or migration of endothelial cells while inhibiting proliferation or migration of vascular smooth muscle cells.
6. The method of claim 1, wherein the composition inhibits the metastasis of melanoma through mimicking an intracellular PrxII activity.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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[0150] The proliferation and the migration of cells are promoted by serum (FBS). The p value in each graph means a statistically significant value.
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DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0152] Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples for illustrative purposes only, and the scope of the present invention is not limited to these examples.
<Synthesis of Epidithiodioxopiperazine Derivative Including Intramolecular Disulfide Bridged Bond>
PREPARATION EXAMPLE 1
Preparation of 2,3-dithia-5,7-diazabicyclo[2.2.2]octane-6,8-dione (A1, Chemical Formula 15)
[0153] ##STR00025##
A. 1,4-dibenzhydrylpiperazine-2,5-dione (2)
[0154] After NaH (60% dispersed in mineral oil, 385 mg, 9.64 mmol) was dispersed in 10 ml of DMF, anhydrous glycine (500 mg, 4.38 mmol) was added thereto in an ice water bath. After the mixture was stirred for 10 minutes, bromodiphenyl methane (2.27 g, 2.1 eq.) was slowly added thereto. The result was reacted overnight at room temperature. Water was added to the reaction solution and the result was stirred. The precipitates were filtered, washed with water, and then dried under reduced pressure to obtain 1,4-dibenzhydrylpiperazine-2,5-dione (2) (1.4 g, yield 71%).
[0155] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 7.39-7.31 (m, 12H), 7.21-7.19 (d, 8H), 7.09 (s, 2H), 3.83 (s, 4H).
B. 7,9-dibenzhydryl-2,3,4,5-tetrathia-7,9-diazabicyclo[4.2.2]decane-8,10-dione (3)
[0156] After sulfur (402 mg, 12.54 mmol) was dispersed in 5 ml of dry THF, LiHMDS (1.0 M in THF, 4.7 ml, 3.0 eq.) was added thereto drop by drop over 2 minutes under argon atmosphere. After the mixture was stirred for approximately 1 minute, a solution in which the compound (2; 700 mg, 1.567 mmol) obtained in Step A was dissolved in 20 ml of THF was added thereto drop by drop. After the result was stirred for approximately 1 minute, LiHMDS (1.0 M in THF, 3.13 ml, 2.0 eq.) was added thereto drop by drop, and then the result was stirred for 30 minutes. After the reaction was complete, the result was extracted twice by adding a saturated NH.sub.4Cl solution and ethyl acetate (EA) (×3). The organic layers were combined, treated with magnesium sulfate, filtered, and then the solvent was removed. The residue was purified using silica gel column chromatography (Hex:EA=2:1) to obtain 7,9-dibenzhydryl-2,3,4,5-tetrathia-7,9-diazabicyclo[4.2.2]decane-8,10-dione (3; 230 mg, yield: 26%).
[0157] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 7.39-7.32 (m, 12H), 7.21 (m, 4H), 7.07 (m, 4H), 6.95 (s, 2H), 5.14 (s, 2H).
C. 3,6-dimercapto-1,4-dibenzhydrylpiperazine-2,5-dione (4)
[0158] After 7,9-dibenzhydryl-2,3,4,5-tetrathia-7,9-diazabicyclo[4.2.2]decane-8,10-dione (3; 230 mg, 0.401 mmol) was dissolved in methanol, NaBH.sub.4 (45 mg, 3.0 eq.) was slowly added thereto in an ice water bath. After the mixture was stirred for 30 minutes and the reaction was complete, the solvent was removed. Water and EA (×3) were added to the crude compound and the result was extracted. The organic layers were combined, treated with magnesium sulfate, filtered, and then the solvent was removed. The residue was dried under reduced pressure to obtain 3,6-dimercapto-1,4-dibenzhydrylpiperazine-2,5-dione (4). The compound was used for the next reaction without further purification. 25
D. 5,7-dibenzhydryl-2,3-dithia-5,7-diazabicyclo[2.2.2]octane-6,8-dione (5)
[0159] After the dithiol crude compound (4) was dissolved in 30 ml of chloroform, 20 ml of a solution in which iodine (101 mg, 0.401 mmol) was dissolved in chloroform was added thereto. After the mixture was reacted for 30 minutes at room temperature, the solvent was removed, and the residue was purified using silica gel column chromatography (EA:Hex=1:2) to obtain 5,7-dibenzhydryl-2,3-dithia-5,7-diazabicyclo[2.2.2]octane-6,8-dione (5) (130 mg, 2 step yield-63%).
[0160] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 7.37-7.28 (m, 12H), 7.27 (m, 4H), 7.14 (m, 4H), 6.77 (s, 2H), 5.31 (s, 2H).
[0161] ESI-MS (M+Na): 531 calculated for C.sub.30H.sub.24N.sub.2O.sub.2S.sub.2 508.
E. 2,3-dithia-5,7-diazabicyclo[2.2.2]octane-6,8-dione (6; A1)
[0162] After the crude compound (5; 50 mg) obtained in Step D was dissolved in trifluoroacetic acid (TFA, 1 mL), triflic acid (CF.sub.3SO.sub.3H, 0.1 mL) was added thereto and reacted in an ice water bath. After the mixture was reacted for 30 minutes, the reaction was terminated by adding ice-cold water to the reaction solution, and the result was stirred. The produced solids were filtered and dried. After that, the solids were stirred in ether, filtered, and dried under reduced pressure to obtain 2,3-dithia-5,7-diazabicyclo[2.2.2]octane-6,8-dione (6; 8 mg, 47%). The NMR data of the obtained final product was shown in
[0163] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 9.69 (br d, 2H), 5.65 (d, 2H).
[0164] ESI-MS (M-1): 175 calculated for C.sub.4H.sub.4N.sub.2O.sub.2S.sub.2 176.
PREPARATION EXAMPLE 2
Preparation of 5,7-dimethyl-2,3-dithia-5,7-diazabicyclo[2.2.2]-octane-6,8-dione (A2, Chemical Formula 7)
[0165] ##STR00026##
A. 3,6-dibromo-1,4-dimethylpiperazine-2,5-dione (2)
[0166] After a sarcosine anhydride (1) (500 mg, 3.52 mmol), N-bromosuccimide (1.87 g, 3.0 eq.) and benzoyl peroxide (85 mg, 0.1 eq.) were dispersed in carbon tetrachloride (50 ml), the mixture was heated under reflux for 2 hours. After the reaction was complete and the reaction material was cooled to room temperature, the obtained succimide was filtered and washed with carbon tetrachloride. The filtrates were combined, dried with magnesium sulfate, and then filtered once again. The solvent was removed, and the residue was dried under reduced pressured to obtain 3,6-dibromo-1,4-dimethylpiperazine-2,5-dione (2). The obtained compound was used as it was for the next reaction without further purification.
[0167] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 6.04 (s, 2H), 3.15 (s, 6H) .
B. 1,4-dimethyl-3,6-dioxopiperazine-2,5-diyl-diethanethioate (3)
[0168] After 3,6-dibromo-1,4-dimethylpiperazine-2,5-dione (2) (crude 1.05 mg, 3.52 mmol) was dissolved in dichloromethane (DCM; 100 ml), potassium thioacetate (1.2 g, 3.0 eq.) was added thereto in an ice water bath. The mixture was stirred overnight at room temperature, and the obtained precipitates were filtered and washed with DCM. The filtrates were combined and the solvent was evaporated under vacuum. The residue was purified using silica gel column chromatography (DCM:ethyl acetate (EA)=5:1) to obtain 1,4-dimethyl-3,6-dioxopiperazine-2, 5-diyl-diethanethioate (3) (white solids; 200 mg, 2 step yield-20%).
[0169] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 5.77 (s, 2H), 2.94 (s, 6H), 2.48 (s, 6H).
C. 3,6-dimercapto-1,4-dimethylpiperazine-2,5-dione (4)
[0170] After dithioacetate (195 mg, 0.67 mmol) was dissolved in ethanol (20 ml), an ethanolic hydrochloric acid solution (prepared by adding 1.5 ml of acetyl chloride to 7 ml of ethanol) was added thereto. The mixture solution was heated under reflux for 2 hours. After the reaction was complete, the solvent was removed, and the residue was dried under reduced pressure to obtain 3,6-dimercapto-1,4-dimethylpiperazine-2,5-dione (4) in bright yellow solids (90 mg, 65%). The obtained compound was used for the next reaction without further purification.
[0171] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 5.00 (s, 2H), 3.09 (s, 6H) .
[0172] ESI-MS (M-1): 205 calculated for C.sub.6H.sub.10N.sub.2O.sub.2S.sub.2 206.
D. 5,7-dimethyl-2,3-dithio-5,7-diazabicyclo[2,2,2]octane-6,8-dione (5; A2)
[0173] A solution in which iodine (63 mg, 1.0 eq.) was dissolved in 10 ml of DCM was added to a solution in which 3,6-dimercapto-1,4-dimethylpiperazine-2,5-dione (50 mg, 0.242 mmol) was dissolved in 10 ml of chloroform. The mixture was reacted for 30 minutes at room temperature. After the reaction was complete, a saturated NaHCO.sub.3 solution containing sodium thiosulfate was poured into the reaction material, and the result was stirred until the color due to iodine disappeared. After the organic layer was separated and the aqueous layer was further extracted twice with DCM, the organic layers were combined and then dried with magnesium sulfate. After that, the organic layer was filtered and vacuum concentrated to give a residue, and the residue was recrystallized using ethanol, filtered, then washed with hexane (Hex), and dried under reduced pressure to otabin 5,7-dimethyl-2,3-dithio-5,7-diazabicyclo[2,2,2]octane-6,8-dione (5) in bright yellow solids (18 mg, 36%). The NMR data of the obtained final product was shown in
[0174] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 5.21 (s, 2H), 3.12 (s, 6H).
[0175] ESI-MS (M+Na): 227 calculated for C.sub.6H.sub.8N.sub.2O.sub.2S.sub.2 204.
PREPARATION EXAMPLE 3
Preparation of 1,5,7-trimethyl-2,3-dithia-5,7-diazabicyclo[2.2.2]octane-6,8-dione (A3, Chemical Formula 8)
[0176] ##STR00027##
A. methyl 2-(2-chloroacetamido)propanoate (2)
[0177] After alanine methyl ester hydrochloride (2 g, 14.3 mmol) was dissolved in 6 ml of water, sodium bicarbonate (NaHCO.sub.3; 2.8 g, 33.6 mmol) was slowly added thereto in an ice water bath. To the reaction material, a chloroacetyl chloride (1.67 ml, 20.9 mmol) solution dissolved in 5 ml of toluene was mixed by being added drop by drop, and the result was vigorously stirred for 3 hours at room temperature. After the reaction was complete, the organic layer was separated and the aqueous layer was further washed with toluene. The organic layers were combined, treated with magnesium sulfate, filtered, and then the solvent was removed. The residue was dried under reduced pressure to obtain crude methyl 2-(2-chloroacetamido)propanoate (2) (2.6 g).
[0178] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 7.23 (br s, 1H), 4.65 (m, 1H), 4.11 (s, 2H), 3.79 (s, 3H), 1.42 (d, 3H).
B. 3-methylpiperazine-2,5-dione (3)
[0179] After methyl 2-(2-chloroacetamido)propanoate (2; 2.6 g, 14.3 mmol) was dissolved in 12 ml of ethanol, 6.1 ml (43.4 mmol) of a 30% aqueous ammonia solution was added thereto. The mixture was reacted for 5 hours at 70° C. After the reaction was complete and the reaction material was cooled to room temperature, ethanol was removed. When solids were formed in the remaining small amount of water, the solids were filtered and then washed with water and hexane. The washed solids were dried under reduced pressure to obtain 3-methylpiperazine-2,5-dione (3) in white solids (420 mg, 2 step yield-23%).
[0180] .sup.1H NMR (400 MHz, DMSO-d6): δ ppm 8.13 (br s, 1H), 7.95 (br s, 1H), 3.84 (q, 1H), 3.72 (s, 2H), 1.26 (d, 3H).
C. 1,3,4-trimethylpiperazine-2,5-dione (4)
[0181] After 3-methylpiperazine-2,5-dione (3; 420 mg, 3.28 mmol) was dispersed in 25 ml of dimethylformamide (DMF), NaH (400 mg, 9.83 mmol) was added thereto in an ice water bath. Methyl iodide (2 ml, 10 eq.) was added thereto, and the mixture was stirred for 4 hours at room temperature. After the reaction was complete, DMF was removed and the residue was purified using silica gel column chromatography (DCM:methano1=20:1 to 9:1) to obtain 1,3,4-trimethylpiperazine-2,5-dione (4) (colorless solids; 440 mg, yield 86%).
[0182] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 4.08 (d, 1H), 3.92 (q, 1H), 3.85 (d, 1H), 2.98 (s, 6H), 1.47 (d, 3H).
D. 6,7,9-trimethyl-2,3,4,5-tetrathia-7,9-diazabicyclo[4.2.2]decane-8,10-dione (5)
[0183] After sulfur (1.64 g, 51.2 mmol) was dispersed in 20 ml of dry THF, NaHMDS (1.0 M in THF, 19.2 ml) was added thereto drop by drop over 2 minutes under argon atmosphere. After the mixture was stirred for approximately 1 minute, a solution in which the compound (4; 1 g, 6.4 mmol) obtained in Step C was dissolved in 20 ml of THF was added thereto drop by drop. After the result was stirred for approximately 1 minute, NaHMDS (1.0 M in THF, 12.8 ml, 2.0 eq.) was added thereto drop by drop, and then the result was stirred for 30 minutes. After the reaction was complete, the result was extracted by adding a saturated NH.sub.4Cl solution and DCM (×3). The organic layers were combined, treated with magnesium sulfate, filtered, and then the solvent was removed. The residue was purified using silica gel column chromatography (EA:hexane=1:1) to obtain 6,7,9-trimethyl-2,3,4,5-tetrathia-7,9-diazabicyclo[4.2.2]decane-8,10-dione (5; 250 mg, yield: 14%).
[0184] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 5.13 (s, 1H), 3.06 (d, 6H), 2.00 (s, 3H).
E. 3,6-dimercapto-1,3,4-trimethylpiperazine-2,5-dione (6)
[0185] After 6,7,9-trimethyl-2,3,4,5-tetrathia-7,9-diazabicyclo[4.2.2]decane-8,10-dione (5) (250 mg, 0.885 mmol) was dissolved in 20 ml of methanol, the result was slowly added to NaBH.sub.4 (167 mg, 4.43 mmol) in an ice water bath. After the mixture was stirred for 30 minutes and the reaction was complete, the solvent was removed. Water and DCM (×3) were added to the crude compound and the result was extracted. The organic layers were combined, treated with magnesium sulfate, filtered, and then the solvent was removed. The residue was dried under reduced pressure to obtain 3,6-dimercapto-1,3,4-trimethylpiperazine-2,5-dione (6). The compound was used for the next reaction without further purification.
F. 1,5,7-trimethyl-2,3-dithia-5,7-diazabicyclo[2.2.2]octane-6,8-dione (7; A3)
[0186] After the dithiol compound (35 mg) obtained above was dissolved in 30 ml of chloroform, 25 ml of a solution in which iodine (249 mg, 1.0 eq.) was dissolved in chloroform was added thereto. After the mixture was reacted for 30 minutes at room temperature, the solvent was removed, and the residue was purified using silica gel column chromatography (EA:Hex=1:1) to obtain 1,5,7-trimethyl-2,3-dithia-5,7-diazabicyclo[2.2.2]octane-6,8-dione (7) in bright yellow solids (24 mg, 2 step yield-12%). The NMR data of the obtained final product was shown in
[0187] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 5.29 (s, 1H), 3.14 (s, 3H), 3.03 (s, 1H), 1.98 (s, 3H).
[0188] ESI-MS (M+Na): 241 calculated for C.sub.7H.sub.10N.sub.2O.sub.2S.sub.2 218.
PREPARATION EXAMPLE 4
Preparation of 1,4-bis(4-methoxybenzyl)-2,3-dithia-5,7-diazabicyclo[2.2.2]octane-6,8-dione (A4, Chemical Formula 9)
[0189] ##STR00028##
A. 1,4-bis(4-methoxybenzyl)piperazine-2,5-dione (2)
[0190] After NaH (1.54 g, 38.5 mmol) was dispersed in 20 ml of DMF, anhydrous glycine (2 g, 17.5 mmol) was added thereto in an ice water bath. After the mixture was stirred for 10 minutes, benzyl chloride (5.94 ml, 2.5 eq) was slowly added thereto over 30 minutes. After the result was reacted for 1 hour at room temperature, water was added thereto and the result was stirred. The precipitates were filtered and then washed with water. The solid compound obtained was stirred in a mixed solvent of EA and hexane (EA:Hex=9:1), filtered and then dried under reduced pressure to obtain 1,4-bis(4-methoxybenzyl)piperazine-2,5-dione (2; 5.1 g, 82%).
[0191] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 7.21 (d, 4H), 6.87 (d, 4H) , 4.51 (s, 4H) , 3.89 (s, 4H) , 3.80 (s, 6H).
B. 7,9-bis(4-methoxybenzyl)-2,3,4,5-tetrathia-7,9-diazabicyclo[4.2.2]decane-8,10-dione (3)
[0192] After sulfur (72 mg, 2.257 mmol) was dispersed in 2 ml of dry THF, NaHMDS (1.0 M in THF, 0.85 ml, 3.0 eq.) was added thereto drop by drop over 2 minutes under argon atmosphere. After the mixture was stirred for approximately 1 minute, a solution in which the compound (2; 100 mg, 0.282 mmol) obtained in Step A was dissolved in 20 ml of THF was added thereto drop by drop. After the result was stirred for approximately 1 minute, NaHMDS (1.0 M in THF, 0.56 ml, 2.0 eq.) was added thereto drop by drop, and the result was stirred for 30 minutes. After the reaction was complete, the result was extracted by adding a saturated NH.sub.4Cl solution and EA (×2). The organic layers were combined, treated with magnesium sulfate, filtered, and then the solvent was removed. The residue was purified using silica gel column chromatography (Hex:EA=4:1 to 2:1) to obtain 7,9-bis(4-methoxybenzyl)-2,3,4,5-tetrathia-7,9-diazabicyclo[4.2.2]decane-8,10-dione (3; 35 mg, yield: 26%).
[0193] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 7.21 (d, 4H), 6.87 (d, 4H), 5.37 (d, 2H), 4.99 (s, 2H), 3.84 (d, 2H), 3.81 (s, 6H).
C. 3,6-dimercapto-1,4-bis(4-methoxybenzyl)piperazine-2,5-dione (4)
[0194] After 7,9-bis(4-methoxybenzyl)-2,3,4,5-tetrathia-7,9-diazabicyclo[4.2.2]decane-8,10-dione (3) (420 mg, 0.873 mmol) was dissolved in 20 ml of methanol, the result was slowly added to NaBH.sub.4 (99 mg, 3.0 eq.) in an ice water bath. After the mixture was stirred for 30 minutes and the reaction was complete, the solvent was removed. Water and EA (×3) were added to the crude compound and the result was extracted. The organic layers were combined, treated with magnesium sulfate, filtered, and then the solvent was removed. The residue was dried under reduced pressure to obtain 3,6-dimercapto-1,4-bis(4-methoxybenzyl)piperazine-2,5-dione (4). The compound was used for the next reaction without further purification.
[0195] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 7.20 (d, 4H), 6.89 (d, 4H), 5.21 (d, 2H), 4.93 (s, 2H), 4.12 (d, 2H), 3.85 (s, 6H), 3.05 (br s, 2H).
[0196] ESI-MS (M+Na): 441 calculated for C.sub.20H.sub.22N.sub.2O.sub.4S.sub.2 418.
D. 5,7-bis(4-methoxybenzyl)-2,3-dithia-5,7-diazabicyclo[2.2.2]octane-6,8-dione (5; A4)
[0197] After the dithiol compound (4; 380 mg) obtained above was dissolved in 50 ml of chloroform, 100 ml of a solution in which iodine (221 mg, 0.873 mmol) was dissolved in chloroform was added thereto. After the mixture was reacted for 30 minutes at room temperature, the solvent was removed, and the residue was purified using silica gel column chromatography (EA:Hex=1:1) to obtain 5,7-bis(4-methoxybenzyl)-2,3-dithia-5,7-diazabicyclo[2.2.2]octane-6,8-dione (5) (120 mg, 2 step yield-33%). The NMR data of the obtained final product was shown in
[0198] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 7.22 (d, 4H), 6.91 (d, 4H), 5.21 (s, 2H), 4.77 (d, 2H), 4.45 (d, 2H), 3.81 (s, 6H).
[0199] ESI-MS (M+Na): 439 calculated for C.sub.20H.sub.20N.sub.2O.sub.4S.sub.2 416.
PREPARATION EXAMPLE 5
Preparation of 5,7-diallyl-2,3-dithia-5,7-diazabicyclo[2.2.2]octane-6,8-dione (A5, Chemical Formula 10)
[0200] ##STR00029##
A. 1,4-diallylpiperazine-2,5-dione (2)
[0201] After NaH (1.3 g, 32.8 mmol) was dispersed in 25 ml of DMF, anhydrous glycine (1.5 g, 13.1 mmol) was added thereto in an ice water bath. After the mixture was stirred for 10 minutes, allyl chloride (3.2 ml, 3.0 eq.) was slowly added thereto over 30 minutes. The result was reacted for 1 hour at room temperature, and then the reaction solution was extracted by adding EA (×2) and a saturated NH.sub.4Cl solution. The organic layers were combined, treated with magnesium sulfate, filtered, and then the solvent was removed. The residue was purified using silica gel column chromatography (DCM:EA=1:1) to obtain 1,4-diallylpiperazine-2,5-dione (2) (1.4 g, yield 56%).
[0202] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 5.79-5.69 (m, 2H), 5.30-5.22 (m, 4H), 4.04 (d, 4H), 3.96 (s, 4H).
B. 7,9-diallyl-2,3,4,5-tetrathia-7,9-diazabicyclo[4.2.2]decane-8,10-dione (3)
[0203] After sulfur (1.32 g, 41.2 mmol) was dispersed in 10 ml of dry THF, NaHMDS (1.0 M in THF, 15.4 ml, 3.0 eq.) was added thereto drop by drop over 2 minutes under argon atmosphere. After the mixture was stirred for approximately 1 minute, a solution in which the compound (2; 1 g, 5.15 mmol) obtained in Step A was dissolved in 20 ml of THF was added thereto drop by drop. After the result was stirred for approximately 1 minute, NaHMDS (1.0 M in THF, 10.3 ml, 2.0 eq.) was added thereto drop by drop, and the result was stirred for 30 minutes. After the reaction was complete, the result was extracted by adding a saturated NH.sub.4Cl solution and EA (×3). The organic layers were combined, treated with magnesium sulfate, filtered, and then the solvent was removed. The residue was purified using silica gel column chromatography (Hex:EA=9:1 to 4:1) to obtain 7,9-diallyl-2,3,4,5-tetrathia-7,9-diazabicyclo[4.2.2]decane-8,10-dione (3; 530 mg, yield: 32%).
[0204] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 5.83-5.73 (m, 2H), 5.40-5.33 (m, 4H), 5.21 (s, 2H), 4.81 (d, 2H), 3.51-3.45 (m, 2H).
C. 3,6-dimercapto-1,4-diallylpiperazine-2,5-dione (4)
[0205] After 7,9-diallyl-2,3,4,5-tetrathia-7,9-diazabicyclo[4.2.2]decane-8,10-dione (3) (530 mg, 1.65 mmol) was dissolved in a methanol/THF mixed solvent, NaBH.sub.4 (187 mg, 3.0 eq.) was slowly added thereto in an ice water bath. After the mixture was stirred for 30 minutes and the reaction was complete, the solvent was removed. Water and EA (×3) were added to the crude compound and the result was extracted. The organic layers were combined, treated with magnesium sulfate, filtered, and then the solvent was removed. The residue was dried under reduced pressure to obtain 3,6-dimercapto-1,4-diallylpiperazine-2,5-dione (4). The compound was used for the next reaction without further purification.
[0206] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 5.80-5.70 (m, 2H), 5.39-5.31 (m, 4H), 5.07 (s, 2H), 4.68 (d, 2H), 3.70-3.64 (m, 2H), 3.09 (br s, 2H).
D. 5,7-diallyl-2,3-dithia-5,7-diazabicyclo[2.2.2]octane-6,8-dione (5; A5)
[0207] After the dithiol compound (4; 430 mg) obtained above was dissolved in 30 ml of chloroform, 60 ml of a solution in which iodine (419 mg, 1.0 eq.) was dissolved in chloroform was added thereto. After the mixture was reacted for 30 minutes at room temperature, the solvent was removed, and the residue was purified using silica gel column chromatography (EA:Hex=1:9 to 1:4) to obtain 5,7-diallyl-2,3-dithia-5,7-diazabicyclo[2.2.2]octane-6,8-dione (5) (110 mg, 2 step yield-26%). The NMR data of the obtained final product was shown in
[0208] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 5.86-5.81 (m, 2H), 5.38-5.34 (m, 4H), 5.30 (s, 2H), 4.32-4.27 (m, 2H), 4.00-3.95 (m, 2H).
[0209] ESI-MS (M+K): 295 calculated for C.sub.10H.sub.12N.sub.2O.sub.2S.sub.2 256.
PREPARATION EXAMPLE 6
Preparation of 5-(4-methoxybenzyl)-2,3-dithia-5,7-diazabicyclo[2.2.2]octane-6,8-dione (A6, Chemical Formula 11)
[0210] ##STR00030##
A. 1,4-bis(4-methoxybenzyl)piperazine-2,5-dione (2)
[0211] 1,4-Bis(4-methoxybenzyl)piperazine-2,5-dione (2) was obtained in the same manner as in Step A of Preparation Example 3.
B. 3,6-dibromo-1,4-bis(4-methoxybenzyl)piperazine-2,5-dione (3)
[0212] After 1,4-bis(4-methoxybenzyl)piperazine-2,5-dione (2; 1 g, 2.82 mmol), N-bromosuccimide (1.05 g, 2.1 eq.) and benzoyl peroxide (6.8 mg, 0.01 eq.) were dispersed in carbon tetrachloride (250 ml), the mixture was reacted for 2 hours while being refluxed. After the reaction was complete and the reaction material was cooled to room temperature, the produced solids were filtered. The solvent was removed, and the result was dried under reduced pressure to obtain 3,6-dibromo-1,4-bis(4-methoxybenzyl)piperazine-2,5-dione (3). The obtained compound was used as it is for the next reaction without further purification.
[0213] .sup.1H NMR(400 MHz, CDCl.sub.3): δ ppm 7.22 (d, 4H), 6.89 (d, 4H), 5.89 (s, 2H), 5.30 (d, 2H), 3.93 (d, 2H), 3.82 (s, 6H).
C. 1,4-bis(4-methoxybenzyl)-3,6-dioxopiperazine-2,5-diyl-diethanethioate (4)
[0214] After 3,6-dibromo-1,4-bis(4-methoxybenzyl)piperazine-2,5-dione (3; 1.44 g, 2.82 mmol) was dissolved in dichloromethane (DCM; 100 ml), sodium thioacetate (966 mg, 3.0 eq.) was added thereto in an ice water bath. The mixture was stirred overnight at room temperature, and the obtained precipitates were filtered, and the solvent was removed. The result was purified using silica gel column chromatography (Hex:EA=2:1 to 1.5:1) to obtain 1,4-bis(4-methoxybenzyl)-3,6-dioxopiperazine-2,5-diyl-diethanethioate (4) (600 mg, 2 step yield-43%).
[0215] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 7.22 (d, 4H), 6.86 (d, 4H), 5.83 (s, 2H), 4.98 (d, 2H), 3.89 (d, 2H), 3.81 (s, 6H), 2.45 (s, 6H).
D. 1-(4-methoxybenzyl)-3,6-dioxopiperazine-2,5-diyl-diethanethioate (5)
[0216] After 1,4-bis(4-methoxybenzyl)-3,6-dioxopiperazine-2,5-diyl-diethanethioate (4; 1.37 g, 2.73 mmol) was dissolved in an ACN/H.sub.2O (90 ml/20 ml) mixed solvent, ceric ammonium nitrate ((NH.sub.4).sub.2Ce(NO.sub.3).sub.6; CAN, 4.5 g, 3.0 eq) was added thereto, and the mixture was stirred for 2 hours. After the organic solvent was removed, the result was extracted by adding DCM (×2) and water. The organic solvent was treated with magnesium sulfate, filtered, and then the solvent was removed. The residue was purified using silica gel column chromatography (Hex:EA=2:1 to 1.5:1) to obtain 1-(4-methoxybenzyl)-3,6-dioxopiperazine-2,5-diyl-diethanethioate (5; 380 mg, 36%).
[0217] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 7.27 (d, 2H), 6.88 (d, 2H), 6.73 (br s, 1H), 5.78-5.74 (m, 2H), 5.12 (d, 1H), 3.81 (s, 3H), 3.78 (d, 1H), 2.46 (s, 6H)
E. 3,6-dimercapto-1-(4-methoxybenzyl)piperazine-2,5-dione (6)
[0218] After the dithioacetate (5; 150 mg, 0.392 mmol) was dispersed in ethanol (20 ml), an ethanolic hydrochloric acid solution (prepared by adding 1.5 ml of acetyl chloride to 7 ml of ethanol) was added thereto. The mixture solution was reacted for 2 hours while being refluxed. After the reaction was complete, the solvent was removed, and the residue was dried under reduced pressure to obtain 3,6-dimercapto-1-(4-methoxybenzyl)piperazine-2,5-dione (6). The obtained compound was used for the next reaction without further purification.
F. 5-(4-methoxybenzyl)-2,3-dithia-5,7-diazabicyclo[2.2.2]octane-6,8-dione (7; A6)
[0219] A solution in which iodine (99.5 mg, 1.0 eq.) was dissolved in 15 ml of DCM was added to a solution in which 3,6-dimercapto-1-(4-methoxybenzyl)piperazine-2,5-dione (6; 117 mg, 0.392 mmol) was dissolved in 20 ml of DCM. The mixture was reacted for 30 minutes at room temperature. After the reaction was complete, the solvent was removed, and the residue was purified twice using silica gel column chromatography (Hex:EA=1:1) to obtain 5-(4-methoxybenzyl)-2,3-dithia-5,7-diazabicyclo[2.2.2]octane-6,8-dione (7) (4 mg). The NMR data of the obtained final product was shown in
[0220] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 7.23 (d, 2H), 7.15 (br s, 1H), 6.89 (d, 2H), 5.39 (d, 1H), 5.14 (s, 1H), 4.84 (d, 1H), 4.45 (d, 1H), 3.81 (s, 3H).
[0221] ESI-MS (M+Na): 319 calculated for C.sub.12H.sub.12N.sub.2O.sub.3S.sub.2 296.
PREPARATION EXAMPLE 7
Preparation of 5,7-dibenzhydryl-2,3-dithia-5,7-diazabicyclo[2.2.2]octane-6,8-dione (A7, Chemical Formula 12)
[0222] ##STR00031##
[0223] 5,7-Dibenzhydryl-2,3-dithia-5,7-diazabicyclo[2.2.2]octane-6,8-dione was obtained in the same manner as in Steps A to D of Preparation Example 1. The NMR data of the obtained final product was shown in
[0224] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 7.37-7.28 (m, 12H), 7.27 (m, 4H), 7.14 (m, 4H), 6.77 (s, 2H), 5.31 (s, 2H).
[0225] ESI-MS (M+Na): 531 calculated for C.sub.30H.sub.24N.sub.2O.sub.2S.sub.2 508.
PREPARATION EXAMPLE 8
Preparation of 5,7-dibutyl-2,3-dithia-5,7-diazabicyclo[2.2.2]octane-6,8-dione (A8, Chemical Formula 13)
[0226] ##STR00032##
A. 1,4-dibutylpiperazine-2,5-dione (2)
[0227] After NaH (430 mg, 10.95 mmol) was dispersed in 10 ml of DMF, anhydrous glycine (500 mg, 4.38 mmol) was added thereto in an ice water bath. After the mixture was stirred for 10 minutes, 1-bromobutane (1.8 g, 13.14 mmol, 3 eq.) was slowly added thereto over 30 minutes. The result was reacted overnight at room temperature, and the reaction solution was quenched with a saturated NH.sub.4Cl solution and then extracted with EA (×2). The organic layers were combined, washed with water, dried with magnesium sulfate, and then filtered. The filtrate was concentrated under reduced pressure and the residue was purified using silica gel column chromatography (DCM:EA=1:1) to obtain 1,4-dibutylpiperazine-2,5-dione (2) (410 mg, yield 41%, yellow oil).
[0228] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 3.95 (s, 4H), 3.39 (t, 4H), 1.54 (m, 4H), 1.34 (m, 4H), 0.94 (t, 6H).
B. 7,9-dibutyl-2,3,4,5-tetrathia-7,9-diazabicyclo[4.2.2]decane-8,10-dione (3)
[0229] After sulfur (450 mg, 14.16 mmol) was dispersed in 5 ml of dry THF, LiHMDS (1.0 M in THF, 5.31 ml, 3.0 eq.) was added thereto drop by drop over 2 minutes under argon atmosphere. After the mixture was stirred for approximately 1 minute, a solution in which the compound (2; 400 mg, 1.567 mmol) obtained in Step A was dissolved in 20 ml of THF was added thereto drop by drop. After the result was stirred for approximately 1 minute, LiHMDS (1.0 M in THF, 3.54 ml, 2.0 eq.) was added thereto drop by drop, and the result was stirred for 30 minutes. After the reaction was complete, the result was extracted by adding a saturated NH.sub.4Cl solution and EA (×3). The organic layers were combined, treated with magnesium sulfate, filtered, and then concentrated under reduced pressure. The result was purified using silica gel column chromatography (Hex:EA=3:1) to obtain 7,9-dibutyl-2,3,4,5-tetrathia-7,9-diazabicyclo[4.2.2]decane-8,10-dione (3; 100 mg, yield: 16%).
[0230] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 5.15 (s, 2H), 3.90 (m, 2H), 3.05 (m, 2H), 1.61 (m, 4H), 1.35 (m, 4H), 0.95 (t, 6H).
C. 3,6-dimercapto-1,4-dibutylpiperazine-2,5-dione (4)
[0231] After 7,9-dibutyl-2,3,4,5-tetrathia-7,9-diazabicyclo[4.2.2]decane-8,10-dione (3; 100 mg, 0.28 mmol) was dissolved in a methanol/THF mixed solvent (1:1), NaBH.sub.4 (32 mg, 0.84 mmol, 3.0 eq.) was slowly added thereto in an ice water bath. After the mixture was stirred for 30 minutes and the reaction was complete, the solvent was removed. A saturated NH.sub.4Cl solution and EA (×3) were added to the crude compound and the result was extracted. The organic layers were combined, dried with magnesium sulfate, filtered, and then concentrated under reduced pressure to obtain 3,6-dimercapto-1,4-dibutylpiperazine-2,5-dione (4). The compound was used for the next reaction without further purification.
[0232] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 5.03 (d, 2H), 3.80 (m, 2H), 3.21 (m, 2H), 1.63 (m, 4H), 1.37 (m, 4H), 0.96 (t, 6H).
D. 5,7-dibutyl-2,3-dithia-5,7-diazabicyclo[2.2.2]octane-6,8-dione (5; A8)
[0233] After the dithiol crude compound (4) obtained above was dissolved in 30 ml of chloroform, 20 ml of a solution in which iodine (86 mg, 0.34 mmol) was dissolved in chloroform was added thereto. The mixture was reacted for 30 minutes at room temperature, and the solvent was removed by being concentrated under reduced pressure, and the residue was purified using silica gel column chromatography (EA:Hex=1:3) to obtain 5,7-dibutyl-2,3-dithia-5,7-diazabicyclo[2.2.2]octane-6,8-dione (5) (26 mg, 2 step yield-32%). The NMR data of the obtained final product was shown in
[0234] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 5.27 (s, 2H), 3.50 (m, 4H), 1.66 (m, 4H), 1.35 (m, 4H), 0.95 (t, 6H).
[0235] ESI-MS (M+Na): 311 calculated for C.sub.12H.sub.20H.sub.2O.sub.2S.sub.2 288.
PREPARATION EXAMPLE 9
Preparation of 5,7-bis(3-methoxypropyl)-2,3-dithia-5,7-diazabicyclo[2.2.2]octane-6,8-dione (A9, Chemical Formula 14)
[0236] ##STR00033##
A. 1,4-bis(3-methoxypropyl)piperazine-2,5-dione (2)
[0237] After anhydrous glycine (0.5 g, 4.38 mmol) and K.sub.2CO.sub.3 (2.12 g, 15.33 mmol) were dispersed in 10 ml of DMSO, the temperature was raised to 80° C., and the mixture was stirred for 15 minutes at the temperature. 1-Chloro-3-methoxypropane (1.43 ml, 13.14 mmol) was slowly added thereto drop by drop. After the result was reacted for 24 hours at 80° C., the reaction was terminated, and the solids were filtered. The filtered solids were purified using silica gel column chromatography (DCM:MeOH=9:1) to obtain 1,4-bis(3-methoxypropyl)piperazine-2,5-dione (2) (350 mg, yield 31%). The residual DMSO was further washed with water.
[0238] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 3.98 (s, 4H), 3.48 (t, 4H), 3.41 (t, 4H), 3.32 (s, 6H), 1.84 (m, 4H).
B. 3,6-dibromo-1,4-bis(3-methoxypropyl)piperazine-2,5-dione (3)
[0239] After 1,4-bis(4-methoxypropyl)piperazine-2,5-dione (2; 200 mg, 0.77 mmol), N-bromosuccimide (276 mg, 1.55 mmol, 2.01 eq.) and azo-bis-isobutyronitrile (AIBN; 6.3 mg, 0.04 mmol, 0.05 eq.) were dispersed in carbon tetrachloride (50 ml), the mixture was reacted for 2 hours while being refluxed. After the reaction was complete and the reaction material was cooled to room temperature, the produced solids were filtered. The solvent was removed, and the result was dried under reduced pressure to obtain 3,6-dibromo-1,4-bis(3-methoxypropyl)piperazine-2,5-dione (3). The obtained compound was used as it is for the next reaction without further purification.
[0240] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 6.21 (s, 2H), 3.92 (m, 2H), 3.41 (m, 4H), 3.32 (s, 6H), 3.25 (m, 2H), 1.87 (m, 4H).
C. 1,4-bis(3-methoxypropyl)-3,6-dioxopiperazine-2,5-diyl-diethanethioate (4)
[0241] After 3,6-dibromo-1,4-bis(4-methoxypropyl)piperazine-2,5-dione (3; 400 mg, 0.96 mmol) was dissolved in 30 ml of DCM, sodium thioacetate (329 mg, 2.888 mmol, 3.0 eq.) was added thereto in an ice water bath. After the mixture was stirred overnight at room temperature, the produced solids were filtered and then the solvent was removed from the solution. The result were purified using silica gel column chromatography (EA:Hex=1:1 to 3:1) to obtain 1,4-bis(4-methoxypropyl)-3,6-dioxopiperazine-2,5-diyl-diethanethioate (4) (65 mg, 2 step yield-6.4%).
[0242] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 5.88 (s, 2H), 3.95 (m, 2H), 3.43 (m, 4H), 3.37 (s, 6H), 2.98 (m, 2H), 2.43 (s, 6H), 1.90 (m, 4H).
D. 3,6-dimercapto-1,4-bis(3-methoxypropyl)piperazine-2,5-dione (5)
[0243] After the dithioacetate (4; 40 mg, 0.1 mmol) was dispersed in ethanol, an ethanolic hydrochloric acid solution (prepared by adding 0.75 ml of acetyl chloride to 3.5 ml of ethanol) was added thereto drop by drop, and the mixture solution was reacted for 2 hours while being refluxed. After the reaction was complete and the reaction material was cooled to room temperature, the organic solvent was removed and the residue was dried to obtain 3,6-dimercapto-1,4-bis(methoxypropyl)piperazine-2,5-dione (5). The obtained compound was used for the next reaction without further purification.
[0244] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 5.08 (d, 2H), 3.89 (m, 2H), 3.41 (m, 4H), 3.35 (s, 6H), 3.23 (m, 2H), 3.18 (d, 2H), 1.93 (m, 4H).
E. 5,7-bis(3-methoxypropyl)-2,3-dithia-5,7-diazabicyclo[2.2.2]octane-6,8-dione (6; A9)
[0245] A solution in which iodine (20.3 mg, 0.08 mmol, 1.0 eq.) was dissolved in 15 ml of DCM was added to a solution in which 3,6-dimercapto-1,4-bis(methoxypropyl)piperazine-2,5-dione (5; 25 mg, 0.08 mmol) was dissolved in 10 ml of chloroform, and the mixture was reacted for 1 hour at room temperature. After the reaction was complete, the solvent was removed and the residue was purified using silica gel column chromatography (Hex:EA=1:1 to 1:2) to obtain 5,7-bis(methoxypropyl)-2,3-dithia-5,7-diazabicyclo[2.2.2]octane-6,8-dione (6) (9 mg, yield 36%). The NMR data of the obtained final product was shown in
[0246] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 5.35 (s, 2H), 3.69 (m, 2H), 3.57-39 (m, 6H), 3.36 (s, 6H), 1.93 (m, 4H).
[0247] ESI-MS (M+Na): 343 calculated for C.sub.12H.sub.20H.sub.2O.sub.4S.sub.2 320.
<Synthesis of Piperazinedione Derivative Including Reduced Dithiol Group>
PREPARATION EXAMPLE 10
3,6-dimercapto-1,4-dimethylpiperazine-2,5-dione (A2R)
[0248] ##STR00034##
[0249] 3,6-Dimercapto-1,4-dimethylpiperazine-2,5-dione was obtained in the same manner as in Steps A to C of Preparation Example 2. The NMR data of the obtained final product was shown in
[0250] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 5.00 (s, 2H), 3.09 (s, 6H) .
[0251] ESI-MS (M-1): 205 calculated for C.sub.6H.sub.10N.sub.2O.sub.2S.sub.2 206.
PREPARATION EXAMPLE 11
3,6-dimercapto-1,4-bis(4-methoxybenzyl)piperazine-2,5-dione (A4R)
[0252] ##STR00035##
[0253] 3,6-Dimercapto-1,4-bis(4-methoxybenzyl)piperazine-2,5-dione was obtained in the same manner as in Steps A to C of Preparation Example 4. The NMR data of the obtained final product was shown in
[0254] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 7.20 (d, 4H), 6.89 (d, 4H), 5.21 (d, 2H), 4.93 (s, 2H), 4.12 (d, 2H), 3.85 (s, 6H), 3.05 (br s, 2H).
[0255] ESI-MS (M+Na): 441 calculated for C.sub.20H.sub.22N.sub.2O.sub.4S.sub.2 418.
PREPARATION EXAMPLE 12
3,6-dimercapto-1,4-diallylpiperazine-2,5-dione (A5R)
[0256] ##STR00036##
[0257] 3,6-Dimercapto-1,4-diallylpiperazine-2,5-dione was obtained in the same manner as in Steps A to C of Preparation Example 5. The NMR data of the obtained final product was shown in
[0258] .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 5.80-5.70 (m, 2H), 5.39-5.31 (m, 4H), 5.07 (s, 2H), 4.68 (d, 2H), 3.70-3.64 (m, 2H), 3.09 (br s, 2H).
EXAMPLE 1
Materials
[0259] Anti-PrxI and PrxII antibodies were purchased from AbFrontier (Seoul, Korea). The sequence of the PrxII-specific siRNA used in the present invention was as follows: 5′-CGCUUGUCUGAGGAUUACGUU-3′ (human PrxII siRNA-1, Prx2-1; sequence number 1), 5′-AGGAAUAUUUCUCCAAACAUU-3′ (human PrxII siRNA-2, Prx2-2; sequence number 2), 5′-ACTCAACTGCCAAGTGATTUU-3′ (human PrxI siRNA, PrxI; sequence number 3) and 5′-AAAUCAAGCUUUCGGACUAUU-3′ (mouse PrxII siRNA-1, mPrx2-1; sequence number 4). The siRNA oligonucleotide duplex was synthesized from Dharmacon. The firefly luciferase siRNA was synthesized and used as control siRNA. The siRNA duplex was transfected according to the protocol of the manufacturer using Lipofectamine RNAi MAX™ (Invitrogen).
[0260] A SMART pool of four siRNA duplexes for rat PrxII (5′-GCAACGCGCACAUCGGAAAUU (sequence number 5), 5′-GAUCACAGUCAACGACCUAUU (sequence number 6), 5′-AGAAUUACGGCGUGUUGAAUU (sequence number 7) and 5′-ACGCUGAGGACUUCCGAAAUU (sequence number 8); Dharmacon Cat. no. D-089973) was used for rat carotid balloon injury experiments. The firefly luciferase siRNA was synthesized and used as control siRNA.
[0261] Gliotoxin, cheatocin and chetomin were purchased from Sigma-Aldrich. Bis(methylthio)gliotoxin was purchased from Santa Cruz Biotechnology. SU-5416 was purchased from Calbiochem. Phospho-PLCγ1 (pY783), PLCγ1, and phospho-VEGFR2 (pY1175) antibodies were purchased from Cell Signaling Technology. Anti-PDGFR-β (M-20) and KDR/Flk-1 (VEGFR2) antibodies were purchased from Santa Cruz Biotechnology. Anti-phosphotyrosine (4G10) and PDGF-BB were purchased from Upstate. VEGF-A (human VEGF.sub.165) were purchased from R&D systems. Mouse anti-rat CD31 antibody was purchased from BD Bioscience. Alexa Fluor 488-conjugated donkey anti-rabbit and Alexa Fluor 568-conjugated donkey anti-mouse secondary antibodies were purchased from Invitrogen. Biotinylated goat anti-rabbit IgG, Avidin-HRP and DAB substrates were purchased from Vector Laboratories. PrxI, PrxII, Prx-SO.sub.2/3, and phospho-PDGFRβ (pY857) rabbit polyclonal antibodies were prepared as described previously [M. H. Choi et al., 2005, Nature, 435: 347].
EXAMPLE 2
Cell Culture
[0262] Human aortic endothelial cells (HAEC) and human aortic smooth muscle cells (HASMC) were purchased from Clonetics-Bio Whittaker (Venders, Belgium). The cells were seeded on a 0.1% gelatin-coated plate and grown at 37° C. in a humidified incubator including 5% carbon dioxide in Endothelial Basal Medium (EBM™-2) and Smooth Muscle Cell Basal Medium (SmBMTM) SingleQuotes° including 10% fetal bovine serum (FBS) and full supplements (Cat no. cc-4176 for HAEC and Cat no. cc-4149 for HASMCs; Clonetics-BioWhittaker), respectively. Cells of passages 5 to 7 were used in the present invention.
[0263] Melanoma cell lines SK-MEL-5, A375 and B16F10 cells were grown at 37° C. in a CO.sub.2 incubator using Dulbecco's Modified Eagle's Medium (DMEM) including 10% fetal bovine serum (FBS). SK-MEL-28 and G361 cells were grown in a RPMI 1640 medium including 10% FBS.
[0264] Human epidermal melanocyte was grown in Medium 254 (Cascade Biologics™) including human melanocyte growth supplements. The melanoma cell lines and the melanocyte used in the present invention were purchased from American Type Culture Collection.
EXAMPLE 3
Peroxidase Activity Assay
[0265] The peroxidase activity assay for the epidithiodioxopiperazine derivatives according to the present invention (hereinafter, referred to as ETP compounds) was performed according to known methods [Korean Patent No. 10-0953326]. A standard peroxidase reaction for spectrophotometric assay was carried out using a 200 μl reaction mixture of 1 mM EDTA-containing 50 mM Hepes-NaOH buffer solution (pH 7.0) including 250 μM NADPH, 3 μM yeast thioredoxin (Trx), 1.5 μM yeast thioredoxin reductase (TR), 25 μM ETP compound and 1.2 ml of hydrogen peroxide. For the comparison with a glutathione (GSH)-dependent peroxidase reaction, GSH (1 mM) and yeast glutathione reductase (GSH reductase; GR) (1 Unit) were added instead of yeast Trx and TR. Each reaction was initiated by adding hydrogen peroxide, and the NADPH oxidation was monitored for 12 minutes at 30° C. according to absorbance decrease at 340 nm using an Agilent UV8453 spectrophotometer (Hewlett Packard, USA). The initial reaction rate was calculated using the linear portion of the curve and expressed as the amount of NADPH oxidized per minute.
EXAMPLE 4
In Vitro Vascular Cell Function Assay
[0266] For cell proliferation assay, HAECs were divided at a concentration of 4000 cells/well in a final volume of 100 μl onto a 96-well plate including a siRNA-transfection reagent mixture. After siRNA-transfected for 24 hours, the cells were serum-starved for 18 hours, and then placed in an EBM-2 basal medium supplemented with VEGF-A165 (25 ng/ml, Cat no. 293-VE, R&D systems) for additional 24 hours. The extent of cell proliferation was measured using a WST-1 cell proliferation assay kit (Roche Diagnostics, USA), and the number of cells was expressed as absorbance at 450 nm, which was averaged from 3 wells after subtracting the turbidity at 600 nm.
[0267] The cell migration assay was performed in a 24-well Transwell culture chamber (Costar; 8-μm pore size). The bottom of the filter was coated with gelatin B (1 mg/ml) and air-dried for 1 hour. HAECs (6×10.sup.3) were added to upper chambers including siRNA-transfection complexes. After 24 hours, the siRNA-transfected HAECs were serum-starved overnight. A solution of VEGF-A (25 ng/ml) was prepared in a basal medium including 0.5% bovine serum albumin (BSA), and added to bottom chambers. The upper chamber wells were each filled with a basal medium including 0.5% BSA. The Transwell chamber was incubated for 8 hours under the condition of 37° C./5% carbon dioxide. After the incubation, the non-migrated cells at the top of the filter were removed, and the cells that migrated onto the bottom of filter were fixed and stained with 0.6% hematoxylin and 0.5% eosin. The stained cells were photographed and counted. The number of migrating cells was averaged from 3 wells.
EXAMPLE 5
Immunoblot Analysis
[0268] The cells were rinsed with an ice-cold phosphate buffered saline (PBS) solution and then lysed in an extraction buffer solution containing 20 mM Hepes (pH 7.0), 1% Triton X-100, 150 mM NaCl, 10% glycerol, 1 mM EDTA, 2 mM EGTA, 1 mM DTT, 5 mM Na.sub.3VO.sub.4, 5 mM NaF, 1 mM AEBSF, aprotinin (5 μg/ml), and leupeptin (5 μg/ml). After being centrifuged at 12,000×g, the purified cell extracts were used for immunoblotting. In order to adjust the loading, the membranes were stripped by shaking them for 30 minutes at 60° C. in 67 mM Tris (pH 6.7), 2% SDS, and 100 mM 2-mercaptoethanol solution, and reprobed with an appropriate pan antibody.
EXAMPLE 6
Immunocytochemistry
[0269] The cells were grown on a glass cover slide, and fixed with a prewarmed 4% paraformaldehyde solution for 15 minutes. The fixed cells were rinsed twice with PBS, and treated with 0.2% Triton X-100 for 30 minutes at room temperature so as to have cell permeability. After that, the cells were blocked for 1 hour using a PBS solution including 2% BSA, and incubated overnight at 4° C. with primary antibodies diluted in a blocking buffer solution: anti-phosphotyrosine (clone 4G10, 1:100), anti-PrxI (1:300), anti-Prx2 (1:300), anti-β-catenin (1:200) and anti-E cadherin (1:200). The cells were rinsed 3 times with a blocking buffer solution, and incubated for 30 minutes with secondary antibodies conjugated with Alexa Fluor 568 or Alexa Fluor 488. The stained cover slip was washed three times with a blocking buffer solution, and mounted. The fluorescence images were recorded using an LSM510 META confocal laser scanning microscope (Zeiss).
EXAMPLE 7
Measurement of Intracellular Hydrogen Peroxide
[0270] The intracellular H.sub.2O.sub.2 level was measured using 5,6-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-DCFH-DA, Invitrogen), an oxidation-sensitive fluorescent dye. Human epidermal melanocyte, SK-MEL-5, SK-MEL-28, A375 and G361 cells (3×10.sup.5) were grown in a 35 mm culure dish and infected with retroviruses for 24 hours. After that, the cells were serum-starved for 18 hours, and stimulated with 20% FBS for 10 minutes in a phenol red-free medium. After stimulated as described above, the cells were quickly rinsed with a Krebs-Ringer solution, and incubated with 5 μM CM-DCFH-DA for 5 minutes. 2′,7′-dichlorodihydrofluorescein (DCF) fluorescence was collected for 10 seconds using an inverted Axiovert 200 fluorescence microscope (Zeiss). The relative DCF fluorescence was calculated by averaging the fluorescence intensities of 60 to 80 cells after subtracting the background fluorescence from each image using an ImageQuant™ software (GE Healthcare). The desorbed sphenoid cells were excluded from quantification.
EXAMPLE 8
Retrovirus Production
[0271] Retroviruses encoding human PrxII were produced using a bicitronic pLXIN vector and a Retro-X Q vector system (Clontech). First, human PrxII wild-type (WT) coding sequences were inserted to pLXIN by PCR cloning. Viruses were produced by stably trasfecting the resultant vector to a stable NIH3T3-based dualtropic packaging cell line RetroPackTM PT67. The viruses were mostly used for PrxII overexpression in the SK-MEL cells. The contentration of the viruses (viral titers) was determined (1×10.sup.6 virus particle/ml), and the viruses were divided and stored at −70° C. For retrovirus infection, the divided viruses were thawed in a warm water bath, and mixed with 10 μg/ml polybrene.
EXAMPLE 9
Proliferation and Migration Assay
[0272] The extent of cell proliferation was measured using a WST-1 cell proliferation assay kit (Roche Diagnostics, USA) according to the protocol of the manufacturer. The number of cells was expressed as an absorbance value at 450 nm averaged from 3 wells after subtracting the turbidity at 600 nm. The chemotactic transmigration assay was performed in a 24-well Transwell culture chamber (Costar; polycarbonate membrane insert with 8-pm pore size). The bottom of the insert was coated with gelatin B (1 mg/ml) and air-dried for 1 hour. Retroviruses or transfection complexes were treated to melanoma cells (6×10.sup.3). After 24 hours, the infected or transfected melanoma cells were serum-starved for 18 hours. A 20% FBS solution was added to bottom chambers including a basal medium containing 0.5% BSA. The upper chamber wells were each filled with a basal medium including 0.5% BSA. The Transwell chamber was incubated for 12 hours in a 37° C./5% CO.sub.2 incubator. After the incubation, the non-migrating cells at the top of the filter were removed. The cells that migrated onto the bottom of filter were fixed and stained with 0.6% hematoxylin and 0.5% eosin. The stained cells were photographed and counted. The number of migrating cells was averaged from 3 wells.
EXAMPLE 10
Wound Healing Assay
[0273] A wound was created on a cell monolayer by scratching with a pipet tip. The wound was washed with PBS to remove cell fragments, and a fresh medium was supplied thereto. For 12 hours after that, the cells were made to be proliferated and migrated to the wound. The migration of the cells to the wounded area was observed under a microscope. The width of the wound was measured using an Image J software.
EXAMPLE 11
Metastasis of B16F10 Melanoma Cells to Lung in Mouse
[0274] After B16F10 cells were temporarily transfected with PrxII siRNA for 24 hours and trypsinized, the cells were resuspended in a Hank's balanced salt solution (HBSS). The cell suspension was injected by intravenous injection to a C57/BL6 mouse (1×10.sup.6 cells per mouse). The mouse was sacrified after 10 days, and the lung was removed after transcardia perfusion-fixation with a heparinized saline solution including 3.7% formaldehyde. The removed lung was paraffin embedded and sectioned using a rotary microtome (Leica RM2255). Two serial tissue sections (4 μm in thickness) were stained with haematoxylin and eosin. The melanoma tumor nodules metastasized from the surface of the lung and the HE-stained tissue sections were counted. In order to verify the effects of gliotoxin (GT), chaetocin and chetomin, the melanoma cells were intravenously injected to the C57/BL6 mouse, and then gliotoxin (300 mg/kg) was intraperitoneally injected for 5 times over 10 days.
EXAMPLE 12
NADPH Oxidase Activity Assay
[0275] Whole cell superoxide production was measured using an enhanced luminescence system (Diogenes, National Diagnostics). For the assay, the serum-starved cells were pre-incubated with a 100 μl Diogene reagent at 37° C. for 5 minutes. After being stimulated by the indicated growth factor, chemiluminescence was detected every second for 10 minutes with a TD-20/20 Luminometer (Turner Biosystems).
EXAMPLE 13
Balloon-Injury Model of Rat Carotid Artery
[0276] Animal experiments were performed in compliance with the guidelines of Institutional Animal Care and Use Committee (IACUC) of the Ewha Womans University. Balloon injury was created using an infiltrated 2F Fogarty balloon catheter in the left common carotid artery as follows: after the ten-week-old male Sprague-Dawley rats were anaesthetized by isoflurane gas (N.sub.2O:O.sub.2/70%:30%) inhalation, the left external carotid artery was exposed, and its branches were electrocoagulated. A catheter was pushed 1 cm in through the transverse arteriotomy of the external carotid artery, and the endothelial denudation was achieved by three passes along the common carotid artery. After the catheter was removed, the punched area was sealed and the clapped common carotid artery was opened for resuming the blood flow. Unless otherwise stated, the rats were recovered in a cage for 10 days, and subjected to histological and immunological analyses.
EXAMPLE 14
Histological Analysis
[0277] Rats were anesthetized, and the common carotid artery was excised after transcardiac perfusion-fixation with a heparinized saline solution containing 3.7% formaldehyde. The vessels were paraffin embedded and sectioned using a rotary microtome (Leica RM2255). Two serial tissue sections (4 pm in thickness) were obtained from the middle area of common carotid arteries, and stained with haematoxylin and eosin (HE). The luminal, internal elastic laminal, and external elastic laminal areas were measured using NIH Image v1.62. The intimal and medial areas were determined by subtracting the luminal area from the internal elastic area and subtracting the internal elastic area from the external elastic area. The values from the two serial sections for each rat were averaged for analysis.
EXAMPLE 15
Local Delivery of siRNA or ETP Compound or Derivative in Carotid Artery
[0278] For the in vivo injection of siRNA, a siRNA mixture specific to rat PrxII (200 nM) was premixed with a siPORT™ NeoFX™ reagent according to the instructions of the manufacturer (Ambion). Immediately after the balloon injury, the siRNA-injected mixture (200 μL) was injected through a catheter after briefly washed with Opti-MEM. After the incubation for 15 minutes, the blood flow was resumed. A siGLO-Red (Dharmacon), which is fluorescent dye-conjugated control siRNA, was used for optimizing in vivo injection efficiency. Similarly, the ETP compound (200 nM in DMSO) was injected through a catheter and incubated for 30 minutes.
EXAMPLE 16
Immunohistochemistry and Immunofluorescence Staining
[0279] Immunohistochemistry for the overoxidized 2-Cys Prxs was performed on the paraffin sections using an anti-Prx-SO.sub.2/3 antibody (1:1000 dilution). Briefly, the sections were de-waxed in xylene and rehydrated in ethanol, and subsequently the antigen retrieval was performed by boiling in a citric buffer solution (pH 6.0). After that, the sections were incubated with a primary antibody for 48 hours at 4° C. After washing three times with a phosphatebuffered saline solution, the sections were incubated with a peroxidase-conjugated secondary antibody and stained with a 3′,3′-diaminobenzidine (DAB) substrate solution. For negative staining, the Prx-SO.sub.2/3 antibody was blocked with the corresponding antigenic peptide (DFTFVC(SO.sub.2/3)PTEI). For indirect immunofluorescence staining, the paraffin sections were blocked with 5% normal rabbit serum (Vector Laboratories) in PBST (a PBS solution of 0.3% Triton X-100) for 1 hour at room temperature. The sections were then incubated overnight at 4° C. with the antigens against rat smooth muscle α-actin (1:300 dilution) and rat CD31 (PECAM-1, 1:200 dilution). The nuclei were labeled with DAPI. After several PBST washes, the samples were incubated for 2 hours at room temperature with Alexa Fluor 568-conjugated donkey anti-mouse and Alexa Fluor 488-conjugated donkey anti-rabbit IgG antibodies. The fluorescence images were recorded on three random fields per tissue section at a screen magnification of 100× using a LSM 510 Meta confocal microscope equipped with argon and helium-neon lasers.
EXAMPLE 17
TUNEL Assay
[0280] The paraffin sections were incubated for 10 minutes in PBS containing 0.1% Triton X-100. After that, TUNEL reactions were performed for 60 minutes at 37° C. using an In Situ Cell Death Detection Kit and Fluorescein (Roche Diagnostics Corp.) according to the instructions of the manufacturer. The cell nuclei were counterstained with DAPI.
EXAMPLE 18
Vascular Permeability Test
[0281] Mice were injected intravenously with 100 μl of 5% Evans blue for 30 minutes and then perfused with PBS for 5 minutes. The common carotid arteries were removed from both uninjured collateral and injured ipsilateral ones. The result was dissected, opened longitudinally, and examined on a phase contrast microscope with a magnification of 20×. For quantification, the Evans blue inflowed into the blood vessel was extracted by being placed in formamide at 55° C. overnight and centrifuged for 10 minutes at 12,000 rpm. The supernatants were collected and the absorbance was measured at 620 nm. The background value of the Evans blue dye was measured at 740 nm and substracted from the values in carotid arteries. For VEGFR2 inhibition, SU5416 (20 mg/kg) was injected intraperitoneally for 3 times (Days −1, 1, and 3) before and after the balloon injury. Control injection was made of 200 μl PBS including a vehicle (5% DMSO).
EXAMPLE 19
Scanning Electron Microscopy (SEM)
[0282] The carotid vessels were taken from animals, opened longitudinally, and fixed with 2.5% glutaraldehyde for 24 hours. The tissues were rinsed with PBS, incubated with 1% osmium tetroxide, and then dehydrated through a series of ethanol dilutions. The tissues were dried to the critical point and mounted on scanning electron microscopy stubs with colloidal silver paste. After sputter-coated with gold/palladium, the specimens were examined with a scanning electron microscope (Hitachi, Japan).
EXPERIMENTAL EXAMPLE 1
Catalytic Activity for Hydrogen Peroxide Reduction
[0283] The chemical feature of the epidithiodioxopiperazine compound of the present invention or its derivatives is the intramolecular disulfide bridge present in the epidithiodioxopiperazine ring moiety. Cellular peroxidase reduces hydrogen peroxide utilizing the electrons derived from NADPH via two electron-transfer routes, that is, Trx/TR or GSH/GR, therefore, spectrophotometric assay was performed for the hydrogen peroxide-reducing activity in the presence of each system, and the results are shown in
[0284] As a result, as shown in
[0285] In addition, the results were compared using the reduced types (A2R, A4R and A5R) of A2, A4 and A5 (
[0286] Meanwhile, A2R, A4R and A5R did not actually show PDGF- and VEGF-dependent cell proliferation and migration regulatory activities as shown in the experiments to be described later, and this may be inferred that these compounds had no PDGF- and VEGF-dependent cell proliferation and migration regulatory activities since the reduced form of the derivatives which have the dithiol in the piperazine ring cannot be introduced into the cells. This indicated that the oxidation-reduction cycle between the intramolecular disulfide bridged bond and the dithiol in the piperazine ring within the ETP derivative was essential for the peroxidase activity. In addition, from the results described above, it was demonstrated that the 2-Cys-Prx-analogous peroxidase activity of the epidithiodioxopiperazine compound or its derivatives according to the present invention was Trx/TR system-dependent.
EXPERIMENTAL EXAMPLE 2
In Vitro and In Vivo Cytotoxicity Experiment
[0287] Most ETP compounds have been known to be cytotoxic to animal cells. Accordingly, the inventors of the present invention determined a safe concentration range of the epidithiodioxopiperazine compound or its derivatives in vascular cells. For this, human aortic smooth muscle cells (HASMCs) and human aortic endothelial cells (HAECs) were pretreated for 2 hours with various concentrations of gliotoxin or chaetocin, and A1 to A9 that are ETP derivatives according to the present invention, and cultured in a fresh medium for 24 hours followed by cell viability assay (
[0288] As a result, the viability of HASMCs and HAECs with respect to gliotoxin and A1 to A9 under a serum-deficient condition were guaranteed at concentrations of 200 nM and 50 nM or less, respectively. Chaetocin was found to be relatively less toxic. The low viability of the two cells at high concentrations was considered to be due to various side effects including NF-κB inhibition. Accordingly, for the experiments described below, the inventors of the present invention used the ETP compounds at a concentration range of 100 nM and 50 nM, a safe dosage for in vitro experiments for HASMCs and HAECs, and for compounds having low tendency, the concentration was increased up to a range of 200 nM and 100 nM.
EXPERIMENTAL EXAMPLE 3
Effects of PrxII Function Replacement by Gliotoxin and Epidithiodiketopiperazine Derivative in Growth Factor-induced Signaling and Cell Proliferation/Migration
[0289] (1) Effects of PrxII Function Replacement by Gliotoxin
[0290] First, the ability of GT to eliminate intracellular hydrogen peroxide in PrxII knocked down HASMCs and HAECs was tested. The cellular hydrogen peroxide production was monitored using an oxidant-sensitive fluorescence dye (2′,7′-dihydro-chlorofluorescein diacetate, H.sub.2DCF-DA). The level of intracellular hydrogen peroxide increased by approximately two fold by PDGF treatment in serum-deficient control HASMCs, and then markedly increased by combining with the PrxII knockdown (
[0291] In addition, the regulatory effects of GT on the PDGFRβ- and VEGFR2-mediated signaling pathway were examined. The GT treatment in HASMCs was quite different from PDGF-induced tyrosine phosphorylation that was enhanced by the PrxII knockdown (
[0292] (2) Effects of PrxII Function Replacement by A1 to A9
[0293] The regulatory effects of the ETP derivatives A1 to A9 on the PDGFRβ- and VEGFR2-mediated signaling pathway were examined. The effects of ETP derivatives on the function of vascular cells, that is, human aortic smooth muscle cells (HASMCs) and human aortic endothelial cells (HAECs) were demonstrated. As described above, each EPT derivative was used in a concentration range of 100 nM and 50 nM or less, a concentration range exhibiting no cytotoxicity for HASMCs and HAECs, respectively, and for several compounds (A4, A7 and A9) having insignificant activities, the concentration was increased up to a maximum of 200 nM and 100 nM.
[0294] As a result, the ETP derivative treatments with increasing concentrations gradually reduced the proliferation and the chemotactic transmigration of HASMCs in response to PDGF, which had been enhanced by the PrxII knockdown (
[0295] Furthermore, A2R, A4R and A5R with exposed thiol groups were used to compare the results. As a result, no significant changes were observed in both HASMCs and HAECs (
[0296] It is noteworthy that nanomolar concentrations of ETP derivatives A1 to A9, which does not exhibit cytotoxicity, are sufficient to regulate RTK signaling and cell function in HASMCs and HAECs. Particularly, it was demonstrated that A1 to A3, A5 and A8 having relatively small substituents or a small number of substitutents exhibited excellent activities, and A6 and A9 exhibited moderate activities. Collectively, the experimental results described above indicate that low concentrations of the ETP derivatives may restore the injured PDGF- and VEGF-induced signaling due to PrxII deficiency.
EXPERIMENTAL EXAMPLE 4
Effects of ETP Compound on NADPH Oxidase (NOX) Activity
[0297] When HASMCs and HAECs were stimulated with PDGF-B and VEGF-A, respectively, the NOX activity increased approximately twofold. However, the treatments with GT and cheatocin did not inhibit the NOX activity until the concentrations of the compounds increased up to 500 nM, which exceeds the toxic limit (
EXPERIMENTAL EXAMPLE 5
Identification of 2-Cys-Prx, Particularly, PrxII Overoxidation for Balloon-injured Carotid Arteries through in vivo Experiments
[0298] The effects of the ETP compounds were identified in vivo using an experimental animal model of vascular injury (balloon-induced injury of rat carotid artery) capable of monitoring the hyperplasia of VSMCs and vascular reendothelialization. When the arteries are injured by the insertion of a balloon catheter, the endothelium is denuded. Therefore, the platelets and macrophages are accumulated in the injured lesion to repair the injured vessel. These cells produce active oxygen radicals including hydrogen peroxide, therefore, the 2-Cys Prx enzymes in neighboring vascular cells are presumed to be inactivated by overoxidation of the active site cysteine residue to sulfinic/sulfonic acids (Cys-SO.sub.2/3). To address this possibility, the overoxidation of 2-Cys Prxs in the balloon-injured rat carotid arteries was examined using an anti-Prx-SO.sub.2/3 antibody. The balloon injury induced the intimal hyperplasia of carotid arteries along with time (
[0299] Next, the inventors of the present invention assessed whether GT orchestrated the proper repair of the injured vessel where the PrxII overoxidation occured. When the GT solution was locally administered at various concentrations to the lumen of the carotid arterial vessels through the catheter after balloon injury, the intimal hyperplasia was notably suppressed in a concentration dependent manner within a nanomolar range that did not induce cell death, as identified by TUNEL staining (
[0300] In addition, as a control experiment, bis(methylthio)gliotoxin having methylated dithiol groups and TR inhibitor (DNCB 5 μM, or Auronafin 0.5 μM) were treated either alone or as a combination with GT, and the HE-stained images for tissues were observed.
[0301] The result showed that bis(methylthio)gliotoxin did not suppress the thickened vascular intimal layer, and TR inhibitors all canceled out the intimal hyperplasia suppression effects of GT (
[0302] Subsequently, experiments to identify whether GT actually promoted the endothelial repair in the injured vessel walls were carried out. The immunofluorescence staining of endothelial marker CD31 clearly revealed that, as in normal carotid vessels, an endothelium monolayer in contact with lumen was formed in GT-treated injured vessels, while the monolayer was not formed in control injured carotid arteries (
[0303] In addition, immunohistochemistry staining was carried out using an overoxidized 2-Cys-Prx (Prx-SO.sub.2/3) antibody in order to identify the effects of GT on the overoxidation of 2-Cys-peroxiredoxin (2-Cys-Prx), and as the DAB-stained images, representative images among 5 different carotid artery samples were shown. As a result, as shown in
[0304] In addition, it was identified whether A1 to A3, A5, A6 and A8, the ETP derivatives according to the present invention, may inhibit the proliferation of aortic smooth muscle cells.
[0305] Specifically, whether the derivative orchestrates the proper repair of the injured vessel where the PrxII overoxidation occurs was identified. The solution including the derivative was locally administered at a concentration of 200nM to the lumen of the carotid arterial vessels through a catheter after the balloon injury, and the HE-stained images for tissues were observed.
[0306] The result, as shown in
[0307] Subsequently, the experiments to identify whether the derivatives A1 to A3, A5, A6 and A8 actually promoted the endothelial repair in the injured vessel walls were carried out. The immunofluorescence staining of the endothelial marker CD31 verified that, while an endothelium monolayer in contact with the lumen was formed in the injured vessels treated with A1 to A3, A5, A6 or A8, the monolayer was not formed in control injured carotid arteries (
EXPERIMENTAL EXAMPLE 6
Vascular Permeability Test
[0308] The permeability of the repaired endothelium was examined by Evans blue inflow. The endothelial denudation by balloon injury resulted in the complete loss of permeability controlling activity, whereas the uninjured collateral carotid arteries showed the intact barrier function of normal endothelium (
[0309] The interendothelial junction on the luminal surfaces of the injured carotid arterial vessels was examined by a scanning electron microscopy. Endothelial cells in the carotid artery of a GT-treated rat were spread and formed a uniform layer with tight junction, whereas those in carotid artery of a control-treated rat were shrunken and patched (
EXPERIMENTAL EXAMPLE 7
Effects of Chaetocin, Chetomin and Other Anti-oxidant Compounds
[0310] The inventors of the present invention tested whether other ETP compounds such as chaetocin and chetomin also induce the proper repair of injured vessels. The efficacy of chaetocin in vascular cell function was first evaluated. The chaetocin treatment with increasing concentrations markedly reduced the proliferation and the chemotactic transmigration of HASMCs in response to PDGF, which had been enhanced by the PrxII knockdown (
[0311] Lastly, the effects of GT, a representative ETP compound, on vascular cells were compared with the effects of other compounds known as anti-oxidant compounds such as N-acetylcysteine or butylated hydroxyanisole, and the results are shown in
EXPERIMENTAL EXAMPLE 8
Identification of Effects of GT on PrxII-deficient Vascular Cells and Mouse
[0312] Aortic vascular smooth muscle (A) and endothelial cells (B) were separated from a normal mouse and a PrxII−/− mouse, and cultured, and then treated with PDGF-BB and VEGF-A for 10 minutes each after either treating with GT or no treating. The activation of PDGFRβ, PLCγ1, VEGFR2 and ERK was analyzed using a phosphorylation-specific binding antibody for each protein. The result verified that, as shown in A and B of
[0313] In addition, in order to identify the effects of GT on vascular thickening in a PrxII−/− mouse, left carotid artery of a PrxII−/− mouse was damaged using a flexible wire. After the injury, a Whatman No. 1 filter paper strip was soaked in a control vehicle or a GT solution, and the paper was attached to the surface of vascular epithelia for 30 minutes for the solution to permeate into the tissue. The mouse was recovered at the 10th day after the injury. As a result, the ratio of intima versus media measured from the HE-stained carotid artery sample was presented as mean+−standard error (n=8 per group, *p<0.01). The result indicated that, as shown in
EXPERIMENTAL EXAMPLE 9
Inhibition Effects of PrxII on Proliferation and Migration of Melanoma Cells, and Identification of Inhibiting Melanoma Cells From in vitro Migration and In Vivo Metastasis to Lung by Treating Melanoma Cells with ETP compound or its derivatives
[0314] (1) Identification of Relation between Proliferation and Migration of Melanoma Cells and PrxII Suppression
[0315] Melanoma cells were immunoscreened for the PrxII protein level using a PrxII-specific antibody. While SK-MEL-5 (SK5) and SK-MEL-28 (SK28) melanoma cells scarcely expressed PrxII, two other cell lines, A375 and G361, expressed PrxII protein (
[0316] Consequently, the basic hydrogen peroxide level appeared to be high in the PrxII-deficient SK-MEL cells while the level was low not only in primary melanocyte in which PrxII was present but also in A375 and G361 (
[0317] Furthermore, in vitro cell activities such as proliferation and migration of 4 melanoma cell types were compared. The serum condition is physiologically related to tumor metastasis involving complex factors, therefore, the cells were in a serum-starved condition and then stimulated with 20% fetal serum in order to maximize the inducement of serum-dependent cell activities. As a result, the PrxII-deficient SK-MEL cells exhibited a higher proliferation activity compared to 2 other PrxII-expressed melanoma cells (
[0318] In addition, the direct regulatory effects of PrxII on melanoma cells regarding the exogenous expression and the specific knockdown of PrxII were identified. The exogenous expression of PrxII in the SK-MEL cells was achieved by the retroviral transduction of human Prdx2 genes (
[0319] (2) Metastasis Increase of Melanoma Cells to Lung Due to PrxII Absence
[0320] In the proliferation and the migration of melanoma cells, in vivo experiments were performed using a metastasis to lung model with B16F10, a mouse melanoma cell, based on the regulatory activity of PrxII. B16F10 cells are known to express wild-type BRAF, therefore, it was identified that PrxII still regulated B16F10 cell activities and Src/ERK pathway. In the B16F10 cells, the PrxII knockdown using mouse PrxII-specific siRNA reduced the level of E-cadherin while increasing the Src/ERK activation and the β-catenin phosphorylation (
[0321] For in vivo experiments, the stable deficiency of PrxII expression was guaranteed during the metastasis to lung assay using siRNA-mediated knockdown of PrxII expression (
(3) In Vitro Migration Inhibition and In Vivo Metastasis to Lung Inhibition of Melanoma Cells by ETP Derivative and Gliotoxin Treatment
[0322] The fungal secondary metabolite known as gliotoxin (GT) is a first natural product known to exhibit a thioredoxin-dependent peroxidase activity represented as a typical Prx activity, therefore, the treatability of GT for melanoma metastasis inhibition was identified. GT treatment at the nontoxic level actually reduced the level of intracellular hydrogen peroxide in the PrxII-deficient SK28 cells (
[0323] In addition, whether chaetocin and chetomin inhibited the activities of melanoma cells were identified.
[0324] Specifically, after chaetocin and chetomin was treated with SK-MEL28 melanoma cells at different concentrations (0, 100, 200, 500 and 1000 nM), the cell viability was checked, and the result is shown in
[0325] Accordingly, chaetocin and chetomin were treated with SK-MEL28 melanoma cells at 100 nM for 1 hours, and the proliferative and migratory activities were checked, and the proliferation and the migration were promoted with serum (FBS).
[0326] As shown in
[0327] In addition, in order to demonstrate the in vivo treatment efficiently, chaetocin and chetomin were injected to a mouse intraperitoneally after B16F10 cells were injected. The treatment with chaetocin and chetomin significantly reduced the metastasis to lung in melanoma cells (
[0328] Accordingly, it may be inferred that the ETP derivatives according to the present invention mimicking in vivo PrxII activity may also exhibit similar activities for melanoma metastasis inhibition through the PrxII activation.
[0329] Metastatic malignancy is the most crucial issue in vertical growth phase melanoma. Despite that major genes or signaling networks relating to melanoma metastasis have been established, there are no evidences regarding which antioxidant enzymes are involved. The inventors of the present invention first established that the level of PrxII is inversely correlated to the metastasis activities of melanoma cells. It was demonstrated that the SK-MEL melanoma cells having silenced PrxII expression had more superior proliferative and migratory activities compared to A375 and G361 cells highly expressing PrxII. While exogenous reexpression of PrxII in the SK-MEL cells reduced the proliferation and the migration of cells, PrxII knockdown in A375 and G361 cells increased both cell activities. At a molecular level, PrxII was regulated in the directions to reduce Src/REK activities, and the sustenance of E-cadherin and Y654 phosphorylation-independent β-catenin were tightened one by one in plasma membrane. The PrxII-deficient mouse melanoma B16F10 cells showed improved metastasis to lung in vivo. Interestingly, gliotoxin, a natural compound exhibiting a Prx-analogous activity, inhibited not only metastasis of PrxII-deficient melanoma cells to lung but also the proliferation and the migration. Accordingly, the present invention demonstrated that PrxII and its small molecule mimetic have a possibility as potential therapeutic agents to be used as an inhibitor for melanoma metastasis.
[0330] From the descriptions above, it will be apparent to those skilled in the art that the present invention may be executed in other specific manners without changing the technological ideas and essential features. Regarding this, it is to be understood that the examples described above are for illustrative purposes in all aspects, and are not limitative. The scope of the present invention needs to be interpreted to include all modifications or modified forms deduced from the meaning, the scope, and the equivalent concepts of Claims described below rather than detailed descriptions made above.