KR101466377B1 - dimethoxyphenyldihydropyrazolylnaphthalenol derivatives, preparing method of the same and Use in anti-cancer agent thereof - Google Patents

Abstract

The present invention relates to dimethoxyphenyl dihydropyrazolyl naphthalenol derivatives, their preparation and their use as anticancer agents, and more particularly to dimethoxyphenyl dihydropyrazolyl naphthalenyl derivatives of human colorectal cancer cells, And to a pharmaceutical composition for an anticancer agent having a progressive inhibitory effect.

Description

Dimethoxyphenyldihydropyrazolylnaphthalenol derivatives, preparation method of the same and Use of anti-cancer agent thereof,

The present invention relates to dimethoxyphenyl dihydropyrazolyl naphthalenol derivatives, their preparation and their use as anticancer agents, and more particularly to dimethoxyphenyl dihydropyrazolyl naphthalenyl derivatives of human colorectal cancer cells, And to a pharmaceutical composition for an anticancer agent having a progressive inhibitory effect.

All cells grow normally through cell cycle control by various mechanisms. The process of cell cycle is divided into interphase and mitotic phase. The former is further divided into three processes: G (1) -phase, where cells synthesize various proteins necessary for cell division, and the mitotic phase, which is the S-phase and DNA differentiation . Various cyclin proteins involved in cell division process regulate cell cycle phase by forming a cyclin / CDK complex with a kinase called cyclin dependent kinase (CDK). Among them, cyclin D1 (Cyclin D1) is a protein that plays an important role in the progression from the G1 stage to the S stage. Cyclin D1 phosphorylates Retinoblastoma protein (pRb) when activated by complexing with CDK4 or CDK6. The retinoblastoma protein binds to the transcription factor E2F protein and causes the cell cycle to stop at G1 stage. When phosphorylated by CycD1, it becomes inactivated and E2F is released. [Seminars Cancer Biol. 6 , 99 (1995). This activates E2F as a transcription factor and facilitates transcription and translation of proteins for cell cycle progression, leading to the cell cycle from G1 to S phase. In normal cells, this cell cycle regulating mechanism occurs normally, and cell division and proliferation are appropriately performed and the cell number is appropriately controlled. On the other hand, it is known that excessive proliferation and growth of cells occurs due to abnormal cell cycle progression and the like in cancer cells. In some cancer cells, cyclin D1 (cyclin D1) gene amplification or protein overexpression was observed. [Am. J. Pathology 147, 545 (1995)]. Therefore, when the expression level of cyclin D1 is decreased, the phosphorylation of pRB is not normally performed. Therefore, the cell cycle is maintained in the G1 stage, so that abnormal cell division and proliferation of cancer cells can be prevented, and thus cancer therapy can be applied.

Korean Patent Application No. 10-2006-0099919 discloses a novel naringenin derivative that regulates apoptosis, a method for producing the same, and a use thereof as an anticancer agent, and a natural flavonoid compound, naringin The present invention relates to novel naringenin derivatives substituted by a hydroxy group, a method for producing the same, and a use thereof as a therapeutic agent for colorectal cancer. The naringenin derivatives are useful for regulating cell signaling, And thus it can be widely used as a therapeutic agent for various cancers including colon cancer.

The present invention has been made in view of the above needs, and an object of the present invention is to provide an effective anticancer agent.

Another object of the present invention is to provide a method for producing an anticancer agent.

It is still another object of the present invention to provide a composition for the prevention and treatment of colorectal cancer.

In order to achieve the above object, the present invention provides a dimethoxyphenyl dihydropyrazolyl naphthalenyl derivative represented by the following formula (1).

[Chemical Formula 1]

In one embodiment of the present invention, the compound preferably has an effect of inhibiting cycling-di 1 expression, but is not limited thereto.

The present invention also provides a composition comprising, as an active ingredient, a compound of the following formula:

[Chemical Formula 1]

In Formula 1, R is a functional group selected from the group consisting of H, p-methoxy, and bism-p-trimethoxy.

The present invention also provides a pharmaceutical composition comprising, as an active ingredient, a compound represented by the following formula (1) and a pharmaceutically acceptable carrier:

[Chemical Formula 1]

In Formula 1, R is a functional group selected from the group consisting of H, p-methoxy, and bism-p-trimethoxy.

The present invention also provides an anticancer pharmaceutical composition comprising, as an active ingredient, a compound represented by the following formula (1) and a pharmaceutically acceptable carrier:

[Chemical Formula 1]

In Formula 1, R is a functional group selected from the group consisting of H, p-methoxy, and bism-p-trimethoxy.

In one embodiment of the present invention, the composition preferably has anticancer effect on colon cancer, but is not limited thereto.

In another embodiment of the present invention, the composition preferably inhibits G1 cell cycle progression of cancer cells, but is not limited thereto.

The present invention relates to a process for the preparation of benzoquinone compounds, comprising the steps of: a) mixing 1'-hydroxy-2'-acetonaphthone with 2,3-dimethoxy-substituted benzaldehyde to obtain a benzocholacone compound, b) After dissolving, obtaining phenyldihydropyrazolylnaphthalenol from the reaction mixture; And c) adding 1-isothiocyanatobenzene to the phenyldihydropyrazolylnaphthalenol to prepare a dimethoxyphenyl dihydropyrazolyl naphthalenyl derivative represented by the following general formula (1).

[Chemical Formula 1]

In Formula 1, R is a functional group selected from the group consisting of H, p-methoxy, and bism-p-trimethoxy.

 The compositions of the present invention may be formulated into a variety of carriers and delivery systems. The amount of therapeutic compound administered and the concentration of the compound will depend on the vehicle or device selected, the clinical condition of the patient, side effects and the formulation stability of the compound. Thus, the physician will use the appropriate formulation containing the appropriate concentration of the therapeutic compound, and will select the dosage form according to clinical experience with the patient or similar patient in question.

In addition, excipients may be used in the formulation. Examples include co-solvents, surfactants, oils, wetting agents, emollients, preservatives, stabilizers and antioxidants. As a pharmaceutically acceptable buffer, for example, a tri or phosphate buffer may be used. Effective amounts of diluents, additives and excipients are amounts effective to obtain pharmaceutically acceptable formulations in terms of solubility, bioactivity, and the like.

Thus, the compositions of the present invention include conventional, pharmaceutically acceptable vehicles for topical, oral or parenteral administration, and therapeutic compounds that can be formulated. The formulations may also contain minor amounts of adjuvants, such as buffers and preservatives, to maintain isotonicity, physiological and pH stability.

The compositions of the present invention may be administered to both human and animal subjects.

The compositions of the present invention may be administered in a composition wherein the active compound is intimately mixed with one or more inactive ingredients and optionally one or more additional active ingredients. The compositions can be used as any composition for human and animal administration known in the art.

The composition of the present invention may be administered according to a proper route depending on the dosage form. For example, the subject can be administered intravenously, intraarterially, subcutaneously, intramuscularly, and the like.

For oral administration, either solid or fluid unit dosage forms may be prepared. The aqueous form may be dissolved in an aqueous vehicle together with a sugar, an aromatic flavor and a preservative to produce a syrup. Elsys are prepared using suitable sweeteners such as aromatic flavors, sugars and saccharin, and hydro-alcoholic (e.g., ethanol) vehicles. Suspensions can be prepared using aqueous vehicles, such as acacia, tragacanth, methylcellulose, and the like. The synthetic compound of the present invention may be formulated with a stabilizing agent, for example, a metal chelator reducing agent such as ethylenediaminetetraacetic acid (EDTA), or a reducing agent such as sodium metabisulfite.

Suitable formulations for parenteral administration will be apparent to those skilled in the art. Generally, the therapeutic compound is prepared in aqueous solution at a concentration of about 1 to about 100 mg / mL. More typically, the concentration is about 10 to 60 mg / mL or about 20 mg / mL. Depending on the stability and efficacy of the compound selected for use, in some cases concentrations below 1 mg / mL may be necessary.

The effective dose of the active ingredient of the present invention will depend, at least, on the nature of the therapeutic condition, toxicity, whether the compound is used prophylactically (with less dosage) or against an active cancer infection, on the delivery method and on its pharmaceutical formulations , Will be determined by the clinician using conventional dose escalation studies. The dose is generally from about 0.0001 to about 100 mg / kg body weight per day, generally from about 0.01 to about 10 mg / kg body weight per day, more usually from about 0.01 to about 5 mg / kg body weight per day, more usually from 1 Day to about 0.05 mg / kg body weight per day. For example, the daily recommended dose for an adult weighing about 70 kg will be from 1 mg to 1000 mg, preferably from 5 mg to 500 mg, and may be single or multiple dosage forms.

The sterile formulations are suitable for a variety of parenteral routes including intraperitoneal, intraarticular, intramuscular, intravascular, intravenous, inhalation and subcutaneous.

A slow or extended release delivery system comprising any of a number of biopolymers (bio-based systems), liposome systems and polymer delivery systems, such as dendrimers, can be utilized in the compositions herein to provide a continuous or long term source Can be provided. Such slow release systems are applicable for topical, ocular, oral and parenteral formulations.

The synthetic compound (s) of the present invention may also be formulated as a nutraceutical or a nutraceutical. For example, the synthetic compound (s) can be formulated into oral use foods such as cereal, fruit juices, alcoholic beverages, bread, and the like.

Hereinafter, the present invention will be described.

In the present invention, a substance that reduces the expression level of Cyc D1 was devised, and it was confirmed that the anticancer effect was excellent, thereby completing the present invention.

In order to find an effective anticancer agent, the present invention was designed by synthesizing dimethoxyphenyl dihydropyrazolylnaphthalenyl derivatives, and observed the effect of inhibiting cell cycle progression of colon cancer cells on these. Dimethoxyphenyl dihydropyrazolyl naphthalenol derivatives inhibit G1 cell cycle progression of colorectal cancer cells.

The dimethoxyphenyl dihydropyrazolyl naphthalenyl derivatives of the present invention exhibit an inhibitory effect on G1 cell cycle progression of colon cancer cells, and thus can be usefully used as a pharmaceutical composition for the prevention and treatment of cancer diseases.

1 is a schematic view showing the structure of a dimethoxyphenyl dihydropyrazolyl naphthalenyl derivative 5- (2,3-dimethoxyphenyl) -3- (1 -hydroxynaphthalen-2-yl) Pyrazole-1-carbothioamide (DK114). ≪ / RTI > (Using a 400 MHz Bruker nuclear magnetic resonance spectrometer)
FIG. 2 is a graph showing the results of the reaction of dimethoxyphenyl dihydropyrazolyl naphthalenyl derivative 5- (2,3-dimethoxyphenyl) -3- (1 -hydroxynaphthalen-2-yl) Carbon atom nuclear magnetic resonance spectroscopy of pyrazole-1-carbothioamide (DK114). (Using a 100 MHz Bruker nuclear magnetic resonance spectrometer)
FIG. 3 is a graph showing the molecular weight distribution of the dimethoxyphenyl dihydropyrazolyl naphthalenyl derivative 5- (2,3-dimethoxyphenyl) -3- (1 -hydroxynaphthalen-2-yl) Pyrazole-1-carbothioamide (DK114). ≪ tb >< TABLE > (Using a JMS700 HREIMS (high-resolution electron impact ionization mass spectrometer) instrument from Jeol Ltd., Tokyo, Japan)
4 is a diagram showing the structure of the dimethoxyphenyl dihydropyrazolyl naphthalenyl derivative 5- (2,3-dimethoxyphenyl) -3- (1-hydroxynaphthalen-2-yl) -N- (4-methoxyphenyl) Is a hydrogen nuclear magnetic resonance spectroscopy spectrum of 4,5-dihydropyrazole-1-carbothioamide (DK115). (Using a 400 MHz Bruker nuclear magnetic resonance spectrometer)
FIG. 5 is a graph showing the molecular weight distribution of the dimethoxyphenyl dihydropyrazolyl naphthalenyl derivative 5- (2,3-dimethoxyphenyl) -3- (1 -hydroxynaphthalen-2-yl) -N- (4-methoxyphenyl) Carbon atom nuclear magnetic resonance spectroscopy of 4,5-dihydropyrazole-1-carbothioamide (DK115). (Using a 100 MHz Bruker nuclear magnetic resonance spectrometer)
6 is a schematic diagram showing the structure of the dimethoxyphenyl dihydropyrazolyl naphthalenyl derivative 5- (2,3-dimethoxyphenyl) -3- (1 -hydroxynaphthalen-2-yl) -N- (4-methoxyphenyl) High-resolution mass spectrometry spectrum of 4,5-dihydropyrazole-1-carbothioamide (DK115). (Using a JMS700 HREIMS (high-resolution electron impact ionization mass spectrometer) instrument from Jeol Ltd., Tokyo, Japan)
7 is a graph showing the results of the reaction of dimethoxyphenyl dihydropyrazolyl naphthalenyl derivative 5- (2,3-dimethoxyphenyl) -3- (1 -hydroxynaphthalen-2-yl) -N- Methoxyphenyl) -4,5-dihydropyrazole-1-carbothioamide (DK119). (Using a 400 MHz Bruker nuclear magnetic resonance spectrometer)
FIG. 8 is a graph showing the results of measurement of the activity of the dimethoxyphenyl dihydropyrazolyl naphthalenyl derivative 5- (2,3-dimethoxyphenyl) -3- (1 -hydroxynaphthalen-2-yl) -N- Methoxyphenyl) -4,5-dihydropyrazole-1-carbothioamide (DK119). (Using a 100 MHz Bruker nuclear magnetic resonance spectrometer)
FIG. 9 is a cell growth inhibitory effect of dimethoxyphenyl dihydro pyrazolyl naphthalenyl derivatives in colorectal cancer cells.
10 is an effect of inhibiting the cyclin-di 1 expression of the dimethoxyphenyl dihydro pyrazolylnaphthalenyl derivative DK115 compound in colorectal cancer cells.
Fig. 11 shows the effect of the dimethoxyphenyl dihydropyrazolylnaphthalenyl derivative DK115 compound on G1 cell cycle progression inhibition in colorectal cancer cells.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example  One. Dimethoxyphenyl dihydro pyrazolyl naphthalenol  Synthesis of derivatives

In the present invention, the dimethoxyphenyl dihydropyrazolyl naphthalenyl derivatives of the present invention were synthesized as follows using the method shown in the following reaction formula.

1, 2'-acetonaphthone (I, 1,864 mg, 10 mmol) and 2,3-dimethoxy-substituted benzaldehyde (II, 10 mmol) were dissolved in 85 mL of ethanol. C down to. Add 10 mL of 50% KOH solution slowly to the above cooling solution, and stir at room temperature for about 40 hours. The reaction mixture is put into about 200 mL of ice water, acidified with 30 mL of 6N HCl, and the resultant solid is filtered. This solid was recrystallized from ethanol to obtain a pure benzocholacone compound (III). After dissolving the benzocholacone compound (III, 1 mmol) and hydrazine (1.2 mmol) in ethanol (30 mL), the reaction mixture was refluxed for about 10 hours. The reaction mixture was cooled to 5 ° C using an ice water bath The resulting solid was filtered and the filtered solid was recrystallized in ethanol to synthesize the pure compound phenyl dihydropyrazolyl naphthalenol (IV). 1-isothiocyanatobenzene (V) was added thereto to obtain the target compound dimethoxyphenyl dihydropyrazolyl naphthalenol derivatives.

Nuclear magnetic resonance spectroscopy experiments were performed to confirm the final product. The device used was a Bruscia 400MHz device. High resolution mass spectrometry was also used to confirm the structure of the derivatives identified by nuclear magnetic resonance spectroscopy. The instrument used was JMS700 HREIMS (high-resolution electron impact ionization mass spectrometer) from Jeol Ltd., Tokyo, Japan. Nuclear magnetic resonance spectroscopy confirmed the nomenclature according to the positions of hydrogen and carbon of the identifiers.

(2)

Dimethoxyphenyl dihydropyrazolyl naphthalenyl derivative 5- (2,3-dimethoxyphenyl) -3- (1 -hydroxynaphthalen-2-yl) -N-phenyl-4,5-dihydropyrazole- Hydrogen nuclear magnetic resonance of 5- (2,3-dimethoxyphenyl) -3- (1-hydroxynaphthalen-2-yl) -N-phenyl- 4,5-dihydropyrazole- 1-carbothioamide DK114 The spectroscopic spectrum and the carbon nuclear magnetic resonance spectrum are as shown in Fig. 1 and Fig. 2, respectively, and the chemical mobility is as follows.

^{1 H NMR (400MHz, DMSO-} d 6) δ 10.54 (bs, 1H, 1'-OH), 10.50 (s, 1H, 7-NH), 8.35 (d, 1H, H-8 ', J = 7.5 Hz 1H), 7.89 (d, 1H, H-5 ', J = 7.3 Hz), 7.62 (m, -3 ', J = 8.8 Hz) , 7.48 (dd, 2H, H-2''' / H-6 ''', J = 1.0, 8.5 Hz), 7.45 (d, 1H, H-4', J = 8.8 Hz), 7.36 (dd , 2H, H-3 '''/H-6''', J = 7.4, 8.5 Hz), 7.19 (td, 1H, H-4 ''', J = 1.0, 7.4 Hz), 7.01 (dd, 1H, H-5 ''', J = 7.6, 8.3 Hz), 6.95 (dd, 1H, H-4'', J = 1.5, 8.3 Hz), 6.65 (dd, 1H , H-6 '', J = 1.1, 7.6 Hz), 6.25 (dd, 1H, H-5, J = 3.6, 11.7 Hz), 4.16 (dd, 1H, H-4, J = 11.7, 18.0 Hz) , 3.84 (s, 3H, 2 '' - OCH 3), 3.81 (s, 3H, 3 '' - OCH 3), 3.26 (dd, 1H, H-4, J = 3.6, 18.0 Hz); ¹³ C NMR (100 MHz, DMSO-d ₆ )? 173.6 (C-6), 158.0 (C-3), 153.6 ), 139.8 (C-1 '''), 136.0 (C-1'') 134.9 (C-10'), 128.2 ''), 127.5 (C-5), 126.1 (C-2 '''/C-6'''), 125.9 '), 124.4 (C-9'), 123.9 (C-5 ''), 122.9 ''), 109.0 (C- 2 '), 59.6 (2''- OCH 3), 58.3 (C-5), 55.6 (3''- OCH 3), 42.6 (C-4).

Dimethoxyphenyl dihydropyrazolyl naphthalenyl derivative 5- (2,3-dimethoxyphenyl) -3- (1 -hydroxynaphthalen-2-yl) -N-phenyl-4,5-dihydropyrazole- 1-carbonyloxy thioamide (DK114) has a molecular formula of _{C 28 H 25 N 3 O 3} S as a new material that is not reported to date. High-resolution mass spectrometry was used to confirm the structure of this compound identified by nuclear magnetic resonance spectroscopy. The compound had a molecular weight of 483.1617 and an experimental molecular weight of 483.1744. Therefore, this compound was found to be 5- (2,3-dimethoxyphenyl) (1-hydroxynaphthalen-2-yl) -N-phenyl-4,5-dihydropyrazole-1-carbothioamide. The high-resolution mass spectrometry spectrum of this compound is shown in FIG.

Dimethoxyphenyl dihydropyrazolyl naphthalenyl derivative 5- (2,3-dimethoxyphenyl) -3- (1-hydroxynaphthalen-2-yl) -N- 2-yl) -N- (4-methoxyphenyl) -4,5-dihydropyrazole-1, 2-dihydroxypyrazole- -carbothioamide; DK115) are shown in FIG. 4 and FIG. 5, respectively, and the chemical mobility thereof is as follows.

^{1 H NMR (400MHz, DMSO-} d 6) δ 10.51 (bs, 1H, 1'-OH), 10.39 (s, 1H, 7-NH), 8.34 (d, 1H, H-8 ', J = 7.4 Hz ), 7.89 (d, IH, H-5 ', J = 7.2 Hz), 7.61 (m, IH, H-6'), 7.58 -3 ', J = 8.7 Hz) , 7.45 (d, 1H, H-4', J = 8.7 Hz), 7.32 (d, 2H, H-2 '''/H-6''', J = 8.8 Hz), 6.99 (m, 1H , H-5 ''), 6.94 (m, 1H, H-4 ''), 6.92 (d, 2H, H-3 '''/H-5''', J = 8.8 Hz), 6.64 (d , 1H, H-6 '', J = 6.8 Hz), 6.23 (dd, 1H, H-5, J = 3.6, 11.7 Hz), 4.13 (dd, 1H, H-4 , J = 11.7, 18.0 Hz) , 3.83 (s, 3H, 2 '' - OCH 3), 3.80 (s, 3H, 3 '' - OCH 3), 3.76 (s, 3H, 4 '''- OCH 3 ), 3.23 (dd, 1H, H-4, J = 3.6, 18.0 Hz); ³ C NMR (100 MHz, DMSO-d ₆ )? 174.1 (C-6), 157.7 (C-3), 156.8 '), 145.0 (C-2''), 136.1 (C-1''), 134.8 C-6 ''), 127.5 (C-5 '), 126.0 (C-7'), 125.7 C-5 ''), 122.8 (C-8 '), 119.2 (C-4'), 117.8 -4 ''), 109.0 (C -2 '), 59.6 (2''- OCH 3), 58.3 (C-5), 55.6 (3''- OCH 3), 55.2 (4''- OCH 3) , 42.4 (C-4).

Dimethoxyphenyl dihydropyrazolyl naphthalenyl derivative 5- (2,3-dimethoxyphenyl) -3- (1-hydroxynaphthalen-2-yl) -N- -Dihydropyrazole-1-carbothioamide (DK115) has a molecular formula of C ₂₉ H ₂₇ N ₃ O ₄ S as a new substance not reported to date. High-resolution mass spectrometry was used to confirm the structure of this compound as confirmed by nuclear magnetic resonance spectroscopy. Since the theoretical molecular weight was 513.1722 and the molecular weight obtained by the experiment was 513.1852, the compound was found to be 5- (2,3-dimethoxyphenyl) 3- (1-hydroxynaphthalen-2-yl) -N- (4-methoxyphenyl) -4,5-dihydropyrazole-1-carbothioamide. The high-resolution mass spectrometry spectrum of this compound is shown in Fig.

Dimethoxyphenyl dihydropyrazolyl naphthalenyl derivative 5- (2,3-dimethoxyphenyl) -3- (1-hydroxynaphthalen-2-yl) -N- (3,4,5-dimethoxyphenyl) ) -4,5-dihydropyrazole-1-carbothioamide (5- (2,3-dimethoxyphenyl) -3- (1-hydroxynaphthalen-2-yl) -N- (3,4,5-trimethoxyphenyl) ) -4,5-dihydropyrazole-1-carbothioamide (DK119) are shown in FIG. 7 and FIG. 8, respectively, and the chemical mobility thereof is as follows.

^{1 H NMR (400MHz, DMSO-} d 6) δ 10.54 (bs, 1H, 1'-OH), 10.39 (s, 1H, 7-NH), 8.36 (d, 1H, H-8 ', J = 7.6 Hz ), 7.89 (d, IH, H-5 ', J = 7.3 Hz), 7.62 (m, IH, H-6'), 7.58 -3 ', J = 8.7 Hz) , 7.45 (d, 1H, H-4', J = 8.7 Hz), 7.01 (dd, 1H, H-5 '', J = 7.5, 8.2 Hz), 6.95 (dd H-4 '', J = 1.1, 8.2 Hz), 6.89 (s, 2H, H-2 ''' J = 1.1, 7.5 Hz), 6.27 (dd, 1H, H-5, J = 3.5, 11.8 Hz), 4.15 (dd, 1H, H-4, J = 11.8, 18.0 Hz), 3.85 (s, 3H, _{2 '' - OCH 3),} 3.81 (s, 3H, 3 '' - OCH 3), 3.77 (s, 6H, 3 '''- OCH 3/5''' - OCH 3), 3.67 (s, 3H , 4 '''- OCH ₃ ), 3.26 (dd, 1H, H-4, J = 3.5, 18.0 Hz); ^{13 C NMR (100MHz, DMSO-} d 6) δ 173.3 (C-6), 157.9 (C-3), 153.3 (C-1 '), 152.6 (C-3''), 152.1 (C-3'' (C-1 ''), 134.0 (C-10 '), 134.8 (C- 4 ''), 128.1 (C-6 '), 127.5 (C-5'), 125.9 C-2 '), 103.6 (C-4'), 122.9 (C-8 '), 119.1 2 '''/ C-6 '''), 60.0 (4 '''- OCH 3), 59.6 (2''- OCH 3), 58.2 (C-5), 55.8 (3''' - OCH 3 / 5 '''- OCH ₃ ), 55.6 (3''- OCH ₃ ), 42.5 (C-4).

Dimethoxyphenyl dihydropyrazolyl naphthalenyl derivative 5- (2,3-dimethoxyphenyl) -3- (1-hydroxynaphthalen-2-yl) -N- (3,4,5-dimethoxyphenyl) ) -4,5-dihydropyrazole-1-carbothioamide (DK119) has a molecular formula of C ₃₁ H ₃₁ N ₃ O ₆ S as a new substance not reported to date. The structure of this compound confirmed by nuclear magnetic resonance spectroscopy was unnecessary for further high resolution mass spectrometry experiments as compared with the structures of DK114 and DK115 mentioned above.

Example  2. In colorectal cancer cells Dimethoxyphenyl dihydro pyrazolyl naphthalenol  Inhibitory effect of derivatives on cell growth

HCT116 colon cancer cells were purchased from ATTC (American Type Culture Collection), DMEM (Invitrogen Life Technologies) culture medium containing 10% FBS (Fetal Bovine Serum, Invitrogen Life Technologies) and Antibiotic-Antimycotic solution The cells were cultured in a 5% CO ₂ incubator at 37 ° C in a 100-mm cell culture dish at a seeding density of 1 × 10 ⁶ . The inhibitory effect of the bromodymethoxycromonyl chalcone derivative compound on cell proliferation was evaluated by the colony forming assay of cancer cells. HCT116 colon cancer cells were divided into 6000 cells per well in a 24-well culture dish, and various dimethoxyphenyl dihydropyrazolyl naphthalenol derivatives (DK114, DK115, DK119) at concentrations of 0, 10, After 7 days, a mixed solution of 6% glutaraldehyde and 0.5% crystal violet solution was added to the cells, followed by reaction for 15 minutes to stain the remaining cells . As a result, as shown in FIG. 9, when the dimethoxyphenyl dihydropyrazolyl naphthalenyl derivative compound was treated at a concentration of 5 μM or more, it was observed that the colony forming ability of cancer cells was drastically reduced. From these facts, it has been confirmed that the dimethoxyphenyl dihydropyrazolyl naphthalenyl derivative (DK114, DK115, DK119) has an effect of inhibiting the proliferation of colon cancer cells.

Example  3. In colorectal cancer cells Dimethoxyphenyl dihydro pyrazolyl naphthalenol  Derivative Cyclin D1  Expression inhibitory effect

The effect of the DK115 compound on the cell cycle regulating effect was confirmed in the dimethoxyphenyl dihydropyrazolylnaphthalenyl derivative of the present invention. After treatment of DK115 with HCT116 cells, cyclin D1 (cyclin D1 cyclin D1) was analyzed by Western blot assay. HCT116 colon cancer cells were purchased from American Type Culture Collection (ATTC) and cultured in DMEM (Invitrogen Life Technologies) containing 10% FBS (Fetal Bovine Serum, Invitrogen Life Technologies) and Antibiotic-Antimycotic solution And incubated at 37 ° C in a 5% CO ₂ incubator with 10 ⁶ seed density.

HCT116 cells were treated with DK1115 at a concentration of 20 [mu] M, and cells were harvested after 24 and 48 hours. Cells were lysed by adding 20 mM HEPES buffer + 1% Triton X-100 + 10% glycerol + 150 mM NaCl + 10 μg / ml leupeptin + 1 mM PMSF to the harvested cells, And centrifuged to harvest only the cell lysate in the upper layer. The cell lysate prepared to contain the same amount of protein was subjected to SDS-polyacrylamide gel electrophoresis to separate the cellular proteins by molecular weight. The electrophoresed proteins were transferred to a nitrocellulose filter, and the primary antibody (purchased from Cell Signaling Technology) for cyclin D1 protein and the GAPDH protein, the protein amount of which was not changed as a control, (Purchased from Santa Cruz Technology) were reacted for 5 hours each, and a secondary antibody (purchased from Cell Signaling Technology), which was a primary antibody, was reacted for 1 hour. The expression of each protein was analyzed on an X-ray film using a chemiluminescence detection system (Amersham Pharmacia Biotech, Piscataway, NJ).

As a result, as shown in Fig. 10, it was confirmed that the treatment of DK1115 of the present invention gradually decreased the expression amount of cyclin D1 protein in a time-dependent manner. At this time, the amount of the control protein GAPDH was not changed. These results suggest that when the DK115 compound of the present invention acts on colon cancer cells, cyclin D1 (cyclin D1) protein expression is reduced and cell cycle progression is inhibited, thereby inhibiting the growth of cancer cells.

Example  4. In colorectal cancer cells Dimethoxyphenyl dihydro pyrazolyl naphthalenol  derivative DK115 of G1  Inhibition of cell cycle progression

Usually, the cell cycle progression is analyzed through measurement of the intracellular DNA content. Among the cell cycle, G1 periodic cells contain 2N DNA, S cells contain between 2N and 4N, G2 and M periodic cells contain 4N DNA. Since the cyclin D1 protein promotes the progression of the G1 cell cycle, whether the cell cycle progression of the cancer cells is impaired by the decrease of the CycD1 protein by DK107 is measured by a flow cytometer (BD Science, USA ).

HCT116 cells were cultured in the same manner as Western blotting method, and treated with DK115 compound, and after 0, 24, and 48 hours, trypsin-EDTA (1%) was added to remove the cells from the incubator. Cells were fixed with 70% . PI (Propidium Iodine) was reacted for 30 minutes to stain the intracellular DNA, and the change in the amount of DNA was measured.

As shown in FIG. 11, in the normally growing HCT116 colorectal cancer cells, about 49.0% of the cells having the G1 cell cycle were increased to 77.7% after 24 hours of treatment with the DK115 compound treated cells, and 80.4% Respectively. In addition, the S period of normal cells was 23.5%, but in the cells treated with DK115 compound, S period cells decreased to 6.4% and 6.3% after 24 and 48 hours of treatment, respectively. G2 / M periodic cells in normal cells were 23.0%, but in DK115 treated cells, G2 / M periodic cells gradually decreased with time at 11.4% and 8.7% after 24 and 48 hours of treatment, respectively. At this time, the amount of sub-G1 cells indicating cell death was not significantly changed by treatment with DK115 compound. From these results, it was confirmed that the DK1157 compound of the present invention inhibits the cell cycle progression from the G1 phase to the S phase of HCT116 colon cancer cells, thereby inhibiting the growth of cancer cells.

Having described specific portions of the present invention in detail, it will be apparent to those skilled in the art that this specific description is only a preferred embodiment and that the scope of the present invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

A compound of formula

[Chemical Formula 1]
In Formula 1, R is a functional group selected from the group consisting of H, p-methoxy, and 3,4,5-trimethoxy. 2. The compound according to claim 1, wherein the compound has a cycling-diI expression inhibiting effect. delete delete An anticancer pharmaceutical composition comprising a compound represented by the following formula (1) and a pharmaceutically acceptable carrier as an active ingredient:

[Chemical Formula 1]
In Formula 1, R is a functional group selected from the group consisting of H, p-methoxy, and 3,4,5-trimethoxy. The anticancer pharmaceutical composition according to claim 5, wherein the composition has anticancer effect on colon cancer. The anticancer pharmaceutical composition according to claim 5 or 6, wherein the composition has an effect of inhibiting G1 cell cycle progression of cancer cells. 1'-hydroxy-2'-acetonaphthone and 2,3-dimethoxy-substituted benzaldehyde to obtain a benzocholacone compound;
Dissolving the benzocholactone compound and hydrazine, and then obtaining phenyldihydropyrazolylnaphthalenol from the reaction mixture;
And adding 1-isothiocyanatobenzene to the phenyldihydropyrazolylnaphthalenol to prepare a dimethoxyphenyl dihydropyrazolyl naphthalenyl derivative represented by the following formula (1).

[Chemical Formula 1]
In Formula 1, R is a functional group selected from the group consisting of H, p-methoxy, and 3,4,5-trimethoxy.

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