WO2011155687A1

WO2011155687A1 - Novel glycolipid derivative compounds and their therapeutic use for vascular smooth muscle cell hyper-proliferation

Info

Publication number: WO2011155687A1
Application number: PCT/KR2011/001122
Authority: WO
Inventors: Yeo-Pyo Yun; Yong Lim; Man Kil Jung
Original assignee: Chungbuk National University Industry-Academic Cooperation Foundation; Industry-Academic Cooperation Foundation, Yonsei University
Priority date: 2010-06-09
Filing date: 2011-02-21
Publication date: 2011-12-15
Also published as: KR101210553B1; KR20110134709A

Abstract

The present invention provides a novel glycolipid derivative compound, a composition for inhibiting vascular smooth muscle cell proliferation using the same and a composition a hyper-proliferative disease of vascular smooth muscle cells using the same. Since the novel glycolipid derivative compound of the present invention has an excellent activity for inhibiting vascular smooth muscle cell proliferation, it may be efficiently utilized in prevention or treatment of not only a hyper-proliferative disease of vascular smooth muscle cells (for example, arteriosclerosis, atherosclerosis, vascular restenosis or vascular stenosis) but also various cardiovascular disorders caused secondarily by the hyper-proliferative disease of vascular smooth muscle cells.

Description

NOVEL GLYCOLIPID DERIVATIVE COMPOUNDS AND THEIR

THERAPEUTIC USE FOR VASCULAR SMOOTH MUSCLE CELL HYPER- PROLIFERATION BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

The present invention relates to a novel glycolipid derivative compound, a composition for inhibiting vascular smooth muscle cell proliferation including the same as an active ingredient, and a composition for preventing or treating a hyper- proliferative disease of vascular smooth muscle cells including the same as an active ingredient.

BACKGROUND OF TECHNIQUE

Cardiovascular diseases are generally called as an ischemic cardiovascular disease, which include: (a) a heart disease such as heart failure, hypertensive heart disease, arrhythmia, congenital heart disease, myocardial infarction and angina pectoris; and (b) a vascular disease such as arteriosclerosis, stroke and peripheral vascular stenosis. These diseases are developed in all ages, and may cause severe sequelae if not appropriately treated, even leading to death. It is a recent trend that attack rate and mortality rate of cardiovascular diseases are rapidly increased due to western pattern diet and lifestyle changes. Unfortunately, no effective therapeutic agents for treating cardiovascular diseases have been yet developed.

Currently, various therapies have been attempted to treat a coronary artery disease,- for example including a drug therapy, a gene therapy, a PTCA (percutaneous transluminal coronary angioplasty and stenting), and a revascularization therapy of CABG (coronary artery bypass graft). However, drug has to be consistently administered in spite of revascularization therapy, and there is any possibility of coronary artery disease recurrence. PTCA has been routinely carried out as a therapy against atherosclerotic patients with a vascular disease since 1977 (Gurentzig, Lancet, 4263, 1978), and albeit highly therapeutic efficacy of PTCA, it has a severe drawback that restenosis has been found in about 40% patients within 3-6 months after surgery (Ryan et al., J. Am. Coll. Cardiol., 22. 2033-2054, 1993). A mechanism of restenosis caused after PTCA is known as follows: (a) various cytokines are produced or secreted from vascular endothelial cells damaged by a balloon, activated platelets or macrophages, leading to promote migration and proliferation of vascular smooth muscle cells; (b) afterwards, enhanced production and secretion of extracellular matrix (ECM) causes thickness of vascular intima, resulting in restenosis development (Godfried et al, Am. Heart J., 129, 203-210, 1995).

Normal vascular smooth muscle cells exhibit no proliferation, but media of endothelial cells damaged by a stent procedure contributes to division, migration and proliferation of vascular smooth muscle cells via diverse signal transduction pathways. For example, a mechanism of vascular smooth muscle cell proliferation includes removal of a proliferation factor and activation of a vascular smooth muscle cell-inducible factor caused by injury of normal vascular smooth muscle cells, proliferation signal transfer via a surface receptor of a proliferation factor, cell cycle alteration by a proliferation-inducible signal transduced into a nuclear of a vascular smooth muscle cell, and so on.

Normal endothelial cells are known to secrete molecules which inhibit vascular smooth muscle cell proliferation. Where endothelial cells are injured, secretion of these molecules is inhibited and vascular smooth muscle cell proliferation is induced by a platelet-derived growth factor (PDGF) secreted from activated platelet and various cytokines in plasma.

Up to now, there have been numerous attempts to prevent restenosis using anti-platelet agents, anti-coagulant agents, cholesterol synthesis inhibitors or antiallergic agents (Lefkovits et al., Progr. Cardiovas. Dis., 40, 141-158, 1997), and recent studies for preventing restenosis with components derived from natural compounds including fish oil, vitamin C or vitamin E. However, it is not yet sufficient to determine whether these drugs have an efficacy on a clinic.

In addition, other drugs have been currently developed as a vascular restenosis inhibitor, for example including taxol (Herdeg et al., Zeischrift fur Kardiologie, 89, 390-397, 1999), heparin, EPA, estrogen, and so like, but have exhibited no significant efficacy on restenosis. Korean Patent No. 10-478671 discloses a pharmaceutical composition for preventing and treating coronary restenosis comprising clotrimazol as an active ingredient, and Korean Patent No. 10- 516026 discloses a pharmaceutical composition for preventing and treating vascular restenosis comprising 3'-deoxyadenosine (cordycepin) as an active ingredient. Additionally, Korean Publication Patent No. 2001-110793 discloses preventives/remedies for angiostenosis comprising a compound with an inhibitory activity for Rho kinase as an active ingredient, and Korean Publication Patent No. 2003-46314 discloses a pharmaceutical composition comprising antithrombin as an active ingredient for prevention and therapy of vasculoproliferative disorders such as transplant vasculopathy, restenosis, in-stent restenosis and pulmonary hypertension. In addition, Korean Publication Patent No. 2005-23249 describes a pharmaceutical composition for preventing and/or treating vascular diseases such as coronary restenosis and/or reocclusion generated after PTCA (percutaneous transluminal coronary angioplasty) using a vascular stent comprising a retinoid and retinoid modulator as an active ingredient, and Korean Publication Patent No. 2005-43183 discloses a pharmaceutical composition for preventing and treating coronary restenosis including curcumin. However, since these vascular restenosis inhibitors not only prevent restenosis but also exhibit diverse side effects (for example, inhibition of scar regeneration, vessel injury, hepatotoxicity, kidney injury, bleeding increase by inhibition of platelet coagulation, and so forth), there have been actively made studies for molecules to inhibit vascular restenosis, which are derived from various natural compounds with safety for human. Unfortunately, any productive results have been not reported yet. Therefore, it has been urgently demanded to develop a novel substance capable of inhibiting vascular restenosis in a safe and effective manner.

Meanwhile, arteriosclerosis, a disease which artery wall is loose in elasticity and is hardened in thickness, has various symptoms depending on invasive internal organs. In common, arteriosclerosis exhibits an ischemia symptom caused by insufficient blood supply in most frequent manner, and often a rupture bleeding symptom due to elasticity loss. Particularly, where blood flow rate is reduced by arteriosclerosis developed in heart coronary artery, angina pectoris causing thoracic pain in exercise is occurred. In addition, where blood flow is more reduced, unstable angina exhibiting pain in rest is progressed, and completely blocked, myocardial infarction in which cardiac muscle is decay is developed, even leading to threaten the life.

Numerous researches for treating arteriosclerosis have been attempted during the past. Since 1970's, there was introduced a therapy that lesions were found using angiography via a tube inserted into heart, followed by physical expansion through balloon insertion. Recently, treatment of a heart disease reaches a turning-point in the senses that surgery success rate by a drug-coating stent is increased even in more extensive situations. Therapies to heart diseases caused by arteriosclerosis have been remarkably progressed as described above, whereas most academic products to prevent and regulate arteriosclerosis in itself are remained to be elucidated, and there have been urgently demanded researches for cause and treatment of arteriosclerosis and studies for detection and application of anti- thrombosis and anti-coagulant agents for inhibiting thrombosis formation in an effective manner.

Throughout this application, various publications and patents are referred and citations are provided in parentheses. The disclosures of these publications and patents in their entities are hereby incorporated by references into this application in order to fully describe this invention and the state of the art to which this invention pertains. DETAILED DESCRIPTION OF THE INVENTION

The present inventors have made intensive studies to develop a substance for effectively treating and preventing a hyper-proliferative disease of vascular smooth muscle cells. As results, we have discovered that a glycolipid derivative compound synthesized in the present invention has an inhibitory activity for vascular smooth muscle cell (VSMC) proliferation experimentally.

Accordingly, it is an object of this invention to provide a novel glycolipid derivative compound.

It is another object of this invention to provide a composition for inhibiting vascular smooth muscle cell proliferation including a glycolipid derivative compound as an active ingredient.

It is still another object of this invention to provide a pharmaceutical composition for preventing or treating a hyper-proliferative disease of vascular smooth muscle cells. Other objects and advantages of the present invention will become apparent from the following detailed description together with the appended claims and drawings.

In one aspect of this invention, there is provided a novel glycolipid derivative compound represented by the following Formula I:

(I) wherein R represents Q-C* straight alkyl substituted with .^cinnamoyl, CrC₁₀ straight alkanal or phenyltriazolyl group.

According to a preferable embodiment, R represents Q-C3 straight alkyl substituted with /^cinnamoyl, CrC₇ straight alkanal or phenyltriazolyl group.

According to more preferable embodiment, the compound of the present invention includes a compound represented by the Following formulae II, III, IV or V:

The glycolipid derivative compounds in the Formula I are a novel compound which is first synthesized by the present inventors. As an exemplified compound of Formula I, the compound 2 (compounds 2a, 2b or 2c) represented by the Formula II may be synthesized by the following reaction Formula I:

In the reaction Formula I, the reactant 'RCOH' represents alkyl or aryl aldehyde; pyrrolidine is a basic catalyst; and anhydrous methylenechloride (M.C.) is utilized as a solvent.

Compound 1 (la, lb or lc), (2 5)-2-(2,4-Di-Obenzyl 3,6-dideoxy-a-L- rhamnopyranosyloxy)-octanone, used as a starting material in the reaction formula I, may be feasibly synthesized by modifying with oxygen and PdClz/CuCI an olefin end group from olefinic glycolipid substituted with Q-C, straight alkyl. The reaction may be carried out in a range from 0°C to room temperature for 18 hrs, and in a reactor that blocks air influx. The reactant aldehyde may be utilized with an equivalent weight of 1.0 to the starting material, compound 1.

As a practical compound of Formula I, the compound 4 (compounds 4a, 4b or 4c) represented by the Formulae III and IV may be synthesized by the following reaction Formula II:

In the reaction Formula II, the reactant -≡' represents ethynyl benzene; CuS0₄ · 5H₂0 and sodium ascorbate are a catalyst; and the mixture (1: 1) of THF/H₂0 is utilized as a solvent. Azide compound (compound 3; 3a, 3b or 3c), [(2 ?,5/¾-2-(/? 6- azidohexan-2-yloxy)-6-methyl-tetrahydro-2H-pyran-3,5-diol], used as a starting material in the reaction Formula II, may be feasibly synthesized by modifying an olefin end group from olefinic glycolipid substituted with d-Q straight alkyl. The reaction may be carried out at room temperature for 10 min to 4 hrs, and in a reactor that blocks air influx. The reactant ethynyl benzene may be utilized with an equivalent weight of 1.0 to the starting material, compound 3.

As an exemplary compound of Formula I, the compound 6 represented by the Formula V may be synthesized by the following reaction Formula III:

(III)

To obtain compound 6 in the reaction Formula III, the compound 5 as a starting material represents a glycolipid aldehyde in which two hydroxyl groups are protected with a benzyol group; NaOMe represents a base to remove a benzoyl protecting group; Amberite IR-120 is an acidic catalyst to neutralize a sodium salt; and CH₃OH is utilized as a solvent.

Aldehyde compound 5, [(2R)-2-(2,4-Di-i>benzyl-3,6-dideoxy-a-L- mannopyranosyloxy)tridecanal], used as a starting material in the reaction formula III, may be feasibly synthesized by modifying an olefin end group from olefinic glycolipid substituted with Ci-Q straight alkyl. The reaction may be carried out at room temperature for 8 hrs, and in a reactor that blocks air influx. The reactant NaOMe may be utilized with an equivalent weight of 3.0 to the starting material, compound 5, to remove a protecting group.

In another aspect of this invention, there is provided a composition for inhibiting vascular smooth muscle cell proliferation including a novel glycolipid derivative compound represented by the above-mentioned Formula I.

According to a preferable embodiment, the compound used as an active ingredient in the composition of the present invention includes a compound in which R represents Q-C₃ straight alkyl substituted with /E^cinnamoyl, Q-C₇ straight alkanal or phenyltriazolyl in the Formula I.

According to more preferable embodiment, the compound used as an active ingredient in the composition of the present invention includes a compound represented by the following Formulae II, III, IV or V.

As the glycolipid derivative compounds of the present invention have an activity for inhibiting vascular smooth muscle cell proliferation, they may be highly useful in various applications in which it is necessary to inhibit hyper-proliferation of vascular smooth muscle cells.

In still another aspect of this invention, there is provided a pharmaceutical composition for preventing or treating a hyper-proliferative disease of vascular smooth muscle cells, including: (a) a therapeutically effective amount of a glycolipid derivative compound represented by the above-mentioned Formula I; and (b) a pharmaceutically acceptable carrier.

The glycolipid derivative compounds of the present invention have an activity for inhibiting vascular smooth muscle cell proliferation.

According to a practical embodiment, the compound of the present invention exhibits an inhibitory efficacy on aortic smooth muscle cell proliferation in white mouse stimulated with PDGF-BB (platelet-derived growth factor-BB).

According to another practical embodiment, the compound of the present invention inhibits DNA synthesis in aorta smooth muscle cells in white mouse stimulated with PDGF-BB, resulting in inhibition of their proliferation.

The term "smooth muscle" used herein refers to a non-striated muscle found in a wall of an organ including tube-like organ, bladder, peritoneal cavity, uterus, male and female reproductive tracts, gastrointestinal tract, respiratory tract, arrector pili of skin, the ciliary muscle and iris of the eye. The term "smooth muscle cell (SMC)" used herein refers to a mononuclear cell consisting of the smooth muscle, which is characterized as arrangement of SMC sheet or bundle which is linked by gap junctions.

The term "vascular smooth muscle cell (VSMC)" used herein means a cell constituting a smooth muscle in a blood vessel wall.

The term "hyper-proliferative disease of vascular smooth muscle cell (VSMC)" used herein refers to a disease or disorder caused by VSMS hyper-proliferation.

The hyper-proliferative disease of VSMC includes: (a) vascular restenosis, vascular stenosis, arteriosclerosis or atherosclerosis directly caused by VSMS hyper- proliferation; and (b) cardiovascular diseases such as heart failure, myocardial infarction, angina pectoris, arrhythmia, hypertensive heart disease, congenital heart disease, stroke or peripheral vascular stenosis, which are secondarily developed by vascular stenosis, vascular restenosis or arteriosclerosis. More preferably, the hyper- proliferative disease of VSMC treated or prevented by the pharmaceutical composition of the present invention includes arteriosclerosis, atherosclerosis, vascular restenosis or vascular stenosis.

Atherosclerosis is a disease that lipid molecule is deposited or fibrosis is generated in internal layer of artery. Meanwhile, vascular restenosis is a disorder that blood vessel's passage is narrow after traumatization is generated in a blood vessel wall. It has been known that vascular restenosis generated after arteriosclerosis progress and stent insertion is caused by proliferation and migration of vascular smooth muscle cells, and secretion of extracellular matrix (Circulation, 1997, 95, 1998-2002; J. Clin. Invest. 1997, 99, 2814-2816; Cardiovasc. Res. 2002, 54, 499- 502). Thereafter, there have been enormously made studies for drug inhibiting vascular smooth muscle cell proliferation to prevent artheriosclerosis development and vascular restenosis. As results, several drugs have been currently utilized for treatment of patients (J. Am. Coll. Cardiol., 2002, 39, 183-193). Therefore, the glycolipid derivative compound of the present invention capable of inhibiting vascular smooth muscle cell proliferation may be effectively utilized for treating a hyper- proliferative disease of vascular smooth muscle cell.

The pharmaceutical composition of this invention may further include a pharmaceutically acceptable carrier in addition to the active ingredient.

The pharmaceutically acceptable carrier contained in the pharmaceutical composition of the present invention, which is commonly used in pharmaceutical formulations, but is not limited to, includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, rubber arable, potassium phosphate, arginate, gelatin, potassium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oils.

The pharmaceutical composition according to the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent and a preservative. Details of suitable pharmaceutically acceptable carriers and formulations can be found in Remington's Pharmaceutical Sciences (19th ed., 1995), which is incorporated herein by reference.

A suitable dose of the pharmaceutical composition of the present invention may vary depending on pharmaceutical formulation methods, administration methods, the patient's age, body weight, sex, severity of diseases, diet, administration time, administration route, an excretion rate and sensitivity for a used pharmaceutical composition.

The pharmaceutical composition according to the present invention may be administered orally or parenterally, and preferably, administered parenterally, e.g., by intravenous, subcutaneous, intramuscular, intra-abdominal or transdermal injection. It is preferable that the administration route is determined depending on a disease to which the pharmaceutical composition of the present invention may be applied. The concentration of the glycolipid compounds included in the composition of the present invention may be determined depending on treatment's purpose, pathological condition of a subject or necessity period, and is not limited to a particular range of concentration. Preferably, the glycolipid derivative compound in the composition of the present invention may be added with a concentration range of 5-50 μΜ, more preferably 10-50 μΜ and much more preferably 20-50 μΜ. In this connection, the glycolipid derivative compound has shortcomings as follows: (a) desired effects of the glycolipid derivative compound may be not obtained in concentrations of 5 μΜ or less; (b) the glycolipid derivative compound used in concentrations of 50 μΜ or more may exhibit no enhanced activity corresponding on increase of concentration; and (c) preparation cost of a medicament including the glycolipid derivative compound of the present invention is increased.

According to the conventional techniques known to those skilled in the art, the pharmaceutical composition may be formulated with pharmaceutically acceptable carrier and/or vehicle as described above, finally providing several forms including a unit dose form and a multi-dose form. Non-limiting examples of the formulations include, but not limited to, a solution, a suspension or an emulsion in oil or aqueous medium, an extract, an elixir, a powder, a granule, a tablet and a capsule, and may further comprise a dispersion agent or a stabilizer.

BRIEF DESCRIPTION OF THE DRAWINGS

Figs, la-lc represent that the glycolipid derivative compound of the present invention with various concentrations has an inhibitory activity for aortic smooth muscle cell proliferation.

Fig. la is a graph assessing to what extent aortic smooth muscle cell proliferation is inhibited. The aortic smooth muscle cells in white mouse were treated with the glycolipid derivative compound (DD189) of the present invention represented by the Formula II with concentrations of 10, 20 and 40 μΜ, respectively, and then with PDGF-BB for proliferation induction.

Fig. lb is a graph measuring to what extent aortic smooth muscle cell proliferation is inhibited. The aortic smooth muscle cells in white mouse were treated with the glycolipid derivative compound (DD204) of the present invention represented by the Formula III with concentrations of 10, 20 and 50 μΜ, respectively, and then with PDGF-BB for proliferation induction.

Fig. lc is a graph determining to what extent aortic smooth muscle cell proliferation is inhibited. The aortic smooth muscle cells in white mouse were treated with the glycolipid derivative compound (DD223) of the present invention represented by the Formula IV with concentrations of 10, 20 and 50 μΜ, respectively, and then with PDGF-BB for proliferation induction.

Figs. 2a-2c represent that the glycolipid derivative compound of the present invention with various concentrations has an inhibitory activity for DNA synthesis in aortic smooth muscle cells.

Fig. 2a is a graph assessing to what extent DNA synthesis in aortic smooth muscle cells is inhibited. The aortic smooth muscle cells in white mouse were treated with the glycolipid derivative compound (DD189) of the present invention represented by the Formula II with concentrations of 10, 20 and 40 μΜ, respectively, and then with PDGF-BB for proliferation induction.

Fig. 2b is a graph measuring to what extent DNA synthesis in aortic smooth muscle cells is inhibited. The aortic smooth muscle cells in white mouse were treated with the glycolipid derivative compound (DD204) of the present invention represented by the Formula III with concentrations of 10, 20 and 50 μΜ, respectively, and then with PDGF-BB for proliferation induction.

Fig. 2c is a graph determining to what extent DNA synthesis in aortic smooth muscle cells is inhibited. The aortic smooth muscle cells in white mouse were treated with the glycolipid derivative compound (DD223) of the present invention represented by the Formula IV with concentrations of 10, 20 and 50 μΜ, respectively^ and then with PDGF-BB for proliferation induction.

Figs. 3a-3c represent that the glycolipid derivative compound of the present invention with various concentrations exerts an inhibitory effect on smooth muscle cell proliferation by arrest in inter-phase between G0/G1 phase and S phase in cell cycle of aortic smooth muscle cells.

Fig. 3a is a graph assessing aortic smooth muscle cell proliferation cycle. The aortic smooth muscle cells in white mouse were treated with the glycolipid derivative compound (DD189) of the present invention represented by the Formula II with concentrations of 10, 20 and 40 μΜ, respectively, and then with PDGF-BB for proliferation induction.

Fig. 3b is a graph measuring aortic smooth muscle cell proliferation cycle. The aortic smooth muscle cells in white mouse were treated with the glycolipid derivative compound (DD204) of the present invention represented by the Formula III with concentrations of 10, 20 and 50 μΜ, respectively, and then with PDGF-BB for proliferation induction.

Fig. 3c is a graph determining aortic smooth muscle cell proliferation cycle. The aortic smooth muscle cells in white mouse were treated with the glycolipid derivative compound (DD223) of the present invention represented by the Formula IV with concentrations of 10, 20 and 50 μΜ, respectively, and then with PDGF-BB for proliferation induction.

The present invention will now be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples.

EXAMPLES

Materials and Methods

EXAMPLE 1: Synthesis of (E)-l-(3,6-dideoxy-a-L-rhamnosyloxy)-9- phenylnon-8-en-7-one [a compound represented by the Formula II]

The compound 1, (2/?)-2-(2,4-Di-Obenzyl 3,6-dideoxy-a-L-rhamnopyranosyl - oxy)-octanone (47 mg, 0.08 mmol), was dissolved in 7 mL MeOH in a round flask, and then NaOCH₃ (6 mg, 0.25 mmol) was gradually added to the solution at 0°C, changing the solution to yellow color depending on to what extent a benzoyl protecting group is removed. After stirring for 12 hrs, the reaction is stopped and Amberlite-IR120 (H⁺) was gradually added into MeOH solvent to determine whether the solution is neutralized using a pH paper. The solution is filtered in neutralization condition to remove resin, and the solvent is eliminated through distillation under reduced pressure, followed by extracting with distilled water and EtOAc (5 mL, 3 times). After drying and filtering the resultant with MgS0₄, the resultant was concentrated under reduced pressure, and then compound 2 (a compound represented by the Formula II) was produced in the amount of 27 mg (yield rate, 88%) using a flash column chromatography (CH₂Cl2/MeOH, 15: 1, v/v). Physiochemical characteristics of the final products were as follows:

C21H30O5, a colorless syrup, Rf = 0.56 (CH₂CI₂ / MeOH, 7: 1, v/v); [a]_D ²⁰ = - 59.17° (c = 0.06, CHCI3); 'H-NMR (250 MHz, MeOD); δ 7.59-7.53 (m, 3H), 7.41-7.38 (m, 2H), 7.33-7.32 (m, 1H), 6.77-6.71 (d, 7 = 15 Hz, 1H), 4.72 (s, 1H) 3.81 (m, 2H), 3.75-3.64 (m, 1H), 3.62-3.48 (m, 1H), 2.70 (t, 2H), 2.13-2.05 (m, 1H), 1.92-1.77 (m, 1H), 1.74-1.66 (m, 2H), 1.64-1.43 (m, 4H), 1.30-1.27 (d, J = 7.5 Hz, 3H), 1.14- 1.12 (d, J = 5 Hz, 3H); ¹³C-NMR (62.9 MHz, MeOD); δ 200.88, 142.85, 134.59, ^•130.62, 129.07, 128.70, 128.42, 126.65, 126.32, 95.90, 71.02, 69.98, 69.42, 68.19, 40.82, 37.11, 35.57, 25.49, 24.44, 19.05, 17.87; IR (film) v_mM 3417, 2963, 2929, 2872, 1656, 1609, 1450, 1408, 1384, 1242, 1124, 1098, 1028, 984 cm^"1; LC Mass ESI probe 385 Da/e, Scan ES+5.61e.

EXAMPLE 2: Synthesis of (3/?,5/¾6fl)-2-methyl-6-((fl)-7-(4-phenyl-l /- 1,2,3-triazol-l-yl) heptan-2-yloxy)-tetrahydro-2 /-pyran-3,5-diol [a compound represented by the Formula III]

(III)

CuSCy5H₂0 (1.5 mg, 0.006 mmol) and sodium ascorbate (2.4 mg, 0.012 mmol) were mixed with 5 ml_ THF/H₂0 (1: 1) solution dissolving azide compound 3 (n = 3), (2 ?,5 ?)-2-( ? 6-azidohexan-2-yloxy)-6-methyl-tetrahydro-2 pyran-3,5-diol (16 mg, 0.06 mmol), and ethynylbenzene (6 mg, 0.06 mmol, 1 eq.). We investigate to what extent reaction is occurred using TLC in the solution with stirring for 10 min at room temperature. After terminating the reaction, the solution was diluted with 20 mL ethylacetate and washed with a saline solution. After drying and filtering the organic layer with MgS0₄, it was concentrated under reduced pressure and then pure compound 4 (a compound represented by the Formula III) was produced in the amount of 19 mg (yield rate, 90%) using a flash column chromatography (CH₂Cl₂/MeOH, 15:1, v/v). Physiochemical characteristics of the final products were as follows:

C₂iH₃iN₃0₄, a white solid material, R = 0.3 (M.C/MeOH, 9:1, v/v); [a]_D ²⁰ = - 88.00° (c = 0.03, MeOH); 'H-NMR (250 MHz, CDCI₃); δ 7.83-7.80 (d, J = 7.5 Hz, 2H), 7.76 (s, 1H), 7.44-7.40 (t, 2H), 7.34-7.31 (t, 1H), 4.69 (s, 1H), 4.44-4.37 (t, 2H), 3.79-3.75 (m, 2H), 2.17 (s, 2H), 2.1-2.04 (m, 1H), 2.00-1.93 (m, 2H), 1.86- 1.83 (m, 1H), 1.56-1.35 (m, 6H), 1.26-1.24 (d, J = 5 Hz, 3H), 1.11-1.09 (d, J = 5 Hz, 3H); ¹³C-NMR (62.9 MHz, CDCI₃); δ 147.85, 130.56, 128.97, 128.30, 125.81, 119.63, 96.17, 71.21, 70.08 , 69.41, 68.09, 50.35, 37.05, 35.37, 31.03, 30.20, 26.60, 24.84, 19.04, 17.86. IR (film) v_mM 3392, 2929, 1739, 1650, 1450, 1383, 1228, 1125, 1099, 1043, 1028, 982, 833, 766, 695 cm^"1 LC Mass ESI probe 412 Da/e, Scan ES+5.7e.

EXAMPLE 3: Synthesis of (3 ?,5 ¾6 ?)-2-methyl-6-(( 0-8-(4-phenyl-l - l,2,3-triazol-l-yl)octan-2-yloxy)-tetrahydro-2/ -pyran-3,5-diol [a compound represented by the Formula IV]

(IV)

CuS0 '5H₂0 (1.5 mg, 0.006 mmol) and sodium ascorbate (2.4 mg, 0.012 mmol) were mixed with 5 mL THF/H₂0 (1: 1) solution dissolving azide compound 3 (n = 4), (2 ?,5 ?)-2-(( ?)-8-azidooctan-2-yloxy)-6-methyl-tetrahydro-2 pyran-3,5-diol (15 mg, 0.04 mmol), and ethynylbenzene (6 mg, 0.06 mmol, 1 eq.). We investigate to what extent reaction occurs using TLC in the solution with stirring for 10 min at room temperature. After terminating the reaction, the solution was diluted with 20 mL ethylacetate and washed with a saline solution. After drying and filtering the organic layer with MgS0₄, it was concentrated under reduced pressure and then pure compound 4 (a compound represented by the Formula IV) was produced in the amount of 11 mg (yield rate, 92%) using a flash column chromatography (CH₂Cl₂/MeOH, 15:1, v/v). Physiochemical characteristics of the final products were as follows:

C₂₂H₃₃N₃0₄, a white solid material, R = 0.3 (M.C/MeOH, 9:1, v/v); [a]_D ²⁰ = - 86.00° (c = 0.05, MeOH); 'H-NMR (250 MHz, MeOD); δ 8.32(s, 1H), 7.83-7.80 (d, J = 7.5 Hz, 2H), 7.45-7.30 (m, 3H), 4.63 (s, 1H), 4.47-4.41 (t, 2H), 3.77-3.68 (m, 2H), 3.65-3.46 (m, 2H), 1.98-1.91 (m, 3H), 1.81-1.70 (m, 1H), 1.6-1.1.32 (m, 8H), 1.21- 1.18 (d, J = 7.5 Hz, 3H), 1.11-1.09 (d, J = 5 Hz, 3H); ¹³C-NMR (62.9 MHz, MeOD); 5148.83, 131.75, 129.98, 129.32, 126.66, 122.177, 97.55, 72.49, 71.17 , 69.94, 68.30, 51.48, 38.23, 35.95, 31.24, 30.03, 27.44, 26.66, 19.36, 18.12. IR (film) v_mM 3395, 2939, 1734, 1645, 1634, 1450, 1383, 1228, 1125, 1099, 1043, 1028, 982, 833, 726, 678cm^"1 LC Mass ESI probe 426 Da/e, Scan ES+4.7e.

EXAMPLE 4: Synthesis of (2/?)-2-(3,6-dideoxy-a-L-mannopyranosyloxy)- tridecanal [a compound represented by the Formula V]

The compound, (2 ?)-2-(2,4-Di-i benzyl-3,6-dideoxy-a-L-mannopyranosyoxy)- tridecanal (40 mg, 0.07 mmol), was dissolved in 5 mL MeOH in a round flask. After NaOCH₃ (14 mg, 0.21 mmol) was added to the solution at 0°C, the reaction temperature was increased to room temperature, changing the solution to yellow color depending on to what extent a benzoyl protecting group is removed. After stirring for 8 hrs, the reaction is stopped and Amberlite-IR120 (H⁺) was gradually added to determine whether the solution is neutralized using a pH paper. The solution is filtered in neutralization condition to remove resin, and the solvent is eliminated through distillation under reduced pressure, followed by extracting with distilled water and EtOAc (20 mL, 3 times). After washing with brine and drying and filtering the organic layer with MgS0₄, it was concentrated under reduced pressure, and then compound 6 (a compound represented by the Formula V) was produced in the amount of 19.5 mg (yield rate, 86%) using a flash column chromatography (CH₂Cl2/MeOH, 15: 1, v/v). Physiochemical characteristics of the final products were as follows:

Ci₉H₃₆0₅, a colorless syrup, Rf = 0.22 (n-Hexane/EtOAc/MeOH, 5:2: 1, v/v/v); [a]_D ²³ = -11.0 (c = 0.05, MeOH); ^!H NMR (250 MHz, CDCI₃); δ 9.68 (s, 1H), 4.61 (s, 1H). 3.73-3.67 (m, 2H), 3.64-3.47 (m, 2H), 2.63 (bs, 2H), 2.38-2.32 (m, 2H), 2.00- 1.95 (m, 1H), 1.80-1.71 (m, 1H), 1.58-1.43 (m, 4H), 1.42-1.18 (m, 17H), 1.05 (d, 3H, J = 5.9 Hz), 1.14 (d, 3H, J= 6.1 Hz); ¹³C NMR (62.9 MHz, CDCI₃); δ 203.1, 96.1, 71.6, 69.8, 69.3, 67.9, 43.9, 37.2, 35.1, 29.6, 29.4, 29.3, 29.1, 25.8, 22.1, 19.0, 17.7; IR (film) i_max 3430, 2927, 2854, 1636, 1558, 1457, 1377, 1253, 1123, 1028 cm^"

1

EXAMPLE 5: Inhibitory effect of the compounds, DD189, DD204 and DD223, on aortic smooth muscle cell proliferation

Effects of synthetic glycolipid derivative compounds of the present invention represented by the formulae 2, 3 and 4 (designated hereinafter as DD189, DD204 and DD223, respectively) in the above-mentioned Examples were determined on inhibition of aortic smooth muscle cell proliferation in white mouse. Briefly, aortic smooth muscle cells (4.0 x 10⁴ cells/mL) were divided into a 12-well plate and cultured for 24 hrs in DMEM supplemented with 0.5%(v/v) fetal bovine serum. Afterwards, the compound DD189 with concentrations of 10, 20 or 40 μΜ, the compound DD204 with concentration of 10, 20 or 50 μΜ, or the compound DD223 with concentration of 5, 20 or 50 μΜ was added to the media, respectively. After further incubating for 24 hrs, the media was treated with PDGF-BB (platelet-derived growth factor-BB) and then the cells were separated from the bottom 24 hrs after trypsinization, followed by counting a separated cell number using a hemocytometer.

In Figs, la-lc, after each compound DD189 (10, 20 or 40 μΜ), DD204 (10, 20 or 50 μΜ) and DD223 (5, 20 or 50 μΜ) was treated to aortic smooth muscle cells in white mouse, it was represented as a graph to assess to what extent cell proliferation is inhibited. According to results in Figs, la-lc, it could be appreciated that the glycolipid derivative compounds of the present invention (DD189, DD204 and DD223) have inhibitory activities on aortic smooth muscle cell proliferation in dose-dependent manner. EXAMPLE 6: Effect of the compounds, DD189, DD204 and DD223, on DNA synthesis of aortic smooth muscle cell

According to the above-described Example 5, it was demonstrated that the compounds (DD189, DD204 and DD223) exert inhibitory effects on aortic smooth muscle cell proliferation. To verify how mechanism works in inhibitory activities of these compounds on aortic smooth muscle cell proliferation, we examined effects of the compounds (DD189, DD204 and DD223) on DNA synthesis in cell proliferation. First, aortic smooth muscle cells (2.0 x 10⁴ cells/mL) were seeded into a 24-well plate and maintained for 24 hrs in DMEM supplemented with 0.5%(v/v) FBS. Afterwards, the compound DD189 with concentrations of 10, 20 or 40 μΜ, the compound DD204 with concentration of 10, 20 or 50 μΜ, or the compound DD223 with concentration of 5, 20 or 50 μΜ was added to the media, respectively. After further incubating for 24 hrs, the media were treated with PDGF-BB (platelet-derived growth factor-BB) and then with 1 μΙ [³H]-thymidine 20 hrs after PDGF-BB treatment. After further incubation for 4 hrs, the cells were washed with PBS two times and treated with TCA reagent for 30 min. TCA reagent was removed and the cells were washed with ethanol/ether (1: 1 v/v) two times. The cells were completely lysed to add IN NaOH (800 μΙ) to each well, and then mixed with 4 mL scintillation cocktail, followed by measuring radioactivity in each well with a liquid scintillation counter.

In Figs. 2a-2c, after each compound DD189 (10, 20 or 40 μΜ), DD204 (10, 20 or 50 μΜ) and DD223 (5, 20 or 50 μΜ) was treated to aortic smooth muscle cells in white mouse, it was represented as a graph to assess to what extent DNA synthesis is inhibited in these cells. According to results in Figs. 2a-2c, it could be appreciated that the glycolipid derivative compounds of the present invention (DD189, DD204 and DD223) have highly inhibitory effects on DNA synthesis of aortic smooth muscle cells in dose-dependent manner. EXAMPLE 7: Effect of the compounds, DD189, DD204 and DD223, on proliferative cell cycle of aortic smooth muscle cells

We investigated changes of cell cycle depending on treatment of the compounds (DD189, DD204 and DD223) of the present invention to aortic smooth muscle cells of which proliferation were induced by PDGF-BB. First, aortic smooth muscle cells (4.0 x 10⁴ cells/mL) were divided into a 6-well plate and cultured for 24 hrs in DMEM supplemented with 0.5%(v/v) FBS. Next, the compound DD189 with concentrations of 10, 20 or 40 μΜ, the compound DD204 with concentration of 10, 20 or 50 μΜ, or the compound DD223 with concentration of 5, 20 or 50 μΜ was added to the media, respectively. After further incubating for 24 hrs, the media were treated with PDGF-BB (platelet-derived growth factor-BB) and then the cells were stained using PI staining after further incubation. Subsequently, the amount of cells was determined using a FACS (fluorescence activated cell sorter) method.

In Figs. 3a-3c, after each compound DD189 (10, 20 or 40 μΜ), DD204 (10, 20 or 50 μΜ) and DD223 (5, 20 or 50 μΜ) was treated to aortic smooth muscle cells in white mouse, it was represented as a graph to assess their effects on proliferative cell cycle of aortic smooth muscle cells. From the results in Figs. 3a-3c, it could be appreciated that the glycolipid derivative compounds (DD189, DD204 and DD223) of the present invention with various concentrations exert an inhibitory effect on proliferative cell cycle progression of aortic smooth muscle cells through arrest in inter-phase between G0/G1 phase and S phase in cell cycle of aortic smooth muscle cells. Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.

Claims

What is claimed is:

1. A glycolipid derivative compound represented by the following Formula I:

wherein R represents Q-Ci straight alkyl substituted with ^cinnamoyl, CrCio straight alkanal or phenyltriazolyl group.

2. A composition for inhibiting vascular smooth muscle cell proliferation, comprising a glycolipid derivative compound represented by the following Formula I:

O'

HO Z¾^

OH _(I)

wherein R represents Q-Q straight alkyl substituted with .F-cinnamoyl, Ci-Ci₀ straight alkanal or phenyltriazolyl group.

3. A pharmaceutical composition for preventing or treating a hyper-proliferative disease of vascular smooth muscle cells, comprising: (a) a therapeutically effective amount of a glycolipid derivative compound represented by the following Formula I; and (b) a pharmaceutically acceptable carrier:

0'" ^^"^-'^

H0 2¾^

OH _(I)

wherein R represents Ci-Q straight alkyl substituted with -cinnamoyl, Q-Cio straight alkanal or phenyltriazolyl group.

4. The composition according to claim 3, wherein the glycolipid derivative compound represented by Formula I comprises a compound represented by the following Formulae II, III, IV or V:

5. The composition according to claim 3, wherein the hyper-proliferative disease of vascular smooth muscle cells comprises vascular restenosis, vascular stenosis, arteriosclerosis, atherosclerosis, heart failure, myocardial infarction, angina pectoris, arrhythmia, hypertensive heart disease, congenital heart disease, stroke or peripheral vascular stenosis.

6. The composition according to claim 3, wherein the compound represented by the formula I is used in a concentration of 5-50 μΜ in the composition.