Pharmaceutical composition including 1,2-naphthoquinone derivative compound for prevention or treatment of solid cancers or blood cancers

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of solid cancers or blood cancers such as acute leukemia or chronic leukemia, including, as an active ingredient, a 1,2-naphthoquinone derivative compound or a pharmaceutically acceptable salt thereof, wherein the 1,2-naphthoquinone derivative compound has excellent effects in killing cancer cells of various solid cancers, acute leukemia, and chronic leukemia, and thus, can be useful as a pharmaceutical composition for the prevention or treatment of cancer, in particular, solid cancers, acute leukemia, or chronic leukemia.

Claims

1. A method for treating solid cancers or blood cancers, comprising, administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising, as an active ingredient, a 1,2-naphthoquinone derivative compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof: ##STR00038## wherein: R.sub.1 and R.sub.2 are each independently hydrogen, halogen, hydroxy, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6 alkyl, C.sub.4-C.sub.10 aryl, C.sub.4-C.sub.10 aryloxy, C.sub.2-C.sub.10 heteroaryl, —NO.sub.2, —NR′.sub.1R′.sub.2, —NR′.sub.1(CO(O)R′.sub.2), —NR′.sub.1(C(O)NR′.sub.1R′.sub.2), —CO(O)R′.sub.1, —C(O)NR′.sub.1R′.sub.2, —CN, —SO(O)R′.sub.1, —SO(O)NR′.sub.1R′.sub.2, —NR′.sub.1(SO(O)R′.sub.2), —CSNR′.sub.1R′.sub.2, or R.sub.1 and R.sub.2 taken together form a cyclic structure of C.sub.4-C.sub.10 aryl or a cyclic structure of C.sub.2-C.sub.10 heteroaryl, wherein R′.sub.1 and R′.sub.2 are each independently hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.8 cycloalkyl, or C.sub.4-C.sub.10 aryl, C.sub.4-C.sub.10 aryloxy, C.sub.1-C.sub.8 heteroaryl, —(CR″.sub.1R″.sub.2).sub.m—C.sub.4-C.sub.10 aryl, —(CR″.sub.1R″.sub.2).sub.m—C.sub.4-C.sub.10 heteroaryl or NR″.sub.1R″.sub.2, the R″.sub.1 and R″.sub.2 are each independently hydrogen, C.sub.1-C.sub.3 alkyl, or R″.sub.1 and R″.sub.2 taken together form a cyclic structure of C.sub.4-C.sub.10 aryl; R.sub.3, R.sub.4 and R.sub.5 are each independently hydrogen, halogen, hydroxy, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.10 alkene, C.sub.1-C.sub.6 alkoxy, C.sub.3-C.sub.8 cycloalkyl, C.sub.2-C.sub.8 heterocycloalkyl, C.sub.4-C.sub.10 aryl, C.sub.4-C.sub.10 aryloxy, C.sub.1-C.sub.8 heteroaryl, —(CR′.sub.5R′.sub.6).sub.m—C.sub.4-C.sub.10 aryl, —(CR′.sub.5R′.sub.6).sub.m—C.sub.4-C.sub.10 aryloxy, —(CR′.sub.5R′.sub.6).sub.m—C.sub.1-C.sub.8 heteroaryl, —(CR′.sub.5R′.sub.6).sub.m—NR′.sub.3R′.sub.4, —(CR′.sub.5R′.sub.6).sub.m—C.sub.3-C.sub.8 heterocycloalkyl, —(CR′.sub.5R′.sub.6).sub.m—OR′.sub.3, —(CR′.sub.5R′.sub.6).sub.m(O)COR′.sub.3, —CO(O)R′.sub.3, —CONR′.sub.3R′.sub.4, —NR′.sub.3R′.sub.4, —NR′.sub.3(C(O)R′.sub.4), —SO(O)R′.sub.3, —SO(O)NR′.sub.3R′.sub.4, —NR′.sub.3(SO(O)R′.sub.4), —CSNR′.sub.3R′.sub.4, —CH.sub.2A where the compound of Chemical Formula 1 is “A”, or -A where the compound of Chemical Formula 1 is “A”, wherein the R′.sub.3 and R′.sub.4 are each independently hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.4-C.sub.10 aryl, —(CR′.sub.5R′.sub.6).sub.m—C.sub.4-C.sub.10 aryl, —(CR′.sub.5R′.sub.6).sub.m—C.sub.4-C.sub.10 aryloxy, —(CR′.sub.5R′.sub.6).sub.m—C.sub.1-C.sub.10 heteroaryl, —CO(O)R″.sub.3, or, R′.sub.3 and R′.sub.4 taken together form a cyclic structure of C.sub.2-C.sub.10 heterocycloalkyl, or a cyclic structure of C.sub.1-C.sub.10 heteroaryl, the R′.sub.5 and R′.sub.6 are each independently hydrogen or C.sub.1-C.sub.3 alkyl, the R″.sub.3 is C.sub.1-C.sub.6 akyl; Q.sub.1 and Q.sub.2 are each independently CO, COR.sub.6, or COR.sub.7, when Q.sub.1 is CO and Q.sub.2 is CO, Q.sub.1 and Q.sub.2 form a single bond, when Q.sub.1 is COR.sub.6 and Q.sub.2 is COR.sub.7, Q.sub.1 and Q.sub.2 form a double bond; R.sub.6 and R.sub.7 are each independently hydrogen, C.sub.1-C.sub.10 alkoxy, C.sub.1-C.sub.6 alkyl, C.sub.4-C.sub.10 aryl, C.sub.4-C.sub.10 aryloxy, C.sub.2-C.sub.10 heteroaryl, —CO(O)R′.sub.7, —C(O)NR′.sub.7R′.sub.8, —SO(O)R′.sub.7, —SO(O)NR′.sub.7R′.sub.8, —SO.sub.3R′.sub.7, —PO.sub.3R′.sub.7, —CSNR′.sub.7R′.sub.8, or R.sub.6 and R.sub.7 taken together form a cyclic structure of C.sub.3-C.sub.10 heterocycloalkyl, or a cyclic structure of C.sub.3-C.sub.10 heteroaryl, the R′.sub.7 and R′.sub.8 are each independently hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.4-C.sub.10 aryl, C.sub.4-C.sub.10 aryloxy, C.sub.1-C.sub.8 heteroaryl, —(CR″.sub.4R″.sub.5).sub.m′—C.sub.4-C.sub.10 aryl, the R″.sub.4 and R″.sub.5 are each independently hydrogen, C.sub.1-C.sub.3 alkoxy; m and m′ are each independently an integer of 1 to 4; the hetero atom is at least one selected from N, O and S; X.sub.1 to X.sub.4 are each independently CH or N(R″.sub.6); X.sub.5 is N, X.sub.6 is C, X.sub.7 is N, wherein R″.sub.6 is hydrogen or C.sub.1-C.sub.3 alkyl; in the Chemical Formula, the notation means a single bond or a double bond, the notation means that a single bond or a bond may not be formed, the notation means that the cyclic structure may be or may not be aromatic; and the substituted means being substituted with one or more substituents selected from the group consisting of hydroxy, halogen element, C.sub.1-C.sub.10 alkyl, C.sub.2-C.sub.10 alkenyl, C.sub.2-C.sub.10 alkynyl, C.sub.1-C.sub.10 alkoxy, C.sub.1-C.sub.10 alkoxycarbonyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.2-C.sub.8 heterocycloalkyl, C.sub.4-C.sub.10 aryl, and C.sub.2-C.sub.10 heteroaryl; wherein the blood cancer is selected from the group consisting of acute leukemia, chronic leukemia and drug-resistant chronic leukemia or refractory acute leukemia; the chronic leukemia is selected from the group consisting of chronic myelogenous leukemia or chronic lymphocytic leukemia; the acute leukemia is selected from the group consisting of acute myelogenous leukemia or acute lymphocytic leukemia; and the solid cancer is selected from the group consisting of lung cancer, uterine cancer, liver cancer and breast cancer.

2. The method according to claim 1, wherein the pharmaceutical composition comprises, as an active ingredient, a 1,2-naphthoquinone derivative compound represented by the Chemical Formula 1 or a pharmaceutically acceptable salt thereof, wherein: R.sub.3 is hydrogen, halogen, hydroxy, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, C.sub.3-C.sub.8 cycloalkyl, (CR′.sub.5R′.sub.6).sub.m—C.sub.4-C.sub.10 aryl, —(CR′.sub.5R′.sub.6).sub.m—OR′.sub.3, —CO(O)R′.sub.3, —CH.sub.2A where the compound of Chemical Formula 1 is “A”, or -A where the compound of Chemical Formula 1 is “A”, wherein R′.sub.3 is hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.4-C.sub.10 aryl, —(CR′.sub.5R′.sub.6).sub.m—C.sub.4-C.sub.10 aryl, —(CR′.sub.5R′.sub.6).sub.m—C.sub.4-C.sub.10 aryloxy, —(CR′.sub.5R′.sub.6).sub.m—C.sub.1-C.sub.10 heteroaryl, —CO(O)R″.sub.3, the R′.sub.5 and R′.sub.6 are each independently hydrogen, C.sub.1-C.sub.6 alkyl, the R″.sub.3 is C.sub.1-C.sub.6 akyl; R.sub.4 is hydrogen, halogen, hydroxy, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.10 alkene, C.sub.1-C.sub.6 alkoxy, C.sub.3-C.sub.8 cycloalkyl, C.sub.2-C.sub.8 heterocycloalkyl, C.sub.4-C.sub.10 aryl, C.sub.4-C.sub.10 aryloxy, C.sub.1-C.sub.8 heteroaryl, —(CR′.sub.5R′.sub.6).sub.m—C.sub.4-C.sub.10 aryl, —(CR′.sub.5R′.sub.6).sub.m—C.sub.4-C.sub.10 aryloxy, —(CR′.sub.5R′.sub.6).sub.m—C.sub.1-C.sub.8 heteroaryl, —(CR′.sub.5R′.sub.6).sub.m—NR′.sub.3R′.sub.4, —(CR′.sub.5R′.sub.6).sub.m—C.sub.3-C.sub.8 heterocycloalkyl, —(CR′.sub.5R′.sub.6).sub.m—OR′.sub.3, —(CR′.sub.5R′.sub.6).sub.m(O)COR′.sub.3, —CO(O)R′.sub.3, —CONR′.sub.3R′.sub.4, —NR′.sub.3R′.sub.4, —NR′.sub.3(C(O)R′.sub.4), -A where the compound of Chemical Formula 1 is “A”, wherein R′.sub.3 and R′.sub.4 are each independently hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.4-C.sub.10 aryl, —(CR′.sub.5R′.sub.6).sub.m—C.sub.4-C.sub.10 aryl, —(CR′.sub.5R′.sub.6).sub.m—C.sub.4-C.sub.10 aryloxy, —(CR′.sub.5R′.sub.6).sub.m—C.sub.1-C.sub.10 heteroaryl, —CO(O)R″.sub.3, or R′.sub.3 and R′.sub.4 taken together form a cyclic structure of C.sub.2-C.sub.10 heterocycloalkyl, or a cyclic structure of C.sub.1-C.sub.10 heteroaryl, the R′.sub.5 and R′.sub.6 are each independently hydrogen or C.sub.1-C.sub.3 alkyl, the R″.sub.3 is C.sub.1-C.sub.6 akyl; R.sub.5 is hydrogen, halogen, hydroxy, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.10 alkene, C.sub.1-C.sub.6 alkoxy, C.sub.3-C.sub.8 cycloalkyl, C.sub.2-C.sub.8 heterocycloalkyl, C.sub.4-C.sub.10 aryl, C.sub.4-C.sub.10 aryloxy, C.sub.1-C.sub.8 heteroaryl, —(CR′.sub.5R′.sub.6).sub.m—C.sub.4-C.sub.10 aryl, —(CR′.sub.5R′.sub.6).sub.m—C.sub.4-C.sub.10 aryloxy, —(CR′.sub.5R′.sub.6).sub.m—C.sub.1-C.sub.8 heteroaryl, —(CR′.sub.5R′.sub.6).sub.m—NR′.sub.3R′.sub.4, —(CR′.sub.5R′.sub.6).sub.m—C.sub.3-C.sub.8 heterocycloalkyl, —(CR′.sub.5R′.sub.6).sub.m—OR′.sub.3, —(CR′.sub.5R′.sub.6).sub.m(O)COR′.sub.3, —CO(O)R′.sub.3, —CONR′.sub.3R′.sub.4, —NR′.sub.3R′.sub.4, —NR′.sub.3(C(O)R′.sub.4), —CH.sub.2A where the compound of Chemical Formula 1 is “A”, wherein R′.sub.3 and R′.sub.4 are each independently hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.4-C.sub.10 aryl, —(CR′.sub.5R′.sub.6).sub.m—C.sub.4-C.sub.10 aryl, —(CR′.sub.5R′.sub.6).sub.m—C.sub.4-C.sub.10 aryloxy, —(CR′.sub.5R′.sub.6).sub.m—C.sub.1-C.sub.10 heteroaryl, —CO(O)R″.sub.3, or R′.sub.3 and R′.sub.4 taken together form a cyclic structure of C.sub.2-C.sub.10 heterocycloalkyl, or a cyclic structure of C.sub.1-C.sub.10 heteroaryl, the R′.sub.5 and R′.sub.6 are each independently hydrogen or C.sub.1-C.sub.3 alkyl, and the R″.sub.3 is C.sub.1-C.sub.6 akyl.

3. The method according to claim 1, wherein the pharmaceutical composition comprises, as an active ingredient, a 1,2-naphthoquinone derivative compound represented by the Chemical Formula 1 or a pharmaceutically acceptable salt thereof, wherein the compound of the Chemical Formula 1 is one of the compounds represented below: ##STR00039## ##STR00040## ##STR00041## ##STR00042## ##STR00043## ##STR00044## ##STR00045## ##STR00046## ##STR00047## ##STR00048## ##STR00049## ##STR00050## ##STR00051## ##STR00052## ##STR00053## ##STR00054## ##STR00055## ##STR00056## ##STR00057## ##STR00058## ##STR00059## ##STR00060## ##STR00061## ##STR00062##

4. The method according to claim 3, wherein the pharmaceutical composition comprises, as an active ingredient, a 1,2-naphthoquinone derivative compound represented by the Chemical Formula 1 or a pharmaceutically acceptable salt thereof, wherein the compound is compound 98 represented below: ##STR00063##

5. The method according to claim 1, wherein the pharmaceutical composition is one formulation selected from the group consisting of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, epidermal formulations, suppositories, and sterile injection solutions.

6. Compound 98 represented by the following structure, an optical isomer thereof, or a pharmaceutically acceptable salt thereof: ##STR00064##

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a graph showing the cell viability of HL60 (FIG. 1A) or U937 (FIG. 1B) cells when treating with the compound 1 of the present invention.

(2) FIG. 2 is a result of confirming the apoptosis or cell necrosis of KG1a cells by FACS (FIG. 2A) and a result of confirming them by Western blot (FIG. 2B), when treating with the compound 1 of the present invention.

(3) FIG. 3 is a result of confirming the apoptosis or cell necrosis of HL60 cells by FACS (FIG. 3A) and a result of confirming them by Western blot (FIG. 3B), when treating with the compound 1 of the present invention.

(4) FIG. 4 is a result of confirming the proliferation inhibitory effect of monocytic cells in peripheral blood (FIG. 4A) or spleen (FIGS. 4B and 4C), when treating with the compound 1 of the present invention in a mouse model of an acute leukemia in which FLT3-ITD is overexpressed.

(5) FIG. 5 is a result of confirming the expression level of the BCR-ABL fusion gene by Western blot by treating K562 cells with the compound 1 of the present invention according to concentration.

(6) FIG. 6 is a graph showing the cell viability of A549 (lung carcinoma) and Hela (cervix adenocarcinoma) cells by treating A549 (lung carcinoma) and Hela (cervix adenocarcinoma) with the compounds 1, 10 and 72 of the present invention.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(7) Hereinafter, preferred examples of the present invention will be described in detail. However, the present invention is not limited to the examples described herein, and can also be embodied in other forms. Rather, the content presented herein will be thorough and complete, and will fully convey the scope of the present invention to those skilled in the art.

Example 1. Synthesis of 1,2-Naphthoquinone Derivative Compound

(8) As the 1,2-naphthoquinone derivative compound of the present invention used for confirming the therapeutic effect on acute leukemia, the compounds 1 to 98 and 175 to 190 were synthesized with reference to the method for synthesizing the compounds disclosed in Korean Patent Application Nos. 10-2014-0193184, 10-2014-0193306, 10-2014-0193370, 10-2015-0043050, and 10-2015-0043068. In addition, among the compounds prepared through the above process, the physicochemical properties for the compound 98 are shown in Table 1 below.

(9) TABLE-US-00001 TABLE 1 Compound No. .sup.1H NMR data .sup.1H NMR (300 MHz, DMSO) δ 7.95-7.86 (m, 2H), 7.70 (t, J = 7.5 Hz, 1H), 7.45 (t, J = 7.5 Hz, 1H), 4.99 (d, J = 5.7 Hz, 2H), 4.5 (d, J = 6.0 Hz, 2H), 1.75 (s, 3H)

Example 2. Preparation of Acute Leukemia Cell Line, Measurement/Confirmation of Cell Viability, Apoptosis or Cell Necrosis Thereof

Example 2-1. Preparation of Acute Leukemia Cell Line

(10) The causative factor of acute leukemia has been found to be very diverse. The constructed cell lines were also diverse. Thus, in order to confirm the applicability to a wide range of therapeutic agents that are not therapeutic agents for specific types of acute leukemia, in the present invention, KG1α cells constructed by selecting only cells having a stem cell phenotype from KG1 cells, which are cell lines obtained from a 59-year-old man with acute myelogenous leukemia, were used. HL60 cells, which are cell lines obtained from a 36-year-old woman with acute promyelocytic leukemia, and U937 cells, which are cell lines obtained from a 37-year-old man with histiocytic lymphoma, were used.

(11) KG1α and HL60 cells were cultured in IMDM medium containing 20% FBS (fetal bovine serum), and U937 cells were cultured in RPMI 1640 medium containing 10% FBS. All cells were cultured in an incubator under conditions of 37° C. and 5% CO.sub.2, and subcultured once every 2 or 3 days and used in the experiment.

Example 2-2. Measurement of Cell Viability in KG1α Cells-WST Assay

(12) KG1α cells are cells resistant to idarubicin and cytarabine, which are the therapeutic agents for acute myeloid leukemia, and are refractory AML cells. KG1α cells cultured in Example 2-1 were cultured into a 96-well plate at 1×10.sup.5 cells/well, and then cultured in an incubator at 37° C. and 5% CO.sub.2 for 16 hours or more for the stabilization of cells. Subsequently, among the compounds synthesized in Example 1, the compounds 1, 10 and 72 were treated at a concentration of 0.1 to 3 μM, respectively. as shown in Table 2 below, and then cultured for 24 hours. At this time, DMSO was used as a control group. After 24 hours, each cell was treated with 10 μl of WST solution, reacted for 2 hours, and then absorbance was measured at 450 nm with a multi-scan machine, and the cell viability of KG1α cells was confirmed as shown in Table 4 below.

(13) TABLE-US-00002 TABLE 2 IC.sub.50 Condition (μM)   0.5 0 0.5 0.7

(14) Table 2 shows the cell viability of KG1α cells when treated with compounds 1, 10 and 72 of the present invention as compared with the control group. The IC.sub.50 value appeared as 0.5 to 1 μM, confirming that the effect of killing acute myeloid cells was excellent.

Example 2-3. Measurement of Cell Viability of HL60 Cells or U937 Cells

(15) The HL60 cells or U937 cells cultured in Example 2-1 were inoculated into a 96-well plate at 1×10.sup.5 cells/well, and then for the stabilization of cells, the cells were cultured in an incubator at 37° C. and 5% CO.sub.2 for 16 hours or more. Subsequently, the compound 1 synthesized in Example 1 was treated at concentrations of 0.1, 0.5 and 1 μM, respectively, and reacted for 24 hours. At this time, DMSO and decitabine having a concentration of 0.1 or 1 μM was used as a control group. After 24 hours, each cell was treated with 10 μl of WST-1 solution, and reacted for 2 hours. The absorbance was then measured at 450 nm with a multi-scan machine. The cell viability of HL60 cells is shown in FIG. 1A, and the cell viability results of U937 cells are shown in FIG. 1B.

(16) Referring to the results of FIG. 1, when HL-60 cells or U937 cells was treated with the compound 1 of the present invention, it was confirmed that it exhibited concentration-dependent cell killing effects in both HL-60 cells or U937 cells. However, when HL-60 cells or U937 cells were treated with a positive control decitabine, it exhibited concentration-dependent cell killing effects in HL60 cells, but in U937 cells, the cell killing effect was remarkably low. Therefore, it was confirmed that the 1,2-naphthoquinone derivative compound of the present invention can be usefully used as a composition for treating a wide range of acute leukemias, rather than a therapeutic agent for a specific type of acute leukemia.

Example 2-4. Confirmation of Apoptosis or Cell Necrosis in Acute Leukemia Cells

(17) Cell death was typically known as apoptosis or cell necrosis. Thus, in order to confirm whether the death of acute leukemia cells induced by the compound 1 of the present invention was due to apoptosis or cell necrosis, fluorescence activated cell sorting (FACS) and western blot were performed.

(18) First, in order to conduct FACS, KG1α cells or HL60 cells cultured in Example 1-2 were inoculated in a 6-well plate at 1×10.sup.7 cells/well, and for the stabilization of cells, the cells were cultured in an incubator at 37° C. and 5% CO.sub.2 for 16 hours or more. Subsequently, the compound 1 was treated at concentrations of 0, 0.1, 0.5, 1 and 2 μM, respectively, and cultured for 24 hours, and then the cells were recovered and washed with cold PBS. Only 1×10.sup.6 cells were taken and resuspended with PBS, and then stained with Annexin-V (green fluorescence), which increases during apoptosis, and Pi (propidium iodide, red fluorescence), which reacts during apoptosis. The degree of the two indicators were quantified by FACS, the FACS results for KG1α cells are shown in FIG. 2A, and the FACS results for HL60 cells are shown in FIG. 3A.

(19) Next, in order to perform western blot, KG1α cells or HL60 cells cultured in Example 2-1 were inoculated into a 6-well plate at 1×10.sup.7 cells/well. In order to stabilize the cells, the cells were cultured for 16 hours or more in an incubator at 37° C. and 5% CO.sub.2. Then, the compound 1 was treated at concentrations of 0, 0.1, 0.5, 1 and 2 μM, respectively, and cultured for 24 hours, and then only cells were recovered. The protein extraction buffer was added to the cells obtained in the above process, and then reacted on ice for 30 minutes to break the cell membrane and centrifuged to take only the supernatant, and then extract the protein. SDS-PAGE was performed using the extracted protein, and then transferred to the nitrocellular membrane. Then, after reacting a specific antibody related to apoptosis, the amount of the protein was confirmed using the ECL buffer, and the results of Western blotting of KG1α cells are shown in FIG. 2B, and the results of Western blotting of HL60 cells are shown in FIG. 3B.

(20) Referring to the results of FIGS. 2 and 3, when U937 cells or A549 cells were treated with the compound 1 of the present invention, in KG1α cells, acute leukemia cells died due to apoptosis, but in HL60 cells, both apoptosis and cell necrosis occurred, confirming that acute leukemia cells died.

Example 3. Confirmation of Changes in Monocyte Cells in Acute Leukemia Model

(21) In order to confirm the change in the expression of the FLT3-ITD gene by Compound 1 of the present invention, peripheral blood and spleen were collected after 8 weeks from the animals of an experimental group in which Compound 1 of the present invention was mixed and administered in a diet at a concentration of 120 mg/kg using a mouse (jackson lab) that systemically overexpressed FLT3-ITD, and a control group to which only a solvent was administered (untreated group).

(22) In the case of peripheral blood, cold 1×PBS was added to a small amount of blood, centrifuged, washed, to which RBC lysis buffer was added and reacted for 5 minutes at room temperature to remove red blood cells. After adding cold 1×PBS again, the pellet from which the supernatant had been removed by centrifugation was resuspended in 1×PBS containing 1% FBS. Only 1×10.sup.6 cells were taken, and Gr-1 and Mac-1 antibodies, which were specifically expressed in granulocytes, and CD3 antibody specifically expressed in monocytes, were simultaneously immuno-stained. The antibodies were reacted on ice at a ratio of 1:200 for 20 minutes, washed with 1×PBS, then resuspended with 1×PBS containing 1% FBS, and quantified by FACS, and shown in FIG. 4A.

(23) In the case of the spleen, the cells were crushed on a mesh having a 40 μm hole, separated into single cells, and then washed with cold 1×PBS to which RBC lysis buffer was added and reacted at room temperature for 5 minutes to remove red blood cells. After washing again with cold 1×PBS, the pellet was resuspended in 1×PBS containing 1% FBS. Only 1×10.sup.6 cells were taken, and Gr-1 and Mac-1 antibodies, which were specifically expressed in granulocytes, and CD3 antibody specifically expressed in monocytes, were simultaneously immuno-stained. The antibodies were reacted on ice at a ratio of 1:200 for 20 minutes, washed with 1×PBS, resuspended in 1×PBS containing 1% FBS, and quantified by FACS, and shown in FIGS. 4B and 4C.

(24) Referring to the results of FIG. 4, when the compound 1 of the present invention was treated in an acute myelogenous leukemia mouse model in which FLT3-ITD was overexpressed, it was confirmed that it had an excellent effect of suppressing the proliferation of monocyte cells (acute leukemia) in both peripheral blood and spleen, and thus had an effect of improving acute myeloid leukemia.

Example 4. Preparation of Chronic Leukemia Cell Line and Measurement of Cell Viability Thereof

Example 4-1. Preparation of Chronic Leukemia Cell Lines

(25) K562 cells obtained from a 53-year-old woman with chronic myeloid leukemia were cultured in an incubator under conditions of 37° C. and 5% CO.sub.2 using RPMI medium containing 10% FBS (fetal bovine serum), and subcultured once every two days and used in the experiment.

Example 4-2. Measurement of Cell Viability in Chronic Leukemia Cells-WST Assay

(26) The K562 cells cultured in Example 4-1 were inoculated into a 96-well plate at 1×10.sup.5 cells/well, and then, for the stabilization of cells, the cells were cultured in an incubator at 37° C. and 5% CO.sub.2 for 16 hours or more. Subsequently, among the compounds synthesized in Example 1, the compounds 1, 10, and 72 and 192 were treated at a concentration of 0.1 to 5 μM and then cultured for 4 hours. At this time, DMSO was used as a control group. After 4 hours of incubation, 10 μl of WST solution was added to each cell and then reacted for 2 hours. The absorbance was then measured at 450 nm with a multi-scan machine, and the cell viability of K562 cells is shown in Table 3 below.

(27) TABLE-US-00003 TABLE 3 IC.sub.50 Condition (μM)   1.6 0.9 1.4

(28) Referring to Table 3 above, when K562 cells were treated with the compounds 1, 10, and 72 of the present invention, it was confirmed that the IC.sub.50 value appeared as 1 to 2 μM, confirming that the effect of killing chronic myelogenous leukemia cells was excellent, as compared with the control group.

Example 5. Confirmation of Expression Level of BCR-ABL Fusion Gene in Chronic Myelogenous Leukemia Cells

(29) The BCR-ABL fusion gene, existing only in chronic myelogenous leukemia, generated and transmitted continuous cell growth signals to induce the growth of cancer cells. Thus, after chronic myeloid leukemia cells were treated with the compound of the present invention, western blot was used to confirm whether the expression level of the BCR-ABL fusion gene was reduced.

(30) First, the K562 cells cultured in Example 4-1 were inoculated into a 60 mm plate at 5×10.sup.6 cells/well, and then for the stabilization of cells, the cells were cultured in an incubator at 37° C. and 5% CO.sub.2 for 16 hours or more. The, the compound 1 was treated at concentrations of 0.5, 1, 1.5, 2 and 2.5 μM, respectively, and cultured for 6 hours, and then the cells were recovered. A protein extraction buffer (RIPA buffer) was added to the cells obtained in the above process, and reacted on ice for 30 minutes to break the cell membrane, which was centrifuged to remove a supernatant, and then the protein was extracted. The extracted protein was developed by electrophoresis using SDS-PAGE, and then transferred to the PVDF membrane. Then, the antibody related to the BCR-ABL fusion gene was reacted, and then the amount of the protein was confirmed using the ECL buffer, and Western blot results for the K562 cell line are shown in FIG. 5.

(31) Referring to the results of FIG. 5, when K562 cells were treated with the compound 1 of the present invention, it was confirmed that the expression level of the BCR-ABL fusion gene, which plays the most important role in the onset of chronic myelogenous leukemia, was decreased in a concentration-dependent manner. In addition, it can be seen that due to the decrease in the expression of the BCR-ABL fusion gene, the expression of phospho-bcr-abl and phospho-stat5, which indicates the activity of the BCR-ABL fusion gene (a signal that induces cell growth), was also decreased.

(32) However, the expression level of the BCR-ABL fusion gene, which is a cancer-causing gene, decreased, whereas in the case of c-abl protein present in normal blood cells, the expression level was not affected. Thus, it was confirmed that the compound of the present invention does not affect the c-abl protein expressed in normal cells, and the composition selectively reduces only the BCR-ABL fusion protein which is a cancer-causing gene existing only in chronic myelogenous leukemia.

Example 6. Preparation of Solid Cancer Cell Lines and Measurement of Cell Viability Thereof

Example 6-1. Preparation of Solid Cancer Cell Lines

(33) To confirm the possibility of use as a therapeutic agent for solid cancer, A549 cell line obtained from a 58-year-old men with lung carcinoma, Hela cell line obtained from a 31-year-old women with cervix adenocarcinoma, HepG2 cell line obtained from a 15-year-old boy with hepatocellular carcinoma, MCF7 cell line obtained from a 69-year-old woman with breast adenocarcinoma, and Beas-2B cell line obtained from a normal lung for comparison with solid cancer cell line were used.

(34) The A549 (lung carcinoma), Hela (cervix adenocarcinoma), HepG2 (hepatocellular carcinoma), MCF7 (breast carcinoma), and Beas-2B (normal lung) cell lines were used in DMEM medium containing 10% FBS, subcultured once every 2 days in an incubator under conditions of 37° C. and 5% CO.sub.2 and used in the experiment.

Example 6-2. Measurement of Cell Viability in Solid Cancer Cells-WST Assay

(35) A549 (lung carcinoma), Hela (cervix adenocarcinoma), HepG2 (hepatocellular carcinoma), MCF7 (breast carcinoma), and Beas-2B (normal lung) cells cultured in Example 6-1 were inoculated into a 96-well plated at 1×10.sup.4 cells/well, and then for the stabilization of cells, the cells were cultured in an incubator at 37° C. and 5% CO.sub.2 for 16 hours or more. Subsequently, among the compounds synthesized in Example 1, the compounds 1, 10 and 72 were treated at a concentration of 1 to 30 μM, and then cultured for 6 hours. At this time, DMSO and β-lapachone were used as the control group. After 4 hours of incubation, 10 μl of WST solution was added to each cell, the reaction was carried out for 2 hours, and the absorbance was measured at 450 nm with a multi-scan machine. The cell viabilities of A549 (lung carcinoma), Hela (cervix adenocarcinoma), HepG2 (hepatocellular carcinoma) and MCF7 (breast carcinoma) cells are shown in Table 4 and FIG. 6 below.

(36) TABLE-US-00004 TABLE 4 IC.sub.50 (μM) Condition A549 Hela HepG2 MCF7 Positive control group 5.0 4.8 12.3 13.2 (beta-rapacon) 5.1 4.6 10.1 14.4 5.5 7.7 12.1 14.1 6.5 9.1 12.6 12.5

(37) Referring to Table 4 and FIG. 6, when A549 (lung carcinoma), Hela (cervix adenocarcinoma), HepG2 (hepatocellular carcinoma), MCF7 (breast carcinoma) and Beas-2B (normal lung) cells were treated with the compounds 1, 10 and 72 of the present invention for 6 hours, it was confirmed that they exhibited a killing effect similar to β-lapachone, and thus, the compounds of the present invention had a therapeutic effect on solid cancer, as compared with the control group.

(38) Further, although not shown in Table 4, when Beas-2B which is a normal lung cell line was treated with the compounds of the present invention, the average cell viability was 80% or more, but when treating A549 which is a lung carcinoma, it exhibited the cell viability of about 50%. Thus, since the 1,2-naphthoquinone derivative compound of the present invention did not exhibit cytotoxicity to normal cells but specifically exhibited apoptosis effect on cancer cells, it can be confirmed that it can be usefully used as a composition for the treatment of various solid cancers.

Example 7. Toxicity Experiment

(39) This experiment was conducted to examine the acute toxicity to the animal body acutely (within 24 hours) when the compound 1 of the present invention was ingested in excess in a short period of time, and to determine the mortality rate. 20 C.sub.57BL/6 mice, which are common mice, were prepared, and ten mice were assigned to each group. Only 0.1% SLS (sodium lauryl sulfate) was administered to the control group, and the compound 1 was orally administered to the experimental group at a concentration of 120 mg/kg, respectively. As a result of examining each mortality rate 24 hours after administration, both the control group and the experimental group to which the compound 1 was administered survived.

Formulation Example 1. Preparation of Pharmaceutical Formulation Containing the Compound of the Present Invention

Formulation Example 1-1. Preparation of Powder

(40) 2 g of the compound 1 of the present invention and 1 g of lactose were mixed and filled in an airtight cloth to prepare a powder.

Formulation Example 1-2. Preparation of Tablets

(41) 100 mg of the compound 1 of the present invention, 100 mg of microcrystalline cellulose, 60 mg of lactose hydrate, 20 mg of low-substituted hydroxypropyl cellulose, and 2 mg of magnesium stearate were mixed, and then the mixture was tableted according to a conventional tablet preparation method to prepare tablets.

Formulation Example 1-3. Preparation of Capsules

(42) 100 mg of the compound 1 of the present invention, 100 mg of microcrystalline cellulose, 60 mg of lactose hydrate, 20 mg of low-substituted hydroxypropyl cellulose, and 2 mg of magnesium stearate were mixed, and then the above ingredients were mixed according to a conventional capsule preparation method and filled into gelatin capsules to prepare capsules.

Formulation Example 1-4. Preparation of Injections

(43) 10 mg of the compound 1 of the present invention, an appropriate amount of injectable sterilized distilled water and an appropriate amount of a pH adjuster were mixed, and then injections were prepared in the amount of the above components per ampoule (2 ml) according to a conventional method for preparing injections.