KR100634232B1 - Novel compounds having anticancer activity, preparation method thereof, and anticancer active pharmaceutical composition containing the compound - Google Patents

Abstract

The present invention is a twin compound of formula (I) having the anti-binding activity synthesized by combining DMNQ with a conventional anticancer agent, a preparation method thereof, and an anticancer activity containing an effective amount of the twin compound and a pharmaceutically acceptable salt thereof. It relates to a pharmaceutical composition having a. Tween compounds of the present invention specifically act on human cancer cell lines A549, HT-1080, SK-OV-3, U937 and the like, and induce apoptosis in two types of A549 mutated p53 of human lung cancer line A549. It was confirmed that, in animal experiments it was confirmed that inhibits tumor growth.

Anticancer, Chlorambucil, A549

Description

Novel compound with anti-cancer activity, method for preparing the same and pharmaceutical composition with anti-cancer activity containing said compound}

1A to 1D show cytotoxic effects in human cancer cell lines of DMNQ and CAM as a control compound, and in two cancer lines of p53 variant of A549.

Figure 2a shows apoptotic bodies through DAPI staining in two species of p53 mutant lung carcinoma treated with twin compounds.

Figure 2b shows the cell death rate through DAPI staining in two species of p53 mutant lung carcinoma treated with twin compounds.

Figure 3a shows the change in body weight according to the twin compound dose to carcinoma prepared by inoculating the rat lung cancer LLC in the rat armpit area.

Figure 3b shows a carcinoma growth inhibitory effect according to the twin compound dose to carcinoma prepared by inoculating the rat lung cancer LLC in the rat armpit area.

4A to 4D show NMR spectra of twin compounds.

The present invention relates to a twin compound having anticancer activity prepared by synthesizing 6- (1-hydropentyl) -5,8-dimethoxy-1,4-naphthoquinone with chloroambucil, an existing anticancer agent, and a method for preparing the same. It is about. The present invention also relates to a pharmaceutical composition having an anticancer activity containing an effective amount of the tween compound and a pharmaceutically acceptable salt thereof.

Cancer is currently the number one cause of death in Korea, and the rate of cancer patients is increasing rapidly due to environmental problems and westernization of diet. Current treatments for cancer are classified into three types: surgical surgery, radiation therapy and drug therapy.

Early stage cancers can be treated by surgical procedures, but if the cancer has advanced or metastasized, surgical surgery alone is difficult to treat and other methods must be used together. In the case of radiation therapy, it is difficult to suppress the spread of new cancers or metastases due to irradiation, and thus is concurrent with drug therapy. The advantage of pharmacotherapy over radiotherapy or surgery is that the drug can reach any part of the body and can treat the metastasized cancer. It plays an important role in improving quality and extending life. Currently used anti-cancer drugs are classified into chemotherapy and biotherapy.

Chemotherapeutic agents include alkylating agents, metabolic antagonists, antibiotics, plant-derived alkaloids and hormonal agents. Biotherapeutic agents include immunotherapies cytokines and recombinant monoclonal antibodies. Alkylating agent, a kind of chemotherapeutic agent, exhibits anticancer activity by inhibiting DNA synthesis through crosslinking of DNA or abnormal base pairing.

As the representative alkylating agent, chloroambucil is an anticancer agent used in the clinic and is mainly used as chemotherapy for ovarian cancer, chronic leukemia, and lymphoma. However, the effect is insignificant, and due to the emergence of resistance to the drug, the maintenance period does not last long, and side effects and toxicity are so strong that its use is limited. In order to overcome the resistance to such drugs, the development of a combination chemotherapy using drugs with different mechanisms of action is being developed. Combination chemotherapy with cyclophosphamide, Adriamycin, and Cisplatin in combination with advanced ovarian cancer has been reported. There have been reports of chloroamsil in combination. Recently, Mark et al., Mark D. Wittman et al, 2001. Synthesis and Antitumor Activity of Novel Paclitaxel-Chlorambucil Hybrids. Bioorganic and Medicinal Chemistry letters, 11; 811-814.] The anti-cancer effect of the material prepared by combining Paclitaxel and alkylating agent anticancer agent was verified.

However, attempts to synthesize compounds having excellent anticancer activity by synthesizing chloroambucil and other anticancer components have not been known. The present invention has been made in view of the above-described circumstances, and has fewer side effects, and shows synergism according to the different mechanism of action between chloroambucil and DMNQ derivatives, and at a lower concentration than when chloroambucil is administered alone. It is an object of the present invention to provide a novel anticancer active compound capable of exhibiting anticancer activity, a method for preparing the same, and a pharmaceutical composition containing the same as an active ingredient.

In the present invention, in order to solve the above problems, naphthazarine derivatives already synthesized by the present inventors as active ingredients to be added to chloroambusil and demonstrated anticancer effects (Song et al., Arch. Pharm, Pharm, Med, Chem ,, 333, 87-92, 2000a; as a starting material of Song et al., Eur. J. Med. Chem., 35, 291-298, 2000b). L1210 demonstrated strong cytotoxic activity against cancer cells (GY Song, Y. Kim, XG Zheng, YY You, DE Sok, BZ Ahn. Bioorg. Med. Chem. Lett ., 1999 , 9, 2407-2412. ) 6- (1-hydroxypentyl) -5,8-dimethoxy-1,4-naphthoquinone) (sometimes referred to as DMNQ) was used.

That is, the present invention provides a compound of formula (I), which is a new compound having anticancer activity by reacting DMNQ having an anticancer effect with chloroambucil, an existing anticancer agent, and the present inventors have the following compounds, 6- { 1- [4- (p-bis (2-chloroethyl) -aminophenyl) -butanoyloxy] pentyl} -5,8-dimethoxy-1,4-naphthoquinone is termed a twin compound It was.

                Formula (Ⅰ)

The present invention also comprises dissolving DMNQ in anhydrous dichloromethane together with dicyclohexylcarbodiimide (sometimes referred to as DCC) and 4-dimethylaminopyridine (sometimes referred to as DDMA), followed by chloroam in the resulting solution. It provides a method for preparing a twin compound of the formula (I) comprising the step of adding insolvent, and separating and purifying the obtained product.

The present invention also provides a pharmaceutical composition having an anticancer activity containing an effective amount of the twin compound of formula (I) and a pharmaceutically acceptable salt thereof.

The present invention also provides an anticancer active pharmaceutical formulation formulated as an injection, liquid or coating.

The present inventors have completed the present invention by finding that the twin compound, which is a red-yellow oily substance prepared through the above steps, expresses anti-cancer effects such as cytotoxicity, cell death induction, and tumor growth inhibition.

 DMNQ (6- (1-hydroxypentyl)), a starting material of the twin compound of the present invention

-5,8-dimethoxy-1,4-naphthoquinone) exhibits DNA topo I inhibition and strong cytotoxic activity against L1210 cancer cells.Chlorambucil is an anticancer agent, an alkylating agent, that synthesizes DNA through crosslinking or abnormal base pairing. By inhibiting it will show anticancer activity. Thus, the twin compounds of the formula (I) obtained by synthesizing DMNQ and Chlorambucil showing anticancer activity in different mechanisms of action show higher anticancer effects than the existing chloroambucil by the different mechanisms of action.

To prepare the tween compound, 6- (1-hydroxypentyl) -5,8-dimethoxy-1,4-naphthoquinone 304 mg (1 mM), dicyclohexylcarbodiimide 247 mg (1.2 mM), After dissolving 146 mg (1.2 mM) of 4-dimethylaminopyridine in 100 mL of anhydrous dichloromethane, 304 mg (1 mM) of chloroambucil (product name: # C0253 from Sigma) was slowly added dropwise at 0 ° C for 30 minutes. The resulting reaction solution was stirred at room temperature for 12 hours and filtered. The residue obtained by concentrating the filtrate under reduced pressure with an evaporator was separated and purified using silica gel, to obtain 6- {1- [4- (p-bis (2-chloroethyl) -aminophenyl) -buta, a desired dark brown liquid substance. Noyloxy] pentyl} -5,8-dimethoxy-1,4-naphthoquinone 468 mg (78%) is obtained.

Such twin compounds (6- {1- [4- (p-bis (2-chloroethyl) -aminophenyl)-

Butanoyloxy] pentyl} -5,8-dimethoxy-1,4-naphthoquinone) is as follows.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments of the present invention. However, the following examples are merely to help the understanding of the present invention, the scope of the present invention is not limited only to the following examples.

<Example 1>

Preparation of Twin Compounds

304 mg (1 mM) of 6- (1-hydroxypentyl) -5,8-dimethoxy-1,4-naphthoquinone in the presence of nitrogen gas, 247 mg (1.2 mM) dicyclopexylcarbodiimide, 4-dimethylamino After dissolving 146 mg (1.2 mM) of pyridine in 100 mL of anhydrous dichloromethane, 304 mg (1 mM) of chloroambucil (product name: # C0253 manufactured by Sigma) was slowly added dropwise at 0 ° C for 30 minutes. The reaction solution was stirred at room temperature using a magnetic stirrer for 12 hours and then filtered after 24 hours. The residue obtained by concentrating the filtrate under reduced pressure with an evaporator was separated and purified using silica gel (30% n-hexane in ethyl acetate) to obtain 6- {1- [4- ( 468 mg (78%) of p-bis (2-chloroethyl) -aminophenyl) -butanoyloxy] pentyl} -5,8-dimethoxy-1,4-naphthoquinone were obtained.

When the NMR spectrum of the obtained twin compound is as shown in Figs. 4A to 4D, the peak of the NMR spectrum was confirmed as follows.

1 H-NMR (CDCl ₃ ) δ 7.24 (s, 1H), 7.03 (d, 2H), 6.76 (s, 2H), 6.61 (d, 2H), 6.12 (m, 1H), 3.91 (s, 6H), 3.70 (s, 4H), 3.59 (s, 4H), 2.37-2.57 (m, 4H), 1.76-1.92 (m, 4H), 1.24-1.39 (m, 4H), 0.86 (s, 3H), IR ( KBr, cm ^-1 ) υ _max : 3050, 2920, 1750, 1620, 1580

<Example 2>

(1) Preparation of Injection

Tween compound (3 μM, 6 μM, 12 μM weight), Tween 20 10% by weight, sterile water 100% by weight The above ingredients were combined in the amounts shown to prepare a vial (100 mg).

(2) Preparation of liquid

Heineanol A-containing liquid formulation was prepared by the following method.

Heineanol A treated with the cells was used by dissolving in an emulsifier (DMSO) at a concentration of 200 mM, and diluted with medium to the concentration used in the following examples as needed.

Experimental Example 1

Culture of Cell Lines

Human cancer cell lines A549, HT-1080, SK-OV-3, and U937 were cultured as follows. The cell lines were cultured in an incubator at 37 ° C., 5 μC ₂ and 95% air in RPMI 1640 (GIBCO-BRL, Richmond, USA) medium containing 10% fetal calf serum.

Experimental Example 2

Cancer cell proliferation inhibitory effect experiment

In order to measure the cancer treatment effect of the twin compounds of the present invention, experiments were carried out on cancer cell proliferation inhibitory effect using human cell lines A549, HT1080, SK-OV-3, and U937 cells. MTT assay was performed to examine cytotoxicity. Each cultured cancer cells were dispensed at about 10,000 per well in a 96 well plate (96well plate), and after 24 hours, 100 μl of the Twin compound solution prepared in Example 2 was passage-diluted for each concentration. After 24 hours after the addition of the drug, MTT solution was added and reacted. After 4 hours of incubation, the absorbance was measured by a microplate reader (wavelength: 570 nm) to measure the survival rate. Figure 1a to 1d shows the results of the survival rate of human lung cancer cell line A549, two types of human cancer cell line and p53 mutation according to the concentration of the twin compounds, respectively, Figure 1a shows the cytotoxicity in the A549 cell line, Figure 1b is Sk- Cytotoxicity in ov-3 cell line is shown and FIG. 1D shows cytotoxicity of A549 treated twin compounds.

 As can be seen in Figure 1, the twin compound expresses a strong cytotoxic effect irrespective of the type of cancer cell line, the higher the concentration of the twin compound was confirmed that the lower the survival rate of cancer cells. In addition, it was confirmed that the cancer cell viability was lowered better than when used as a starting material DMNQ, chloroambusil alone.

Experimental Example 3

Apoptosis Identification Experiment-Staining of DAPI (4 ', 6-Diamidino-2-phenylindole)

Experiments are performed on slides smeared with 0.01% weight of poly-L-lisine. In the slide smearing method of the 0.01% weight poly-L-lysine, the slide glass is washed with methanol for 24 hours and then washed again with PBS (phosphate buffered saline pH 7-7.2), followed by washing with water. After soaking for 10 minutes in 0.01% by weight of poly-L- lysine (poly-L-lisine, Sigma Diagnostrics, INC cat. # P8920) and dried at 37 ℃ for 24 hours.

P53 mutants were treated by subdividing two compounds of A549 in 1 × 10 ⁵ cells by subdiluting the concentration of Twin compounds by concentration, and then smeared with 0.01% by weight of poly-L-lisine. Smear on slides and dry at room temperature. Fix with 4% weight of paraformaldehyde at 4 ° C. for 25 minutes. After reacting with Triton X-100 at 0.2% by weight for 10 minutes, it is washed three times with PBS (phosphate buffered saline pH 7-7.2). DAPI (4 ', 6-diamidino-2-phenylindole, VECTOR, VECTASHELD MOUNING MEDIUM with DAPI Cat. # H-1000) was stained with 4 μg / μl of weight and covered with a cover glass to confirm apoptosis by fluorescence microscopy. It was confirmed that apoptosis was induced in a concentration-dependent manner by the twin compound as shown in Figure 2a, Figure 2b.

Experimental Example 4

Carcinoma Inhibitory Effect

The lung cancer cell line (LLC) was implanted subcutaneously in the left armpit of the rat, and then injected intraperitoneally with the twin compound prepared in Example 2 for 14 days once daily from the third day. Body weights were measured at two-day intervals after cancer cell transplantation. Tumors were removed on day 17 to determine their size and weight. Figure 3a was administered in a concentration of 6 μM, 12 μM of the control group and the positive control group Adriamycin 1 mg / ml and the negative control Chlorambucil, DMNQ, respectively, 6 μM, 12 μM, the content of 3 μM, 6 μM, 12 μM Figure 14 is a graph of the weight change between the subjects administered the twin compound of 14 days, Figure 3b is the result of measuring the size of the tumor. As can be seen in Figures 3a and 3b, it was confirmed that the carcinoma inhibitory effect is expressed in the individual administered the twin compound, it was confirmed that the carcinoma suppression effect increases in proportion to the dose.

 As described above, the twin compounds prepared by the present invention are excellent in cytotoxicity, apoptosis-inducing effect, tumor renal suppression effect, etc., and can be usefully used for cancer treatment and prevention. Also, DMNQ, Chlorambucil alone It can be seen that even at a lower concentration than the effect shows a significant effect. That is, by synthesizing the two substances it was confirmed that each component shows a synergistic effect than the effect contained in the original single substance.

One embodiment of the present invention described above should not be construed as limiting the technical spirit of the present invention. The scope of protection of the present invention is limited only by the matters described in the claims, and those skilled in the art can change and change the technical idea of the present invention in various forms. Therefore, such improvements and modifications will fall within the protection scope of the present invention, as will be apparent to those skilled in the art.

Claims (6)

6- {1- [4- (p-bis (2-chloroethyl) -aminophenyl) -butanoyloxy] pentyl} -5,8-dimethoxy-1, having the anticancer activity, 4-naphthoquinone compound.    Formula (I)

(a) dissolving dicyclohexylcarbodiimide with 4-dimethylaminopyridine in anhydrous dichloromethane, (b) adding chloroambucil to the obtained solution, (c) 6- {1- [4- (p-bis (2-chloroethyl) -aminophenyl) -butanoyloxy] pentyl} -5,8-dimethoxy- comprising separating and purifying the obtained product. Method for producing a 1,4-naphthoquinone compound. (a) dissolving dicyclohexylcarbodiimide with 4-dimethylaminopyridine in anhydrous dichloromethane, (b) adding chloroambucil to the obtained solution, (c) 6- {1- [4- (p-bis (2-chloroethyl) -aminophenyl) -butanoyloxy] pentyl} -5,8-dimethoxy- comprising separating and purifying the obtained product. In the method for producing a 1,4-naphthoquinone compound, The separation and purification step includes the step of stirring the obtained product at room temperature, filtration it under reduced pressure and concentration, and purifying the residue by silica gel column chromatography. A pharmaceutical composition having anticancer activity, comprising the compound according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient. The pharmaceutical composition having anticancer activity according to claim 4, wherein the dose of the compound is 6 µM / kg. The method of claim 5, A pharmaceutical composition having an anticancer activity, which is formulated as an injection or liquid.

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