WO2013077709A1 - Pharmaceutical composition for preventing or treating restenosis comprising epidithiodioxopiperazine compounds or derivatives thereof, or pharmaceutically acceptable salts thereof - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating restenosis comprising epidithiodioxopiperazine (ETP) compounds or the derivatives thereof, or the pharmaceutically acceptable salts thereof. The epidithiodioxopiperazine (ETP) compounds or the derivatives thereof according to the present invention may imitate the function of particularly Prx II from among 2-Cys-Prx in injured blood vessels to suppress the migration and proliferation of PDGF-derived vascular smooth muscle cells to thus inhibit vascular intimal hyperplasia while promoting the migration and proliferation of VEGF-derived vascular endothelial cells to thus improve re-endothelialization. Thus, the epidithiodioxopiperazine compounds or the derivatives thereof according to the present invention may be valuably used as a pharmaceutical composition for preventing or treating restenosis.

Description

Epidithiodioxopiperazin compounds or derivatives thereof; Or a pharmaceutical composition for preventing or treating vascular restenosis including a pharmaceutically acceptable salt thereof

The present invention relates to epidithiodioxopiperazine (ETP) compounds or derivatives thereof; Or it relates to a pharmaceutical composition for the prevention or treatment of vascular restenosis comprising a pharmaceutically acceptable salt thereof.

Atherosclerosis is a condition in which fatty substances (plaque) containing cholesterol, phospholipids, and calcium accumulate in the vascular lining, causing inflammation, and the arteries lose their elasticity and become narrowed, which impedes blood supply or increases pressure, causing rupture or detachment. Say. In particular, the resulting narrowing of the arteries reduces blood supply, resulting in a lack of nutrients and oxygen, which is a major cause of vascular disease. The vascular diseases generally include heart diseases such as atherosclerosis, heart failure, hypertensive heart disease, arrhythmia, myocardial infarction and angina, and vascular diseases such as stroke and peripheral vascular disease.

Methods for overcoming such vascular stenosis include arterial graft surgery and percutaneous angioplasty, a method of expanding blood vessels without surgery. Such percutaneous angioplasty includes percutaneous vascular balloon dilation and percutaneous vascular stent insertion. Percutaneous vascular balloon dilatation is performed by inserting a guide conduit through the femoral or arm arteries, placing it at the entrance of the vessel with the lesion, and placing a catheter with a balloon attached to the end of the vessel through the inside of the conduit. By expanding the balloon, the plaque is compressed to expand the narrowed blood vessel, thereby improving blood flow. In addition, stent implantation is to place the wire mesh coated balloon on the stenosis, and then expand the balloon to coat the inner wall of blood vessels. Such stents have a lower incidence of restenosis than when only balloon dilatation is performed. It is used for the treatment of complications that occur when the balloon is expanded because it has the property of serving as a support for the balloon. Interventional interventions using such angioplasty are simpler than surgical procedures, reduce the risk of general anesthesia, and have a high success rate.

Angiography (restenosis) is a diagnosis by angiography that refers to a case where the stenosis of the endoscopic diameter is more than 50% after angiography. Although the incidence of restenosis decreased with the increase of the use of stents at a rate of about 70%, vascular restenosis still occurs in about 30% of patients who underwent angioplasty (balloon dilation and stent insertion). . Although the mechanism of this restenosis has not yet been precisely defined, growth factors and cytokines are secreted locally due to vascular endothelial cell damage during the procedure, which leads to the proliferation and migration of vascular smooth muscle, resulting in narrowing of the arterial lumen. It is known to lead to stenosis. Thus, the proliferation of smooth muscle cells has recently been a major clinical problem that limits the efficiency of angioplasty. Therefore, an improved stent releasing drug or substance that inhibits the proliferation of vascular smooth muscle cells was developed and used in clinical practice. However, drugs currently used for this purpose are highly toxic to prevent endothelial hyperplasia through a mechanism that kills vascular smooth muscle cells, and their toxicity leads to the death of not only smooth muscle cells but also endothelial cells. Revealing. Therefore, there is an urgent need to develop drugs that can selectively inhibit the growth of vascular smooth muscle cells and promote the repair of damaged endothelial cell layers.

Meanwhile, gliotoxin (GT), a representative substance having epidithiodioxopiperazine (ETP) structure, has been reported to have various pharmacological activities such as immunosuppression, anticancer and antiviral activity (D. M. Gardiner).et al., 2005,Microbiology, 151: 1021). In addition, the epidithiodioxopiperazine derivatives chitocin (chaetocin) and chitomin (chetomin) are also reported to have anticancer activity (Y. M. Leeet al., 2011,Hepatology53: 171; C. R. Ishamet al., 2007,Blood109: 2579; A. L. Kunget al., 2004,Cancer cell, 6: 33).

As such, numerous efforts have been made to find molecular targets in order to utilize epidithiodioxopiperazine compounds as therapeutic agents. As a result, glyotoxins inhibit several enzymes such as NF-κB, patesyltransferase and phagocytic NOX2 (H. L. Pahlet al., 1996,J. Exp. Med., 183: 1829; S. Nishidaet al., 2005,Infect. Immun.73: 235; D. M. Vigushinet al., 2004,Med. Oncol, 21:21) Chitocin has been shown to inhibit thioredoxin reductase or histone methyltransferase (J. D. Tibodeau).et al., 2009,Antioxid Redox Signal.11: 1097; D. Greineret al., 2005,Nat. Chem. Biol., 1: 143). Chitomin has also been shown to inhibit the interaction between hypoxia-inducible factor-1 (HIF-1) and p300 (A. L. Kunget al., 2004,Cancer cell, 6: 33). Although some cellular activities of the ETP compounds have been revealed, it is difficult to infer logical correlations between their chemical structures and cellular activities. In particular, the cellular function of the epidithiodioxopiperazine ring structure, which is the core structure nucleus of ETP compounds and their derivatives, is not known.

We first found that the dithiol group of epidithiodioxopiperazine (ETP) compounds or derivatives thereof exhibit 2-Cys-Prx-like activity in vitro and in vivo. In addition, the compounds or derivatives thereof inhibit PDGF-induced proliferation and migration in vascular smooth muscle cells and promote VEGF-induced proliferation and migration in vascular endothelial cells, thereby inhibiting endovascular thickening due to excessive proliferation of vascular smooth muscle cells. In addition, vascular endothelial layer recovery, that is, re-endothelization was improved, and finally, it was confirmed that the present invention can be usefully used as a pharmaceutical composition for preventing or treating vascular restenosis and completed the present invention.

The present invention is an epidithiodioxopiperazine compound represented by the formula (1) or derivatives thereof; Or to provide a pharmaceutical composition for preventing or treating vascular restenosis comprising a pharmaceutically acceptable salt thereof.

In addition, the present invention comprises the steps of determining whether the compound containing at least one epidithiodioxopiperazine ring represented by the formula (20) exhibits 2-Cys- peroxredoxin (2-Cys-Prx) activity; And if the NADPH oxidation reaction or H ₂ O ₂ reduction reaction occurs to provide a method for screening or preventing vascular restenosis comprising the step of determining the compound as a preventive or therapeutic agent for vascular restenosis.

The present invention also provides a drug delivery device for topical administration comprising the pharmaceutical composition for preventing or treating vascular restenosis.

Epidithiodioxopiperazin compounds or derivatives thereof of the present invention mimic the function of 2-Cys-Prx, especially PrxII isoform, in the damaged blood vessels, thereby inhibiting vascular endothelial thickening by inhibiting migration and proliferation of PDGF-induced vascular smooth muscle cells. On the other hand, the migration and proliferation of VEGF-induced vascular endothelial cells may be promoted to enhance re-endothelialization. Therefore, the epidithiodioxopiperazine compound or derivatives thereof of the present invention can be usefully used as a pharmaceutical composition for the prevention or treatment of vascular restenosis.

1 is hydrogen peroxide of compound (A-1) of formula (2), compound (A-2) having a dithiol group in the compound of formula (2) having a dithiol group, and compound (A-3) of formula (3) according to the present invention. Figure shows the catalytic activity for the reduction. (a) to (c) are graphs showing the results of A-1 to A-3, respectively.

2 is a diagram showing the catalytic activity of the hydrogen peroxide reduction of the ETP compound of the present invention. (a) to (c) show the hydrogen peroxide reducing power of gliotoxin, chitocin and chitomin, respectively. In vitro peroxidase reactions were performed by adding 25 μM of ETP compounds, respectively, in the presence of the coupling redox system indicated in the graph. (d) to (f) show typical Trx-dependent peroxidase activity of ETP compounds. The reaction was performed by adding the elements of the Trx / TR coupling system indicated in the graph. Representative response curves of three experiments are shown. (g) is a diagram showing the concentration-dependent peroxidase activity of an ETP compound. Three independent experiments were performed to show the initial velocity as the mean ± standard error. As a negative control group, bis (methylthio) glytoxin with reduced and disulfide crosslinking was used.

Figure 3 is a diagram showing the results of in vitro and in vivo cytotoxicity test of GT and chitocin. (a) and (d) are micrographs of human aortic smooth muscle cells (HASMCs) and human aortic epithelial cells (HAECs) treated with the concentrations of GT indicated in the graph. (b) and (c) are diagrams showing the survival rate according to the concentration when GT or chitocin was treated to HASMCs, respectively, and (e) and (f) are the diagrams showing the survival rate when GT or chitocin was treated to HAECs, respectively. to be. At this time, each ETP compound was pretreated with serum-depleted cells in a basal medium containing 0.5% FBS at a corresponding concentration for 2 hours, and then cells were recovered and stained with trypan blue to confirm survival rate. Survival is expressed as the percentage of unstained live cells relative to the total cell numbers. (g) is a diagram showing in vivo cytotoxicity of GT in rat carotid artery. GT was injected into the lumen of the balloon-damaged carotid artery at the indicated concentration and incubated for 30 minutes. DMSO was used as a control. Representative HE-stained images are shown and the ratio of intima to media measured from HE-stained carotid arterial samples is expressed as mean ± standard error (n = 7 per group). Cell death was confirmed (left) by TUNEL staining of paraffin sections of balloon-injured carotid artery vessels and calculated as the percentage of TUNEL-positive cells (right) per DAPI-positive cell.

Figure 4 is a compound (A-1) of formula (2), disulfide cross-linking is reduced in the compound of formula (2) having a dithiol group (A-2) and compound (A-3) of vascular smooth muscle cells (SMC) Is a graph showing the results of cytotoxicity test for% in living cells.

FIG. 5 shows vascular endothelial cells (EC) of Compound (A-1) of Formula 2, Compound (A-2) having a dithiol group, and Compound (A-3) having a dithiol group in the compound of Formula 2; Is a graph showing the results of cytotoxicity test for% in living cells.

6 shows the ability of reciprocal regulation of PDFT- and VEGF-dependent signaling by gliotoxin in HASMCs and HAECs injected with PrxII siRNA and pretreated for 2 hours with DMSO (control) or GT. (a) and (b) show intracellular hydrogen peroxide levels in HASMCs and HAECs, respectively, relative to DCF fluorescence images. The graph shows the relative DCF fluorescence intensity averaged from 50 to 80 cells as mean ± standard error (n = 3, * p <0.01, ** p <0.001).

Figure 7 shows the effect of GT and chitocin on NOX activity in HASMCs and HAECs. HASMCs (a) and HAECs (b) were depleted of serum in basal medium containing 0.5% FBS, pretreated with GT or chitocin at that concentration for 2 hours and treated with PDGF or VEGF for 10 minutes. The data indicate the percentage of peroxide amount in stimulated vs. unstimulated cells as mean ± standard error (n = 3, * p <0.05, ** p <0.001).

Figure 8 (a) is an immunoblot analysis of the effect of GT on PDGF-induced tyrosine phosphorylation in PrxII knocked down HASMCs, Figure 8 (b) is VEGF-induced tyrosine phosphorylation in PrxII knocked down HAECs The effect of GT is shown by immunoblot analysis. Total tyrosine phosphorylation (pTyr) was repeated three times with anti-phosphotyrosine antibody (4G10) to show a representative blot. In addition, activation of PDGFR-β and PLCγ1 in HASMCs and VEGFR and Erk in HAECs are shown as representative blots.

Figure 9 shows the effect of GT on growth factor-induced proliferation and migration of PrxII knocked down vascular cells. (a) and (b) show the effect of GT on proliferation and migration of HASMCs in response to PDGF, and (c) and (d), respectively, on proliferation and migration of HAECs in response to VEGF. The fold increase of each cell was expressed as mean ± standard error (n = 3, * p <0.05, ** p <0.001).

10 is a diagram showing the effect of the compound of formula (A-1) on growth factor-induced proliferation and migration of PrxII knocked down vascular cells. (a) and (b), respectively, for the proliferation and migration of HASMCs in response to PDGF, and (c) and (d) for the proliferation and migration of HAECs in response to VEGF, respectively (A-1) This is a diagram showing the influence of.

11 is a diagram showing the effect of the compound (A-2) having a dithiol group in the disulfide cross-linking is reduced in the compound of formula 2 on the growth factor-induced proliferation and migration of PrxII knocked down vascular cells. (a) and (b) show the effect of A-2 on proliferation and migration of HASMCs in response to PDGF, and (c) and (d) on the proliferation and migration of HAECs in response to VEGF, respectively. to be.

12 is a diagram showing the effect of compound (A-3) of formula 3 on the growth factor-induced proliferation and migration of PrxII knocked down vascular cells. (a) and (b) show the effects of A-3 on the proliferation and migration of HASMCs in response to PDGF, and (c) and (d) on the proliferation and migration of HAECs in response to VEGF, respectively. .

FIG. 13 is a diagram showing the effects of GT on endovascular hyperplasia and re-endothelialization in balloon-injured carotid arteries. (a) is a representative HE-staining image showing endovascular thickening with time during recovery in balloon-injured carotid artery. The ratio of intima to media measured from HE-stained carotid artery sections is expressed as mean ± standard error (n = 4 per group). (b) shows peroxidation of 2-Cys-Prx in carotid artery vessels induced by balloon injury, and immunohistochemistry was performed using anti-peroxide 2-Cys-Prx (Prx-SO _2/3 ) antibody. . Normal carotid artery vessels stained with hydrogen peroxide solution for 10 minutes as a positive control. Blocking antigen peptides eliminate immune-positive signals. Representative DAB-stained images from four independent samples are shown. (c) shows the results of immunoblot analysis for peroxidation of 2-Cys-Prx in cells using damaged carotid artery extract. For comparison, extracts from normal carotid artery vessels treated with hydrogen peroxide were loaded together.

14 is a diagram showing the effect of promoting endovascular thickening in balloon-damaged carotid artery by in vivo injection of PrxII siRNA. (a) shows representative HE-stained images of the effect of PrxII knockdown on endovascular thickening induced by balloon injury. The ratio of intima to media measured from HE-stained carotid artery sections is expressed as mean ± standard error (n = 7 per group, * p <0.01). (b) is the Western blotting result of rat PrxII of the damaged carotid artery extract. (c) is the result of immunofluorescence staining of rat PrxII on damaged carotid artery slices. This represents intrinsic PrxII knockdown by siRNA injection in balloon-damaged carotid arteries.

15 shows the effect of GT on endovascular thickening and endothelial regeneration in balloon-damaged rat carotid arteries. (a) is a diagram showing the endometrium of balloon-damaged rat carotid artery after control and GT treatment. Representative HE-stained images obtained by incubation for 30 minutes with DMSO and GT treatment, respectively, are shown. Arrows indicate enlarged endovascular layers. Intra-membrane ratios measured from HE-stained carotid artery samples are expressed as mean ± standard error (n = 9 per group, * p <0.01). (b) shows the results of immunofluorescence staining of smooth muscle α-actin (SMA) and CD31 (n = 3). Normal carotid artery vessels show a typical endothelial monolayer (arrow). DAPI staining revealed nuclei.

FIG. 16 shows HE-stained images following treatment with Bis- (methylthio) GT alone or GT + TR inhibitor (DNCB, auranofin) for endovascular thickening induced by balloon injury.

Figure 17 shows the immunohistochemical staining results confirming the effect of GT on 2-Cys-Prx peroxidation.

FIG. 18 shows the effect of GT on vascular permeability and lumen surface condition in balloon-damaged rat carotid arteries. (a) shows the extent of Evans Blue influx in control and GT treated balloon-damaged rat carotid arteries. Representative images are shown on the left side, and the graph on the right side shows the absorbance at 620 nm as the average ± standard error by extracting Evans blue introduced into the blood vessel (n = 7 per group, * p <0.01). (b) shows the results of scanning electron microscopy of the control and GT treated balloon-damaged rat carotid vascular lumen surfaces. Three independent experiments were performed and representative zoom-in images were shown.

19 is a diagram showing the effect of chitocin treatment on cell proliferation and migration. PrxII knocked down HASMCs ((a) and (b)) and HAECs ((c) and (d)) were pretreated with chitocin for 2 hours and stimulated with PDFG or VEGF for 24 hours. The fold increase is shown as mean ± standard error (n = 3, * p <0.01, ** p <0.005, #p <0.001).

20 is a diagram showing the effects of chitocin and chitomin on endovascular thickening induced by balloon injury. Balloon-damaged rat carotid arteries were incubated with chitocin or chitomin for 30 minutes. DMSO was used as a control and representative HE-stained images ((a) and (b)) were shown. Arrows indicate thickened endovascular layers. Intra-membrane ratios measured from HE-stained carotid artery samples are expressed as mean ± standard error (n = 9 per group, * p <0.01). (c) and (d) show the results of immunofluorescence staining of smooth muscle α-actin (SMA) and CD31. Arrows indicate endothelial faults.

21 shows the effect of various antioxidant compounds on VEGF signaling in HAECs. PrxII siRNA was injected into VEC and pretreated for 2 hours with increased concentrations of the indicated compounds (N-acetylcysteine; NAC, butylated hydroxyanisole; BHA). Cells were treated with VEGF for 10 minutes and analyzed by immunoblotting.

22 is a diagram showing Western blot results ((a), (b)) and HE-staining results (C) confirming the effects of GT on PrxII deficient vascular cells and mice.

23 is a diagram showing an NMR data result of Compound (A-1) of Chemical Formula 2.

24 is a diagram showing a result of NMR data of compound (A-1) of formula (2).

FIG. 25 is a diagram showing an NMR data result of Compound (A-1) of Formula 2.

26 is a diagram showing an NMR data result of Compound (A-3) of Chemical Formula 3.

27 is a diagram showing a NMR data result of a compound (A-2) having a dithiol group after disulfide crosslinking in a compound (A-1) represented by the formula (2).

28 is a diagram showing an NMR data result of a compound (A-2) having a dithiol group after disulfide crosslinking in a compound (A-1) represented by the formula (2).

The present invention is an epidithiodioxopiperazine compound represented by the following formula (1) or derivatives thereof; Or it provides a pharmaceutical composition for preventing or treating vascular restenosis comprising a pharmaceutically acceptable salt thereof:

[Formula 1]

The derivative of the epidithiodioxopiperazine compound refers to a compound containing an epidithiodioxopiperazine ring as the parent nucleus showing activity. The derivative may be a compound substituted with a variety of substituents of the kind known in the art to the NH group or the CH group in the compound ring represented by the formula (1), or may include a compound having a structure in which a number of compounds apparent to those skilled in the art combined It is not limited thereto. Modification and substitution of the structure of the compound of Formula 1 may be easily performed by those skilled in the art, for example, by the following procedure.

Preferably, the derivative of epidithiodioxopiperazine may be any one of the compounds represented by the following Chemical Formulas 2 to 19.

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

[Formula 8]

[Formula 9]

[Formula 10]

[Formula 11]

[Formula 12]

[Formula 13]

[Formula 14]

[Formula 15]

[Formula 16]

[Formula 17]

[Formula 18]

[Formula 19]

The compound of Formula 2 and compound of Formula 3 may be synthesized and used by those skilled in the art by referring to known methods. The present inventors synthesized the compound of Formula 2 and the compound of Formula 3 in one specific embodiment and the specific synthesis method was prepared by the method described in Example 1.

The compound of Formula 4 is a representative ETP compound called gliotoxin (GT), Aspergillus fumigatus , Trichoderma virens , Penicillium spp. Or Candida albicans and the like, its culture, metabolites, or secondary metabolites can be isolated from Kirby and Robins, 1980, The Biosynthesis of Mycotoxins , New York: Academic Press; Shah and Larsen, 1991, Mycopathologia , 116: 203-208.

The compound of Formula 5 is an ETP compound called sirodesmin , a bacterium such as Leptosphaeria maculans or Sirodesmium diversum , a culture solution, a metabolite, or a secondary metabolism thereof. Can be isolated from a sieve [Curtis et al ., 1977, J. Chem. Soc. Perkin Trans . 1 , 180-189; Ferezou et al ., 1977, Nouv. J. Chim. , 1: 327-334.

The compound of Chemical Formula 6 is an ETP compound called hyalodendrin, and may be isolated from bacteria such as Hyalodendron sp. , Its culture, metabolites, or secondary metabolites [Stillwell et al . , 1974, Can. J. Microbiol ., 20: 759-764.

The compound of Formula 7 is an ETP compound called sporidesmin A and may be isolated from bacteria such as Pithomyces chartarum , cultures, metabolites, or secondary metabolites thereof. and Robins, 1980, The Biosynthesis of Mycotoxins , New York: Academic Press.

The compound of Chemical Formula 8 may be separated from Chaetomium globosum by an ETP compound called chetomin (Sekita et al ., 1981, Can. J. Microbiol ., 27: 766-772.

The compound of formula 9 may be isolated from the chitoium spp. As an ETP compound called chitocin (chaetocin) [Sekita et al ., 1981, Can. J. Microbiol ., 27: 766-772.

The compound of Formula 10 may be isolated from Verticillium spp. Or Penicillium sp. As an ETP compound called verticillins [Byeng et al ., 1999, Nat. Prod. Lett ., 13: 213-222; Joshi et al ., 1999, J. Nat. Prod. 62: 730-733; Kirby and Robins, 1980, The Biosynthesis of Mycotoxins , New York: Sekita et al ., 1981, Can. J. Microbiol ., 27: 766-772.

The compound of Formula 11 may be isolated from Leptosphaetia sp. As an ETP compound called leptosin (Takahashi et al ., 1994, J. Antibiot ., 47: 1242-1249).

The compound of Formula 12 may be isolated from Aspergillus spp. As an ETP compound called emesttrin (Seya et al ., 1986, Chem. Pharm. Bull ., 34: 2411-2416.

The compound of Formula 13 may be isolated from Xanthoparmelia scabrosa with an ETP compound called scabrosin (Ernst-Russell et al ., 1999, Aust. J. Chem ., 52: 279-283; Moerman et al ., 2003, Toxicol. Appl. Pharmacol ., 190: 232-240.

The compound of Formula 14 may be isolated from Aspergillus silvaticus as an ETP compound called dithiosilvatin (Kawahara et al ., 1987, J. Chem. Soc. Perkin Trans . 1 , 2099-2101.

The compound of Formula 15 may be separated from Sterum hirsutum , Epicoccum purpurascens or Epicoccum nigrum with an ETP compound called epicorazine [ epicorazine ] [ Deffieux et al ., 1977, Acta Christallogr., B33: 1474-1478; Kleinwachter et al ., 2001, J. Antibiot ., 54: 521-525.

The compound of Formula 16 may be isolated from Arachniotus aureus or Aspergillus terreus with an ETP compound called Aranotin (Neuss et al ., 1968, Antimicrob). . Agents Chemother ., 8: 213-219.

The compound of Formula 17 may be isolated from Aspergillus heterothallicus as an ETP compound called emethallicin (Kawahara et al ., 1989, Chem. Pharm. Bull ., 37: 2592-2595.

The compound of Formula 18 may be isolated from Verticillium spp. With an ETP compound called verticillins (Byeng et al ., 1999, Nat. Prod. Lett ., 13: 213-222; Joshi et al ., 1999, J. Nat. Prod. 62: 730-733; Kirby and Robins, 1980, The Biosynthesis of Mycotoxins , New York: Sekita et al ., 1981, Can. J. Microbiol ., 27: 766-772.

The compound of Formula 19 may be isolated from Gliocladium catenulatum [Byeng et al ., 1999, Nat. Prod. Lett ., 13: 213-222; Joshi et al ., 1999, J. Nat. Prod. 62: 730-733; Kirby and Robins, 1980, The Biosynthesis of Mycotoxins , New York: Sekita et al ., 1981, Can. J. Microbiol ., 27: 766-772.

The epidithiodioxopiperazine compounds or derivatives thereof are isolated from natural sources, obtained from natural sources, prepared by chemical modification, or chemically synthesized by one of ordinary skill in the art with reference to known production methods, or commercially Can be purchased and used. Preferably, the epidithiodioxopiperazine compound or derivatives thereof are used separately from the bacteria, culture medium or metabolite thereof according to methods known in the art or synthesized by the production method described in the Examples of the present invention. Can be used.

A feature of the epidithiodioxopiperazine compounds or derivatives thereof of the present invention lies in the presence of intermolecular disulfide bridges. This characteristic chemical structure is involved in cellular uptake, which, once introduced into a cell, accumulates at a much higher intracellular concentration than expected and is confined within the cell [P. H. Bernardoet al.,J. Biol. Chem., 2003, 278: 46549. For this reason, the epidithiodioxopiperazine compounds or derivatives thereof according to the present invention can effectively prevent or treat vascular restenosis despite being administered at very low concentrations to avoid inherent cytotoxicity.

[Formula 20]

According to a specific embodiment, the compound of Formula 2, the compound of Formula 3, glyotoxin, chitosine and chitomin showed hydrogen peroxide-reducing activity in the presence of the Trx / TR system, while inhibiting the proliferation and migration of vascular smooth muscle cells. At the same time, it was confirmed that there is an activity that promotes the proliferation and migration of vascular endothelial cells. However, derivatives which methylated thiol groups exposed by reduction of disulfide bridges did not show hydrogen peroxide-reducing activity, nor did they exhibit cell function regulation. In the case of a compound having a dithiol group reduced by disulfide crosslinking, hydrogen peroxide-reducing activity was shown, but the cell function control action was not shown. This is because compounds with a reduced form of disulfide bridges are not absorbed into cells, whereby the hydrogen peroxide-reducing activity of the compounds or derivatives according to the invention is due to the presence of disulfide bonds, especially in the epidithiodioxopiperazin ring in the compound. It could be seen that.

Epidithiodioxopiperazine compounds or derivatives thereof of the present invention may be used in the form of pharmaceutically acceptable salts. In addition, the compounds of the present invention or derivatives thereof may be used alone or in combination or in combination with other pharmaceutically active compounds.

As used herein, the term "pharmaceutically acceptable salts" refers to all salts that retain the desired biological and / or physiological activities of the compound or derivatives and exhibit undesirable minimal toxicological effects. In the present invention, any type of salt can be used without limitation as long as it maintains a diketopiperazine ring containing a disulfide bridge in the molecule. As salts are acid addition salts formed with pharmaceutically acceptable free acids. Acid addition salts are prepared by conventional methods, for example by dissolving a compound in an excess of aqueous acid solution and precipitating the salt using a water miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. Equivalent molar amounts of the compound and acid or alcohol (eg, glycol monomethyl ether) in water can be heated and the mixture can then be evaporated to dryness or the precipitated salts can be suction filtered. In this case, inorganic acids and organic acids may be used as the free acid, and hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, tartaric acid, and the like may be used as the inorganic acid, and methane sulfonic acid, p-toluene sulfonic acid, acetic acid, and triacid may be used as the organic acid. Fruoroacetic acid, maleic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid ( glycollic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, etc. Can be used, but not limited to these.

Bases can also be used to make pharmaceutically acceptable metal salts. Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving a compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and then evaporating and drying the filtrate. In this case, as the metal salt, it is particularly suitable to prepare sodium, potassium or calcium salt, but is not limited thereto. Corresponding silver salts may also be obtained by reacting alkali or alkaline earth metal salts with a suitable silver salt (eg, silver nitrate).

Pharmaceutically acceptable salts of epidithiodioxopiperazin compounds or derivatives thereof according to the invention include all salts of acidic or basic groups which may be present, unless otherwise indicated. For example, pharmaceutically acceptable salts may include sodium, calcium and potassium salts of the hydroxy group, and other pharmaceutically acceptable salts of the amino group include hydrobromide, sulfate, hydrogen sulphate, phosphate, hydrogen phosphate, Hydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p -toluenesulfonate (tosylate) salts; and the like through the methods for preparing salts known in the art. Can be prepared.

The pharmaceutical composition according to the present invention can be used without limitation in the prevention or treatment of vascular restenosis caused by vascular transplantation, vascular cutting, arteriosclerosis, intravascular fat accumulation, hypertension, vascular inflammation, angioplasty. The reason for vascular restenosis does not become clear, but it is a growth factor secreted from surrounding cells as a mechanism for restoring vascular endothelial injury caused by vascular injury or a device inserted during angioplasty for various reasons. Or cytokines are known to cause vascular smooth muscle cells to migrate abnormally and proliferate to cause endometrial thickening. The blood vessels include, but are not limited to, carotid arteries, coronary arteries, peripheral arteries, renal arteries, and the like.

Epidithiodioxopiperazine compounds represented by the following Chemical Formula 1 according to the present invention or derivatives thereof; Alternatively, the pharmaceutical composition comprising a pharmaceutically acceptable salt thereof is characterized by inhibiting the proliferation or migration of vascular smooth muscle cells and at the same time promoting the proliferation or migration of vascular endothelial cells.

The epidithiodioxopiperazine compounds or derivatives thereof have the characteristics of mimicking 2-Cys-Prx activity. As used herein, the term "PrxII (peroxiredoxin II)" refers to one of the peroxidase 2-Cys Prx to reduce the intracellular hydrogen peroxide, PDGFRβ by reducing the hydrogen peroxide produced by PDGF in vascular smooth muscle cells It inhibits cell signaling amplification through inhibition of phosphorylation in a site-specific manner at -PLCγ1. Through this mechanism, the PrxII has the activity of inhibiting the migration and proliferation of smooth muscle cells in the damaged blood vessels and inhibiting the endovascular thickening (MH Choi et al, 2005, Nature, 435: 347-353). In vascular endothelial cells, PrxII protects VEGFR2 from oxidative inactivation and activates VEGF-induced signaling [DH Kang et al. , 2011, Mol Cell 44: 545-558. The present inventors confirmed that the epidithiodioxopiperazine compound or derivatives thereof according to the present invention can substitute for the cellular function of PrxII by using PrxII siRNA-injected cells and vascular damage animal models.

On the other hand, the antioxidant activity of the epidithiodioxopiperazine compound or derivatives thereof that mimic the 2-Cys-Prx is different from the simple antioxidant activity, according to a specific embodiment of the present invention, N-acetylcystine, a non-specific antioxidant compound And butylated hydroxyanisole could not achieve the same activity.

The inventors have found for the first time that the epidithiodioxopiperazine compounds or derivatives thereof can mimic intracellular 2-Cys-Prx activity, so that epidithiodioxopiperazine compounds known to be highly toxic to PDGF- It has been shown that it is possible to inhibit the migration and proliferation of induced vascular smooth muscle cells while promoting the migration and proliferation of VEGF-induced vascular endothelial cells.

According to an embodiment of the present invention, the compound of formula 2, the compound of formula 3, gliotoxin, chitocin and chitomin among the compounds according to the present invention inhibits PDGF-induced migration and proliferation of vascular smooth muscle cells While preventing the enlargement, it was confirmed that VEGF-induced migration and proliferation of vascular endothelial cells enhances re-endotheliality and suppresses vascular restenosis without side effects of thinning blood vessels. In addition, the recovered endothelial was confirmed through specific examples that the functional and structurally intact endothelial.

The pharmaceutical composition according to the present invention is preferably provided at a concentration lower than the concentration causing the death of vascular cells. As used herein, the term "concentration lower than the concentration causing vascular cell death" is a concentration that does not cause death of vascular smooth muscle cells and vascular endothelial cells in vivo, and may be determined by a method known in the art. In vitro MTT [3- (4,5-Dimethylthiazol-2-yl) -2,5-Diphenyltetrazolium bromide] analysis, tunnel analysis, Trypan blue analysis, LDH released into culture medium (lactate dehydrogenase) measurement, FACS (fluorescence-activated cell sorter) analysis, CCK-8 (cholecystokinin octapeptide) analysis by using any one method selected from the group consisting of vascular cell death after determining the concentration, and then Levels at the site of administration in human patients may be determined and provided according to methods known in the art to achieve the concentrations determined above.

Preferably an epidithiodioxopiperazine compound or derivatives thereof according to the present invention; Or a pharmaceutical composition comprising a pharmaceutically acceptable salt thereof may be provided such that an effective intracellular concentration is about 100 pmoles / 10 ⁵ cell concentration after administration.

Specifically, after treating vascular smooth muscle cells and vascular endothelial cells with 25 nM of glycotoxin and then purifying these methanol extracts by HPLC, the concentration of intracellular glycotoxin was 53 ± 2.7 pmoles / 10 ⁵ in vascular smooth muscle cells. , 63.8 ± 9.6 pmoles / 10 ⁵ in vascular endothelial cells. Considering the average volume per cell, the actual concentration of glycotoxin per cell is about 100 μM, which is comparable to about 10 to 100 μM, the average cell concentration of 2-Cys-Prxs evaluated in various cell lines.

According to an embodiment of the present invention, treatment of glytoxin at low nanomolar concentrations (50 nM for smooth muscle cells and 25 nM for endothelial cells) without cytotoxicity results in the migration of vascular smooth muscle cells by PDGF in vitro and It inhibited proliferation and promoted VEGF-induced migration and proliferation of vascular endothelial cells (FIG. 9). Endothelial thickening to concentrations of about 1 to 2 microM in rat models injured carotid arteries damaged by balloon catheters. Was inhibited and induced endothelial recovery (FIG. 15). In addition, scanning electron microscopy and vascular permeability test confirmed that during gliotoxin treatment, the endothelial layer was uniformly remodeled in the damaged carotid artery and its function was also restored (FIG. 18). In addition, chitocin and chitomin were also found to have the effect of controlling cell migration and proliferation in the same manner, blocking endometrial thickening and promoting endothelial remodeling in damaged blood vessels (FIGS. 19 and 20).

The composition according to the invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. The composition is sterile or sterile, and such solutions are sterile or sterile, and when applied to water, buffers, isotonic agents or animals or humans, do not cause allergies or other harmful reactions known to those skilled in the art. It may include other ingredients.

As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial agents, antifungal and isotonic agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. In addition to conventional media or agents incompatible with the active ingredient, their use in therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

The composition may be prepared in a formulation such as a liquid, an emulsion, a suspension or a cream, and may be used parenterally. The amount of the composition can be used in a conventional amount for preventing vascular restenosis, it is preferable to be applied differently depending on the age, sex, condition of the patient, the absorption of the active ingredient in the body, the inactivation rate and the drug used in combination.

The present invention also provides a method for preventing or treating vascular restenosis, comprising administering the pharmaceutical composition to a subject in need thereof.

In the present invention, the term "prevention" refers to any action that inhibits vascular restenosis or delays the onset by administration of the pharmaceutical composition according to the present invention, and "treatment" means vascular by administration of the pharmaceutical composition. Any action that improves or beneficially changes the symptoms caused by restenosis.

In the present invention, the term "individual" means all animals including humans having or may develop vascular restenosis, and the vascular restenosis can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to an individual. . In addition, the pharmaceutical composition of the present invention may be administered in parallel with known therapeutic agents for vascular restenosis.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term “pharmaceutically effective amount” means an amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to the medical treatment and not causing side effects, wherein the effective dose level is the sex, age and weight of the patient. Factors, including health conditions, severity of disease, drug activity, drug sensitivity, method of administration, time of administration, route and route of administration, duration of treatment, combination or drug used simultaneously, and other well-known factors in the medical field. Thus it can be easily determined by those skilled in the art.

Known therapeutic agents that can be used in combination with the pharmaceutical composition of the present invention may include paclitaxel or sirolimus, and the pharmaceutically effective amount of the known therapeutic agents is known in the art and is a symptom. The amount can be adjusted by the treating physician in consideration of various conditions such as the degree of coexistence and the co-administration with the composition of the present invention. Thus, by combining and administering a known therapeutic agent, not only the side effect of a known therapeutic agent can be reduced by the composition of this invention, but also a synergistic therapeutic effect can be anticipated. These known therapeutic agents may optionally be administered in combination or simultaneously, or at timed intervals with the compositions of the invention.

The term "administration" of the present invention means introducing a predetermined substance into a patient in an appropriate manner, and the route of administration of the composition may be administered via any general route as long as it can reach the target tissue. The method of administration is not limited thereto, but parenteral administration is preferred, and more preferably, it can be topically administered to the lesion, and a double balloon catheter, a dispatch or a microporous balloon may be used for topical drug administration. In particular, stents or sustained release microparticles can be used to deliver drugs for long periods of time. Most preferably, the compositions of the present invention can be applied directly to the stenosis site by application in a stent.

In addition, the present invention is the epidithiodioxopiperazine (ETP) compound containing one or more epidithiodioxopiperazine ring represented by the formula (20) is 2-Cys- peroxredoxin (2-Cys -Prx) confirming whether or not the activity; And it provides a method of producing a preventive or therapeutic vascular restenosis comprising the step of mass-producing a compound identified in the step to exhibit 2-Cys-peroxredoxin (2-Cys-Prx) activity.

In addition, the present invention (a) to determine whether the compound containing at least one epidithiodioxopiperazine ring represented by the formula (20) exhibits 2-Cys- peroxredoxin (2-Cys-Prx) activity Doing; And (b) when the NADPH oxidation or H ₂ O ₂ reduction reaction provides a screening method for preventing or treating vascular restenosis comprising the step of determining the compound as a preventive or therapeutic agent for vascular restenosis.

Determination of whether or not the 2-Cys- peroxredoxin (2-Cys-Prx) activity in the step (a), the compound containing at least one epidithiodioxopiperazine ring represented by the formula (20) Mixing with oredoxin (Trx), thioredoxin reductase (TR), NADPH, buffer and H ₂ O ₂ to determine whether NADPH oxidation or H ₂ O ₂ reduction occurs; And when NADPH oxidation or H ₂ O ₂ reduction reaction may be carried out by the step of determining that it exhibits 2-Cys- peroxredoxin (2-Cys-Prx) activity.

Specifically, the candidate compound containing epidithiodioxopiperazin ring is first reacted with Trx, TR, NADPH, and EDTA in a buffer solution, mixed with H ₂ O ₂ to perform an oxidation reaction to prepare an experimental group, and a sample Except that the same reaction can be carried out to prepare a control group and then monitored by comparing the decrease in absorbance at each 340 nm. In this case, the control group exhibits a constant absorbance, and when a candidate compound exhibiting 2-Cys-Prx activity is included, a decrease in absorbance with time is observed, and activity is proportional to the degree of decrease in absorbance. Therefore, the candidate compound including the epidithiodioxopiperazine ring, in which a decrease in absorbance is observed by performing the above steps, may be determined as a substance having vascular restenosis prevention or therapeutic activity. The Trx and TR may be derived from yeast, human or rat, preferably may be derived from yeast. Confirmation of 2-Cys-peroxredoxin (2-Cys-Prx) activity can be performed according to the method described in Korean Patent Publication No. 10-2006-0020140, the contents of which are disclosed herein Included by reference.

The present invention also provides a drug delivery device for topical administration comprising the pharmaceutical composition for preventing or treating vascular restenosis. The topical drug delivery device may include a double balloon catheter, a dispatch, a micro-balloon, a stent, and the like, but is not limited thereto, and preferably, may be a stent.

In the present invention, the term "stent" refers to a general apparatus for endoluminal application, for example, in a blood vessel, as mentioned above, and the disease occurs in a place where blood flow should be smooth. When the flow is disturbed, it means a cylindrical medical material that is inserted into the narrowed or clogged blood vessel area under X-ray fluoroscopy without surgically open surgery. For example, vascular stents are described by Eric J Topol in "Textbook of Interventional Cardiology", Saunders Company, 1994. Preferably, the sustained release drug stent.

As the method for coating the pharmaceutical composition of the present invention on the stent, a conventional coating method known to those skilled in the art may be applied, and for example, dip-coated and There is a method of coating with a polymer (polymer coated), the dip coating method is the simplest coating method, it is easy to observe the biological effect of the drug only because the pharmaceutical composition is coated, but is not limited thereto. Preferably, the stent of the present invention may be prepared by mixing the composition with a polymeric material in a drug-release stent so as to slowly release the composition according to the present invention. Polymeric materials that can be used in drug release stents are well known in the art and include, for example, polyurethane, polyethylene terephthalate, PLLA-poly-glycolic acid copolymer (PLGA), polycaprolactone, poly- (hydr) Oxybutyrate / hydroxy valerate) copolymer, polyvinylpyrrolidone, polytetrafluoroethylene, poly (2-hydroxyethylmethacrylate), poly (etheruretan urea), silicone, acrylic, epoxide, Polyesters, urethanes, palene, polyphosphazine polymers, fluoropolymers, polyamides, polyolefins and mixtures thereof, but are not limited thereto.

The stent is composed of or comprises one or more substances selected from polysaccharides, heparin, gelatin, collagen, alginate, hyaluronic acid, arginine acid, carrageenan, chondroitin, pectin, chitosan and derivatives thereof and copolymers thereof. It may be further coated with a layer. Suitably, these materials can be incorporated into a biosynthetic top coat, as described in US 2006/0083772. Methods for forming stents from mixtures of polymers and drug compounds are described in Blindt et al ., 1999, Int. J. Artif. Organs , 22: 843-853.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention more specifically, but the scope of the present invention is not limited by these examples.

Experimental Method

Example 1 Synthesis of Epidithiodioxopiperazin Compound

1-1. Preparation of the compound of formula 2 (5,7-dimethyl-2,3-dithia-5,7-diazabicyclo [2.2.2] -octane-6,8-dione)

Compound of Formula 2 was prepared through the following scheme.

A. 3,6-dibromo-1,4-dimethylpiperazine-2,5-dione (2)

A mixture of salcosine anhydride (1) (500 mg, 3.5 mmol), N-bromosuccinimide (1.87 g, 10.5 mmol), and benzoyl peroxide (85 mg, 0.35 mmol) in carbon tetrachloride (50 mL) was added for 2 hours. Heated to reflux and cooled before succinimide was filtered and washed with carbon tetrachloride. The filtrates were combined, dried over magnesium sulfate and filtered again. The residue was evaporated in vacuo to give 3,6-dibromo-1,4-dimethylpiperazine-2,5-dione (2) (crude: 900 mg, 82%). ¹ HNMR (400 MHz, CDCl ₃ ): δ ppm 6.04 (s, 2H), 3.15 (s, 6H).

B. 1,4-Dimethyl-3,6-dioxopiperazin-2,5-diyl-diethanethioate (2006.01)

3,6-Dibromo-1,4-dimethylpiperazine-2,5-dione (2) (crude 900 mg, 3.0 mmol) was dissolved in dichloromethane (100 mL) and potassium thioacetate (1.03 g, 9.0 mmol). The mixture was stirred at rt overnight and the obtained precipitates were filtered and washed with dichloromethane. The filtrates were combined and the residue was evaporated in vacuo. Silicagel column chromatography (DCM: EA = 5: 1) gave 1,4-dimethyl-3,6-dioxopiperazin-2,5-diyl-diethanethioate (3) as a white solid (200). Mg, 23%). ¹ H NMR (400 MHz, CDCl 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)

Ethanol hydrochloric acid solution (prepared by adding 1.5 ml of acetyl chloride to 7 ml of ethanol) was added dropwise to a stirred solution of dithioacetate (195 mg, 0.67 mmol) in ethanol (20 ml). The resulting solution was then heated to reflux for 1.5 hours. After cooling, the volatiles were removed under reduced pressure and the residue was purified by recrystallization (EA / Hex) to convert 3,6-dimercapto-1,4-dimethylpiperazine-2,5-dione (4) into a light yellow solid. Obtained (90 mg, 65%). ¹ H NMR (400 MHz, CDCl ₃ ): δ ppm 5.00 (s, 2H), 3.09 (s, 6H)

D. 5,7-Dimethyl-2,3-dithio-5,7-diazabicyclo [2,2,2] octane-6,8-dione (5)

A solution of iodine (63 mg, 0.246 mmol) in 10 ml of chloroform was added to 3,6-dimercapto-1,4-dimethylpiperazine-2,5-dione (50 mg, 0.242 mmol) in 10 ml of chloroform at room temperature. Was added. It was left to stir for 1 hour and saturated NaHCO ₃ solution containing sodium thiosulfate was poured and stirred until discolored by iodine. The organic layer was separated and the aqueous layer was extracted twice with dichloromethane and dried over magnesium sulfate. Filtration and concentration in vacuo gave a residue which was recrystallized from ethanol to give a light yellow solid 5,7-dimethyl-2,3-dithio-5,7-diazabicyclo [2,2,2] octane-6 , 8-dione (5) was obtained. ¹ H NMR (400 MHz, CDCl ₃ ): δ ppm 5.21 (s, 2H), 3.12 (s, 6H). NMR data of the obtained final product is shown in Figs.

1-2. Preparation of compound of formula 3 (1,5,7-trimethyl-2,3-dithia-5,7-diazabicyclo [2.2.2] octane-6,8-dione)

Compound of Formula 3 was prepared through the following scheme.

A. Methyl 2- (2-chloroacetamido) propanoate (2)

Alanine methyl ester hydrochloride (2 g, 14.3 mmol) was dissolved in 6 mL of water. The aqueous solution was cooled to 0 ° C. and mixed with sodium hydrogen carbonate (2.8 g, 33.6 mmol). To the reaction was added dropwise a solution of chloroacetyl chloride (1.67 mL, 20.9 mmol) in 5 mL of toluene and stirred for 3 hours. After phase separation, the aqueous layer was further extracted with toluene. The combined organic layers were removed with solvent to obtain crude methyl 2- (2-chloroacetamido) propanoate (2). ¹ H NMR (400 MHz, CDCl 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)

Methyl 2- (2-chloroacetamido) propanoate (2) was dissolved in 15 mL of ethanol and mixed with 6.1 mL of 30% aqueous ammonia solution. The reaction was stirred at 70 ° C. for 5 hours, cooled and the precipitated solid was filtered off. The mother liquor was evaporated to dryness and the residue was dissolved in a small amount of water, the remaining precipitate was combined by filtration and dried in vacuo to afford 3-methylpiperazine-2,5-dione (3) as a white solid (420 mg, yield 23%). ¹ H 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)

To a solution of 3-methylpiperazine-2,5-dione (3) 420 mg, 3.27 mmol) in 20 ml of dry dimethylformamide was slowly added NaH (60% in oil, 400 mg, 9.83 mmol) at 0 ° C. 1 ml of methyl iodide was added little by little with stirring and the mixture was stirred at room temperature for 4 hours. The solvent was evaporated and the remaining residue was extracted with dichloromethane and water. The aqueous layer was washed twice with dichloromethane and the combined organic layers were dried over magnesium sulfate and filtered. The residue was evaporated in vacuo and silica gel column chromatography (DCM: MeOH = 20: 1-9: 1) gave 1,3,4-trimethylpiperazine-2,5-dione (4) as a colorless solid (440). Mg, yield 86%). ¹ H NMR (400 MHz, CDCl 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)

A suspension of elemental sulfur (1.64 g, 51.2 mmol) in 20 mL of THF was added dropwise every 2 minutes to NaHMDS (1.0 M in THF, 19.2 mL) under argon. During the addition, insoluble yellow S ₈ quickly changed color, from initially dark green to dark orange and finally to bright orange solution. The solution was stirred for another 1 min and 1,3,4-trimethylpiperazine-2,5-dione (4) (1 g, 6.4 mmol) in 20 mL of THF was added and the resulting mixture was stirred at 25 ° C. for 0.5 h. . The reaction was quenched with saturated aqueous NH ₄ Cl solution and extracted twice with ethyl acetate. The combined organic layers were dried over magnesium sulfate, filtered and concentrated to give a residue. 6,7,9-trimethyl-2,3,4,5-tetrathia-7,9-diazabicyclo [4.2.2] decane-8 via silica gel column chromatography (EA: Hex = 1: 1) , 10-dione (5) was obtained (250 mg, yield: 14%). ¹ H NMR (400 MHz, CDCl ₃ ): δ ppm 5.13 (s, 1H), 3.06 (d, 6H), 2.00 (s, 3H).

E. 1,5,7-trimethyl-2,3-dithia-5,7-diazabicyclo [2.2.2] octane-6,8-dione (6)

Step 1: 6,7,9-trimethyl-2,3,4,5-tetrathia-7,9-diazabicyclo [4.2.2] decane-8,10-dione (5) in 20 mL methanol 250 mg, 0.885 mmol) was added to NaBH ₄ (167 mg, 4.4 mmol) at 0 ° C. under argon. The resulting mixture was stirred for 45 minutes to reach the appropriate temperature. Solvent was evaporated and the residue was extracted with EA and saturated NH ₄ Cl solution. The aqueous layer was washed twice with EA and the combined organic layers were dried over magnesium sulfate and filtered. The residue was dried in vacuo.

Step 2: Iodine (249 mg, 0.885 mmol) in 20 mL of chloroform was added to a solution of the dithiol compound in 30 mL of chloroform at room temperature. The solution was left stirring for 1 hour and the mixture was stirred until a saturated NaHCO ₃ solution containing sodium thiosulfate was poured out and discolored by iodine. The organic layer was separated and the aqueous layer was extracted twice with dichloromethane. The combined organic layers were dried over magnesium sulfate, filtered and concentrated in vacuo to give a residue. 1,5,7-trimethyl-2,3-dithia-5,7-diazabicyclo [2.2.2] octane-6,8-dione via silica gel column chromatography (EA: Hex = 1: 1) (6) was obtained as a light yellow solid (24 mg, 12%). ¹ H NMR (400 MHz, CDCl ₃ ): δ ppm 5.29 (s, 1H), 3.14 (s, 3H), 3.03 (s, 1H), 1.98 (s, 3H). NMR data of the obtained final product is shown in FIG.

Comparative Example 1: Preparation of a compound in which a dithiol group was exposed by reducing disulfide bridge in the compound of Formula 2

The compound of formula 2 prepared by the method of Example 1 was used to reduce the disulfide bridge in the compound using a known reducing agent to prepare a compound exposed to the dithiol group, NMR data of the obtained final product is shown in Figure 27 and 28 .

Example 2: Materials

Gliotoxin, chitocin and chetomin were purchased from Sigma-Aldrich. Bis (methylthio) gliotoxin was purchased from Santa Cruz Biotechnology. SU-5416 was purchased from Calbiochem. Phosphoric acid-PLCγ1 (pY673), PLCγ1 and phosphoric acid-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 VEFG₁₆₅) Was purchased from R & D Systems. Mouse anti-rat CD31 antibodies were purchased from BD Biosciences. Alexa Fluor 488-conjugated donkey anti-rabbit and Alexa Fluor 568-conjugated donkey anti-mouse secondary antibody were purchased from Invitrogen. Biotinylated goat anti-rabbit IgG, avidin-HRP and DAB substrates were purchased from Vector Laboratories. PrxI, PrxII, Prx-SO_2/3 And phosphate-PDGFR-β (pY857) rabbit polyclonal antibodies were prepared as previously described [M. H. Choiet al., 2005,Nature, 435: 347.

The sequences of the two siRNA dimers for human PrxII are 5'-CGCUUGUCUGAGGAUUACGUU-3 '(PrxII-1, SEQ ID NO: 1) and 5'-AGGAAUAUUUCUCCAAACAUU-3' (PrxII-2, SEQ ID NO: 2). PrxII-1 siRNA dimers were mainly used for in vitro experiments. SMART pool (5'-GCAACGCGCACAUCGGAAAUU (SEQ ID NO: 3), 5'-GAUCACAGUCAACGACCUAUU (SEQ ID NO: 4), 5'-AGAAUUACGGCGUGUUGAAUU (SEQ ID NO: 5), of 4 siRNA dimers for rat carotid balloon injury experiments) 5'-ACGCUGAGGACUUCCGAAAUU (SEQ ID NO: 6); Dharmacon Cat. No. D-089973) was used. Drosophila luciferase siRNA was synthesized as a control siRNA.

Hereinafter, the compound of Formula 2 prepared in Example 1-1 is A-1, and the compound of Formula 2 prepared in Comparative Example is reduced in disulfide bridge to expose a compound having a dithiol group in A-2, Example 1-2. The compound of Formula 3 prepared in the A-3, Gliotoxin is referred to as GT abbreviation.

Example 3: Cell Culture

Human aortic endothelial cells (HAECs) and human aortic smooth muscle cells (HASMCs) were purchased from Clonetics-BioWhittaker (venders, Belgium). 10% fetal bovine serum (FBS) and full supplement (Cat. No. Cc-4176 for HAECs) in a wet incubator containing 5% carbon dioxide at 37 ° C. seeded in a 0.1% gelatin coated dish. , and for HASMCs Cat no cc-4149;. . Clonetics-BioWhittaker) endothelial basal medium (endothelial Basal medium, including; EBM -2 ^TM) and smooth muscle cell basal medium (Smooth muscle cell Basal medium; smBM TM) SingleQuotes ® by Each was incubated. In the present invention, cells of five to seven passages were used.

Example 4: Peroxidase Activity Assay

Peroxidase activity assays of epidithiodioxopiperazine compounds or derivatives thereof (hereinafter referred to as ETP compounds) according to the present invention were carried out according to known methods [Republic of Korea 10-2006-0020140]. . 50 mM Hepes-containing 1 mM EDTA with 250 μM NADPH, 3 μM yeast thioredoxin (Trx), 1.5 μM yeast thioredoxin reductase (TR), 25 μM ETP compound and 1.2 ml hydrogen peroxide Standard peroxidase reactions for spectrophotometric analysis were performed with 200 μl of the reaction mixture in NaOH buffer (pH 7.0). For glutathione (GSH) -dependent peroxidase reactions, GSH (1 mM) and yeast Glutathione Reductase (GR) (1 Unit) were added instead of yeast Trx and TR. The reaction was initiated by the addition of hydrogen peroxide, and NADPH oxidation was monitored following a decrease in absorbance at 340 nm with an Agilent UV8453 spectrophotometer (Hewlett Packard, USA) at 30 ° C. for 12 minutes. Initial reaction rates were calculated using the linear portion of the curve and expressed as the amount of NADPH oxidized per minute.

Example 5: In Vitro Cell Function Analysis

For cell proliferation analysis, HAECs were seeded in 96-well plates containing siRNA-transfection reagent mixture at a final volume of 100 μl at a concentration of 4000 cells / well. After siRNA-injection for 24 hours, cells were serum depleted for 18 hours and added to EBM-2 basal medium with VEGF-A165 (25 ng / ml, Cat. No. 293-VE, R & D systems) for an additional 24 hours. Put it. The extent of cell proliferation was measured using the WST-1 Cell Proliferation Assay Kit (Roche Diagnostics, USA) and the cell number was expressed as absorbance at 450 nm, with turbidity at 600 nm and averaged in three wells.

Cell migration assays were 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 naturally dried for 1 hour. HAECs (6 * 10 ^ 3) were added to the upper chamber containing the siRNA injection complex. After 24 hours, siRNA injected HAECs were serum-depleted overnight. VEGF-A (25 ng / ml) solution was prepared in basal medium containing 0.5% bovine serum albumin (BSA) and added to the bottom chamber. The upper chamber wells were filled with each basal medium containing 0.5% BSA. The transwell chamber was incubated for 8 hours under 37 ° C./5% carbon dioxide conditions. After incubation, the non-mobile cells above the strainer were removed and the cells migrated to the bottom of the strainer were fixed and stained with 0.6% hematoxylin and 0.5% eosin. Stained cells were photographed and counted. The number of migrated cells was averaged from three wells.

Example 6: Immunoblot Analysis

After appropriate treatment or siRNA injection, cells are washed once with ice cold phosphate buffer solution, 20 mM Hepes (pH 7.0), 1% Triton X-100, 150 mM sodium chloride, 10% glycerol, 1 mM EDTA, 2 mM It was lysed with an extraction buffer containing EGTA, 1 mM DTT, 5 mM Na3VO4, 6 mM NaF, 1 mM AEBSF, aprotinin (5 μg / ml) and lupetin (5 μ / ml). After centrifugation at 12,000 g, purified cell extracts were used for immunoblot. Cell extracts were heated in SDS sample buffer and separated on SDS denaturing gels. If necessary, the membranes were stirred for 60 minutes at 37 ° C. with 67 mM Tris (pH 6.7), 2% SDS, 100 mM 2-mercaptoethanol, and redetected with appropriate pan antibodies.

Example 7: Measurement of Hydrogen Peroxide Concentration in Cells

After stimulation, cells were washed with phenol red-free basal medium and washed with 5 μM 5,6-chloromethyl-2 ', 7'-dichlorodihydrofluorescein diacetate (CM-H ₂ DCFDA) (Molecular Probe). Incubate at 5 ° C. for 5 min. Fluorescence images were recorded with a fluorescence microscope (Axiovert 200 Basic standard, Zeiss, Germany). Relative DCF fluorescence was calculated by averaging fluorescence levels from 50 to 80 cells after removing background fluorescence.

Example 8 NADPH Oxidase Activity Assay

Whole cell superoxide production was measured using an enhanced luminescence system (Diogenes, National Diagnostics). For analysis, serum-depleted cells were preincubated with 100 μl of Diogene reagent for 5 minutes at 37 ° C. After stimulation with the indicated growth factors, chemiluminescence was detected every second for 10 minutes with a TD-20 / 20 Luminometer (Turner Biosystems).

Example 9: balloon-injury model of rat carotid artery

Animal testing was conducted according to the regulations of the Institutional Animal Care and Use Committee (IACUC) of Ewha Womans University. Balloon damage was generated using a 2F Fogarty balloon catheter infiltrated into the left common carotid artery as follows: 10-week-old male Sprague-Dawley rats were made of isoflurane gas (N ₂ O: O ₂ /70%: 30%) by anesthesia, exposed to the left external carotid artery, and the branches were electro-coagulated. The catheter was pushed 1 cm through transverse arteriotomy of the external carotid artery and passed three times along the total carotid artery to dissociate the endothelium. After catheter removal, the carotid artery was sealed and added to open the carotid artery. Unless stated otherwise, rats were recovered from cages for 10 days and used for histological and immunological analysis.

Example 10 Histological Analysis

Rats were anesthetized and the total carotid artery was dissected after transcardiac perfusion-fixation with heparinized saline solution containing 3.7% formaldehyde. Blood vessels were depressed with paraffin and cut with a rotating microtom (Leica RM2255). Two serial tissue fragments (4 μm thick) were obtained from the central portion of the general carotid artery and stained with hematoxylin and eosin (HE). The lumen, inner elastic plate, and outer elastic plate portions were measured using NIH Image v1.62. The inner membrane and the inner portion were determined by removing the lumen portion from the inner elastic portion and removing the inner elastic portion from the outer elastic portion. Values from two consecutive fragments were averaged for each rat for analysis.

Example 11: Local Delivery of siRNA or ETP Compounds in the Carotid Artery

For in vivo injection of siRNA, siRNA mixtures specific for rat PrxII (200 nM) were premixed with siPORT ^™ NeoFX ^™ reagent according to the manufacturer's instructions. Immediately after balloon damage, siRNA injection mixture (200 μl) was injected via catheter after a brief wash with Opti-MEM. After 15 minutes incubation, blood flow was resumed. SiGLO-Red (Dharmacon), a fluorescent dye-binding control siRNA, was used to optimize siRNA injection efficiency in vivo. Similarly, ETP compound (200 nM in DMSO) was injected through the catheter and incubated for 30 minutes.

Example 12 Immunohistochemistry and Immunofluorescence Staining

Immunohistochemistry was performed on overoxidized 2-Cys Prx in paraffin fragments using anti-Prx-SO _2/3 antibody (1: 1000 dilution). Briefly, the fragments were stripped in xylene and rehydrated in ethanol and then heated in citric acid buffer (pH 6.0) to carry out antibody retrieval. Sections were then incubated with the primary antibody for 48 hours at 4 ° C. After washing three times with phosphate buffer solution, the sections were incubated with peroxidase-binding secondary antibody and stained with 3 ', 3'-diaminobenzidine (3', 3'-diaminobenzidine (DAB) substrate solution). It was. For negative staining, Prx-SO _2/3 antibody was blocked with the corresponding antigen peptide (DFTFVC (SO _2/3 ) PTEI). For indirect immunofluorescence staining, paraffin sections were blocked with 5% normal rabbit serum (Vector Laboratories) in PBST (PBS solution of 0.3% Triton X-100) for 1 hour at room temperature. The sections were then incubated overnight at 4 ° C. with antigens for rat smooth muscle α-actin (1: 300 dilution) and rat CD31 (PECAM-1, 1: 200 dilution). Nuclei were labeled with DAPI. After washing several times with PBST, the samples were incubated with Alexa Fluor 568-conjugated donkey anti-mouse and Alexa Fluor 488-conjugated donkey anti-rabbit IgG antibodies for 2 hours at room temperature. Fluorescence images were recorded in three random areas per tissue section at a screen magnification of 100 × using an LSM 510 Meta confocal microscope equipped with argon and helium-neon lasers.

Example 13: TUNEL Analysis

Paraffin sections were incubated for 10 minutes in PBS containing 0.1% Triton X-100. Thereafter, the TUNEL reaction was performed at 37 ° C. for 60 minutes using the In situ Cell Death Detection Kit, Fluorescein (Roche Diagnosrics Corp.) according to the manufacturer's instructions. Cell nuclei were counterstained with DAPI.

Example 14 Vascular Permeability Experiment

Mice were injected intravenously with 100 μl of 5% Evans blue for 30 minutes and perfused with PBS for 5 minutes. The total carotid artery was removed from both the intact side branches and the damaged ipsilateral. It was cut out and opened vertically and observed at a magnification of 20 times in a phase contrast microscope. For quantitation, Evans Blue introduced into blood vessels was extracted by placing in formamide overnight at 55 ° C. and centrifuged at 12,000 rpm for 10 minutes. The supernatant was collected and the absorbance was measured at 620 nm. The background value of Evans blue dye was measured at 740 nm and removed from the value in the carotid artery. For VEGFR2 inhibition, SU5416 (20 mg / kg) was injected intraperitoneally three times before and after balloon injury (Day-1, 1 and 3). Control injections used 200 μl of PBS with vehicle (5% DMSO).

Example 15 Scanning Electron Microscopy (SEM)

Carotid arteries were taken from the animals and opened longitudinally and fixed for 2.5 hours glutaraldehyde for 24 hours. Tissues were washed with PBS and incubated with 1% osmium tetraoxide and dehydrated through serial ethanol dilution. The tissue was dried to the critical point and fixed in a scanning electron microscope stub with a colloidal silver paste. After sputter-coating with gold / palladium, the specimens were observed with a scanning electron microscope (Hitachi, Japan).

< Experimental Results>

Experimental Example 1: Catalytic Activity for Hydrogen Peroxide Reduction

The chemical feature of the epidithiodioxopiperazine compounds or derivatives thereof of the present invention is the internal disulfide bridge in the epidithiodioxopiperazine ring moiety. Peroxidases in cells reduce hydrogen peroxide using electrons derived from NADPH via two electron-transfer pathways, Trx / TR or GSH / GR, so spectrophotometry for hydrogen peroxide-reducing activity in the presence of each system An analysis was performed and the results are shown in FIGS. 1 and 2.

As a result, as shown in Figures 1 and 2 A-1, A-3, GT, chitosine and chitomin all showed excellent hydrogen peroxide-reduction activity in the presence of the Trx / TR system. Interestingly, the activities of chitocin and chitomin were 8.18 ± 0.24 and 8.62 ± 0.51 nmol / min, respectively, about 2 times higher than GT (3.72 ± 0.53 nmol / min). This corresponds to the number of piperazine rings comprising disulfide bridges present in the compound.

In the presence of the GSH / GR system, chitocin and chitomin showed 1.51 ± 0.39 and 2.53 ± 0.79 nmol / min, respectively, but weak compared to the Trx / TR system. Thus, as shown in FIGS. 2D-2F, the hydrogen peroxide-reducing activity of the ETP compound required all three elements of the Trx / TR / NADPH system, which was proportional to concentration (FIG. 2G).

As a control, disulfide bonds were reduced in the diketopiperazine ring, so that the two thiol groups exposed A-2 and the exposed thiol group methylated bis (methylthio) glytoxin were used. As a result, in vitro, it was found to have peroxidase activity in vitro (FIG. 1B), and in the case of bis (methylthio) glytoxins in which thiol groups were methylated, no peroxidase activity was observed. (Figure 2g). In the experiments described below, A-2 does not actually exhibit PDGF- and VEGF-dependent cell proliferation and migration control activity, which is incapable of entering into cells in the state of A-2 with thiol groups exposed to PDGF- and VEGF- It was assumed that there was no activity to regulate dependent cell proliferation and migration. In addition, in the case of bis (methylthio) glytoxin in which a thiol group is substituted with a methyl group, disulfide crosslinking due to an oxidation-reduction reaction or the like is almost impossible, and thus it was found that there is no peroxidase activity. From this, it was found that the oxidation-reduction cycle of dithiol in the ring in the compound is essential for peroxidase activity. From the above results, it was confirmed that 2-Cys-Prx-like peroxidase activity of the epidithiodioxopiperazine compound or derivatives thereof according to the present invention is Trx / TR system dependent.

Experimental Example 2: In Vitro and In Vivo Cytotoxicity Experiments

In the past, most ETP compounds are known to be toxic to animal cells. The present inventors therefore determined the safe concentration range of epidithiodioxopiperazine compounds or derivatives thereof against vascular cells. To this end, human aortic vascular smooth muscle cells (HASMCs) and aortic vascular endothelial cells (HAECs) were pretreated with various concentrations of GT or chitosine for 2 hours and subjected to a water cell viability assay incubated for 24 hours in fresh medium (FIG. 3). To 5).

As a result, the viability of HASMCs and HAECs for A-1, A-3 and GT under serum-deficient conditions was ensured at concentrations of 200 nM and 50 nM or less, respectively. Chitocin has been found to be relatively less toxic. The low viability of both cells at high concentrations is thought to be a variety of side effects, including NF-κB inhibition. Therefore, the inventors used ETP compounds at concentrations of 50 nM and 25 nM, which are safe doses for in vitro experiments on HASMCs and HAECs for the following experiments.

Experimental Example 3 Substitution Effect Test of PrxII Function by GT in HASMCs and HAECs

Among the members of 2-Cys-Prx, PrxII inhibits the proliferation and migration of vascular smooth muscle cells while promoting the proliferation and migration of vascular endothelial cells (Choi et al, 2005, Nature, 435: 347-353; Kang DH et. al, 2011, Mol Cell., 44: 545-558, we tested the ability of PrxII functional substitution of ETP compounds. To this end, we studied the biological effects of ETP compounds on RTK signaling and vascular cell function using GT as a representative ETP compound. First, the ability of GT to remove hydrogen peroxide in PrxII knocked down HASMCs and HAECs was tested. Hydrogen peroxide production of the cells was monitored using an oxidant-sensitive fluorescent dye (2 ', 7'-dihydro-chlorofluorescein diacetate, H ₂ DCF-DA). Intracellular hydrogen peroxide levels were approximately doubled by PDGF treatment in serum-deficient control HASMCs and significantly increased in combination with PrxII knockdown (FIG. 6A). However, GT treatment completely offset the increased intracellular hydrogen peroxide level in a concentration-dependent manner (FIG. 6A). In addition, basal levels of intracellular hydrogen peroxide in HAECs significantly increased by PrxII knockdown were also completely removed by GT treatment (FIG. 6B).

Experimental Example 4: Effect of ETP Compounds on NOX Activity

Nishida reported that GT inhibited NADPH oxidant (NOX) activity in neutrophils [S. Nishidaet al., 2005,Infect. Immun., 73: 235. Therefore, the present inventors measured NOX activity in vascular cells to rule out these side effects. When HASMCs and HAECs were stimulated with PDGF-B and VEGF-A, respectively, NOX activity was increased by about 2-fold. However, GT and chitocin treatment did not inhibit NOX activity until the concentration of the compounds increased to 500 nM above the toxic limit (FIG. 7). Therefore, these ETP compounds act as hydrogen peroxide reducing agents that can replace PrxII in both vascular cells, but the mechanism does not inhibit hydrogen peroxide generator NOX.

Experimental Example 5: Replacement effect of PrxII function by GT on growth factor-induced signaling and cell proliferation / migration

The regulatory effect of GT on PDGFRβ- and VEGFR2-mediated signaling pathways was tested. GT treatment in HASMCs is distinct from PDGF-induced tyrosine phosphorylation increased by PrxII knockdown (FIG. 8A). Specifically, PDGFRβ and PLCγ1 activation levels were reduced to the levels of stimulated control cells. In contrast, GT treatment in HAECs restored VEGF-dependent activation of VEGFR2 and ERK, which was reduced by PrxII knockdown (FIG. 8B). Furthermore, the effect of GT on vascular cell function was confirmed. Increased concentrations of GT treatment gradually reduced proliferation and chemotactic migration of HASMCs in response to PDGF, increased by PrxII knockdown (FIGS. 9A and 9B). In contrast, the same treatment for HAECs enhanced VEGF-induced proliferation and chemotactic migration that was impaired by PrxII knockdown (FIGS. 9C and 9D).

In addition, the effects on PDGFRβ- and VEGFR2-mediated signaling pathways for A-1 and A-3 were confirmed in the same manner as GT, and A-2 exposed with a thiol group was used as a control. As a result, A-1 and A-3, like GT, gradually reduced the proliferation and chemotactic migration of HASMCs in response to PDGF (FIGS. 10A and 10B, 12A and 12B), and the same treatment for HAECs resulted in PrxII knockdown. Enhanced VEGF-induced proliferation and chemotactic migration that were impaired by (Figs. 10C and 10D, 12C and 12D). However, it was confirmed that the control group A-2 did not cause a significant change in both HASMCs and HAECs (FIGS. 11A to 11D).

It is noteworthy that low nanomolar concentrations of A-1, A-3 and GT can sufficiently regulate RTK signaling and cellular function regulation in HASMCs and HAECs. In conclusion, these results indicate that low concentrations of A-1, A-3 or GT can restore PDGF- and VEGF-induced signaling impaired by PrxII deficiency.

Experimental Example 6: Confirmation of peroxidation of 2-Cys-Prx, especially PrxII, through in vivo experiments on balloon-damaged carotid arteries

A vascular injury experimental animal model (rat carotid balloon-induced injury), which can monitor VSMCs overproliferation and re-endothelialization of blood vessels, was used to confirm the effects of ETP compounds in vivo. When the artery is damaged by the insertion of a balloon catheter, the endothelium is detached. Thus, platelets and macrophages accumulate in the damaged lesion to repair the damaged blood vessels. Since they produce active oxygen radicals including hydrogen peroxide, it is believed that 2-Cys Prx enzymes of neighboring vascular cells are inactivated by peroxidation of active site cystine residues (Cys-SO _2/3 ) to sulfinic acid / sulfonic acid. To illustrate this possibility, anti-Prx-SO _2/3 antibody was used to confirm 2-Cys Prx peroxidation of balloon-damaged rat carotid arteries. Balloon injury causes endovascular thickening of the carotid artery over time (FIG. 13A), and immunohistochemical analysis shows that 2-Cys Prx is severely peroxidated in both the inner and endovascular layers on days 5 and 7 after injury (FIG. 13A). 13b). Hydrogen peroxide-treated normal blood vessels were blocked with the corresponding antigen peptides to confirm immuno-reactive signaling specificity. Immunoblot analysis supports the induction of peroxidation of the major 2-Cys Prx by balloon injury. When PrxI and PrxII were isolated by immunoprecipitation with an anti-PrxI antibody, it was confirmed that peroxidation of PrxII was predominantly increased due to endovascular thickening compared to PrxI or PrxIII (FIG. 13C). This is in contrast to hydrogen peroxide-treated control vessels with pronounced PrxI peroxidation. The negative regulatory role of PrxII in PDGF-dependent growth of VSMCs demonstrates that inactivation of PrxII by peroxidation contributes to SMC hyperplasia in damaged vascular walls. Practically, siRNA-mediated knockdown of PrxII expression in the carotid artery wall exacerbates endovascular thickening induced by balloon injury (FIG. 14).

Next, it was tested whether GT coordinates proper repair of damaged vessels where PrxII peroxidation occurs. When the GT solution was administered to the lumen of carotid artery vessels at various concentrations through a catheter after balloon injury, TUNEL staining revealed that endovascular thickening was clearly inhibited in a concentration-dependent manner in the nanomolar range that did not induce cell death. 3G and 15A). This indicates that GT inhibits SMC hyperplasia.

In addition, as a control experiment, bis (methylthio) glytoxin and a TR inhibitor (DNCB 5 μM, or Auronafin 0.5 μM) with methylated dithiol groups were treated alone or in combination with GT and HE-stained images of the tissues were observed. It was. As a result, it was confirmed that bis (methylthio) glytoxin did not inhibit the enlarged vascular endothelial layer, and all TR inhibitors canceled the endovascular thickening inhibitory effect of GT (FIG. 16). As a result, it was found that bis (methylthio) glytoxins in which the thiol group was methylated had no effect on vascular damage recovery, and GT was found to exhibit vascular damage recovery effect through TR regulation.

Next, it was tested whether GT substantially promoted endothelial repair in the damaged vascular wall. Immunofluorescence staining of endothelial marker CD31 was confirmed that the endothelial monolayers in contact with the lumen were formed in GT-treated injured blood vessels, such as normal carotid artery vessels, while not in the control injured carotid arteries (FIG. 15B). Co-immunostaining with α-smooth muscle actin (SMA) also showed that the endothelial layer was covered on the vascular endothelium SMC layer.

In addition, immunohistochemical staining was performed using peroxidized 2-Cys-Prx (Prx-SO2 / 3) antibody to determine the effect of GT on 2-Cys-peroxredoxin (2-Cys-Prx) peroxidation. DAB-staining plots show representative images of five different carotid artery samples. As a result, as shown in Figure 17, there was no significant difference in the sample data between the control group and the GT treatment group. This result supports that GT does not prevent the oxidation of 2-Cys-Prx but replaces the function of 2-Cys-Prx in cells after peroxidation.

Experimental Example 7: Vascular Permeability Test

Permeability of the endothelium recovered with the Evans blue influx was tested. Endothelial detachment due to balloon damage causes a complete loss of permeability control. In contrast, the intact side carotid artery has intact mesenteric function of the normal endothelial (FIG. 18A). GT treatment for balloon injured carotid arteries shows about 2/3 reduced Evans Blue influx compared to control carotid (DMSO) treated carotid arteries. For greater certainty, endothelial function recovery induced by GT is blocked when treated with VEGFR2 kinase-specific inhibitors. This suggests that the effect of GT protects VEGFR2 function from oxidative inactivation induced by peroxidation of PrxII. Endothelial cell junctions at the luminal surface of the damaged carotid artery were identified by scanning electron microscopy. Carotid endothelial cells of GT-treated rats spread widely to form a uniform layer with firm seams, while in control-treated rats they contracted and were sparsely present (FIG. 18B). As a result, it was found that the endothelium recovered by GT treatment was functionally and structurally intact and therefore sufficient to control vascular permeability.

Experimental Example 8: Effect of chitocin, chitomin and other antioxidant compounds

The present inventors have confirmed that other ETP compounds such as chitocin and chitomin can also induce proper recovery of damaged blood vessels. First, the efficacy of chitocin in vascular cell function was confirmed. Increased concentrations of chitocin treatment significantly reduced proliferative and chemotactic migration of HASMCs in response to increased PDGF by PrxII knockdown (FIGS. 19A and 19B). In contrast, the same treatment markedly improved VEGF-induced proliferation and chemotactic migration of HAECs impaired by PrxII knockdown (FIGS. 19C and 19D). As mentioned in Experiment 2, since chitocin has lower cytotoxicity than GT, the efficacy of chitocin to regulate the function of VASMCs and HAECs is important even at higher concentrations. Along with this in vitro activity, the treatment of chitocin markedly blocked endovascular thickening in the injured artery wall and concomitantly promoted re-endothelialization (FIGS. 20A and 20C). This indicates that, in addition to GT, chitocin also acts as an opposing regulator for VSMCs and VECs. Chitomin also showed a similar effect in the repair of damaged arterial walls (FIGS. 20B and 20D). In conclusion, it was confirmed that ETP-based compounds could act as an ideal therapeutic agent for the repair of vascular damage.

Finally, the effect of GT, a representative ETP compound, on vascular cells is compared with other compounds known as antioxidants, such as N-acetylcysteine or butylated hydroxyanisole. The results are shown in FIG. 21. The HAECs in which siRNA was injected and knocked down PrxII were pretreated with each compound at the concentrations indicated in the figure for 2 hours, followed by immunoblotting by treatment with VEGF for 10 minutes. As a result, N-acetylcystine and butylated hydroxyanisole did not induce VEGFR2 and downstream ERK activation even when used at concentrations of 1000 to 10000 times that of GT. From the comparative experiments we concluded that the distinct cellular action of the ETP compound is apparently due to PrxII-like activity and additionally due to the special chemical properties of the dithioketopiperazine ring.

Experimental Example 9: Confirmation of the effect of GT on PrxII deficient vascular cells and mice

Aortic vascular smooth muscle (A) and vascular endothelial cells (B) were isolated and cultured in normal and PrxII-/-mice, and treated with PDGF-BB and VEGF-A for 10 minutes, respectively, with or without GT. Activation of PDGFRβ, PLCγ1, VEGFR2 and ERK were analyzed using phosphorylation-specific binding antibodies to proteins, respectively. As a result, as shown in A and B of FIG. 22, PDGF signaling and VEGF signaling in vascular smooth muscle cells (VSMC) and vascular endothelial cells (VEC) isolated from Prx-/-mice, respectively. The opposite control effect of GT on delivery was confirmed.

In addition, in order to confirm the effect of GT on vascular hypertrophy in PrxII-/-mice, the left carotid artery of PrxII-/-mice was damaged using a flexible wire. After injury, a control vehicle or GT solution was moistened on a Whatman No. 1 filter paper strip and allowed to soak into the tissue for 30 minutes on the surface of the vessel envelope. Mice recovered 10 days after injury. The results show the ratio of intima to media measured from HE-stained carotid arterial samples as mean +-standard error (n = 8 per group, * p <0.01). As a result, as shown in Fig. 22C, GT was found to significantly reduce blood vessel enlargement.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (10)

An epidithiodioxopiperazine compound represented by Formula 1 or a derivative thereof; Or a pharmaceutically acceptable salt thereof. A pharmaceutical composition for preventing or treating vascular restenosis. [Formula 1]

The pharmaceutical composition of claim 1, wherein the derivative is any one of the compounds represented by the following Chemical Formulas 2 to 19. [Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

[Formula 8]

[Formula 9]

[Formula 10]

[Formula 11]

[Formula 12]

[Formula 13]

[Formula 14]

[Formula 15]

[Formula 16]

[Formula 17]

[Formula 18]

[Formula 19]

The pharmaceutical composition of claim 1, wherein the blood vessel is a carotid, coronary, peripheral, or renal artery. The pharmaceutical composition of claim 1, wherein the vascular restenosis is by angioplasty, angioplasty, arteriosclerosis, vascular fat accumulation, hypertension, angiogenesis or angioplasty. The pharmaceutical composition of claim 1, wherein the composition promotes proliferation or migration of vascular endothelial cells while inhibiting proliferation or migration of vascular smooth muscle cells. (a) determining whether a compound including at least one epidithiodioxopiperazine ring represented by Formula 20 exhibits 2-Cys-peroxredoxin (2-Cys-Prx) activity; And (b) when the NADPH oxidation reaction or H ₂ O ₂ reduction reaction occurs, comprising the step of determining the compound as a preventive or therapeutic agent for vascular restenosis prevention or therapeutic screening method. [Formula 20]

The method according to claim 6, wherein the confirmation of 2-Cys-peroxredoxin (2-Cys-Prx) activity, the compound comprising at least one epidithiodioxopiperazine ring represented by the formula (20) Mixing with thioredoxin (Trx), thioredoxin reductase (TR), NADPH, buffer and H ₂ O ₂ to determine whether NADPH oxidation or H ₂ O ₂ reduction occurs; And deciding to show 2-Cys-peroxredoxin (2-Cys-Prx) activity when NADPH oxidation or H ₂ O ₂ reduction occurs. A topical drug delivery device comprising the pharmaceutical composition of any one of claims 1 to 5. The device of claim 8, wherein the drug delivery device for topical administration is a stent. The device of claim 8, wherein the topical drug delivery device delivers the pharmaceutical composition to the sustained release.

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