CYCLIC PEPTIDE COMPOUND CONTAINING PIPERAZIC ACID, METHOD OF PRODUCING SAME, AND USES OF SAME

Abstract

Provided are novel piperazic acid containing peptide compound stereoisomer, solvate, or pharmaceutically acceptable salt thereof, method of preparing the same, and use thereof. The peptide compound, stereoisomer, solvate, or pharmaceutically acceptable salt thereof has activities of anticancer, anti-metastasis, and growth inhibition of resistant tumors, and thus may be used to prevent or treat various cancers or metastases of cancer.

Claims

1. A peptide compound represented by Formula 1, stereoisomer, solvate, or pharmaceutically acceptable salt thereof: ##STR00007## wherein Y is piperazic acid, X.sub.1 is an amino acid selected from the group consisting of Ser, Thr, Cys, and Tyr, and N in a peptide bond of the amino acid is unsubstituted or substituted with a C.sub.1 to C.sub.20 alkyl group, X.sub.2 is an amino acid selected from the group consisting of Leu, Gly, Ala, Val, Ile, and Met, X.sub.3 is an amino acid of Phe or Trp, and N in a peptide bond of the amino acid is unsubstituted or substituted with a C.sub.1 to C.sub.20 alkyl group, X.sub.4 is an amino acid selected from the group consisting of Ala, Gly, Val, Leu, Ile, and Met, X.sub.5 is an amino acid selected from the group consisting of Val, Gly, Ala, Leu, Ile, and Met, and N in a peptide bond of the amino acid is unsubstituted or substituted with a C.sub.1 to C.sub.20 alkyl group. X.sub.6 is a beta(β)-amino acid or aspartic acid (Asp), X.sub.7 is an amino acid selected from the group consisting of Ala, Gly, Val, Leu, Ile, and Met, and N in a peptide bond of the amino acid is unsubstituted or substituted with a C.sub.1 to C.sub.20 alkyl group, X.sub.8 is an amino acid selected from the group consisting of Ile, Val, Gly, Ala, Leu and Met, and X.sub.9 is an amino acid of Phe or Trp, and N in a peptide bond of the amino acid is unsubstituted or substituted with a C.sub.1 to C.sub.20 alkyl group.

2. The peptide compound, stereoisomer, solvate, or pharmaceutically acceptable salt thereof of claim 1, wherein X.sub.1 is Ser or N-methyl-Ser.

3. The peptide compound, stereoisomer, solvate, or pharmaceutically acceptable salt thereof of claim 1, wherein X.sub.2 is Leu.

4. The peptide compound, stereoisomer, solvate, or pharmaceutically acceptable salt thereof of claim 1, wherein X.sub.3 is Phe or N-methyl-Phe.

5. The peptide compound, stereoisomer, solvate, or pharmaceutically acceptable salt thereof of claim 1, wherein X.sub.4 is Ala.

6. The peptide compound of claim 1, wherein X.sub.5 is Val or N-methyl-Val, stereoisomer, solvate, or pharmaceutically acceptable salt thereof.

7. The peptide compound, stereoisomer, solvate, or pharmaceutically acceptable salt thereof of claim 1, wherein X.sub.6 is β-Ala or Asp.

8. The peptide compound, stereoisomer, solvate, or pharmaceutically acceptable salt thereof of claim 1, wherein X.sub.7 is Ala or N-methyl-Ala.

9. The peptide compound, stereoisomer, solvate, or pharmaceutically acceptable salt thereof of claim 1, wherein X.sub.8 is Ile or Val.

10. The peptide compound, stereoisomer, solvate, or pharmaceutically acceptable salt thereof of claim 1, wherein X.sub.9 is Phe; Trp; or N-methyl-Trp.

11. The peptide compound, stereoisomer, solvate, or pharmaceutically acceptable salt thereof of claim 1, wherein the C.sub.1 to C.sub.20 alkyl group is a methyl group.

12. The peptide compound, stereoisomer, solvate, or pharmaceutically acceptable salt thereof of claim 1, wherein the peptide compound represented by Formula 1 is represented by any one of Formulae 2 to 6: ##STR00008## ##STR00009##

13. Streptomyces sp. PC5 strain capable of producing the peptide compound of claim 1, stereoisomer, solvate, or pharmaceutically acceptable salt thereof and deposited in the KCTC Korean Collection for Type Culture) under accession number KCTC14117BP

14. A method of preparing the peptide compound of claim 1, stereoisomer, solvate, or pharmaceutically acceptable salt thereof, the method comprising: culturing a Streptomyces sp. PC5 strain deposited under accession number KCTC14117BP; and separating the peptide compound of claim 1, stereoisomer, solvate, or pharmaceutically acceptable salt thereof, from the culture.

15. A pharmaceutical composition for preventing or treating cancer or metastasis of cancer, comprising the peptide compound of claim 1, stereoisomer, solvate, or pharmaceutically acceptable salt thereof.

16. The pharmaceutical composition of claim 15, wherein the cancer is selected from the group consisting of lung cancer, colorectal cancer, breast cancer, liver cancer, stomach cancer, and leukemia.

17. The pharmaceutical composition of claim 15, wherein the peptide compound, stereoisomer, solvate, or pharmaceutically acceptable salt thereof is effective in cell cycle regulation of cancer cells, apoptosis induction of cancer cells, inhibition of cancer metastasis, or a combination thereof.

18. The pharmaceutical composition of claim 15, wherein the cancer has resistance to an anticancer agent.

19. The pharmaceutical composition of claim 15, wherein the pharmaceutical composition further comprises an anticancer agent.

20. A method of preventing or treating cancer or cancer metastasis, comprising: administering the pharmaceutical composition of claim 15 to a subject in need thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0064] FIG. 1 is a photograph of a solid medium on which a Streptomyces sp. PC5 strain was cultivated;

[0065] FIG. 2A is graphs showing cell cycles of a colorectal cancer cell line HCT116 depending on the concentration (μM) of lenziamide A, with the cell cycle measured by flow cytometry, and

[0066] FIG. 2B is a graph showing the cell cycle distribution (%) of HCT116 depending on the concentration of lenziamide A (μM);

[0067] FIG. 3A is flow cytometry graphs showing results of Annexin V/propidium iodide dual-stain analysis, and

[0068] FIG. 3B is a graph showing cell stages depending of the concentration of lenziamide A;

[0069] FIG. 4A is a graph showing tumor volumes (mm.sup.3) with the administration of lenziamide A,

[0070] FIG. 4B is a graph showing body weights (g) of xenograft animal models, and

[0071] FIG. 4C is a graph showing final tumor weight (g) depending on the amount of lenziamide A administered;

[0072] FIG. 5A is a graph showing tumor volumes (mm.sup.3) in resistant cancer cell line injected xenograft animal models depending on the administration of lenziamide A, 5-FU, or combination thereof, and

[0073] FIG. 5B is a graph showing the weights (g) of the resistant cancer cell line injected xenograft animal models;

[0074] FIG. 6A is an image of immunoblotting analysis showing the expression of metastasis related biomarkers in a metastatic colorectal cancer cell line depending on the administration of lenziamide A,

[0075] FIG. 6B is a graph showing tumor volumes (mm.sup.3) in metastatic colorectal cancer cell injected xenograft animal models with the administration of lenziamide A, and

[0076] FIG. 6C is a graph showing body weights of metastatic colorectal cancer cell injected xenograft animal models.

DETAILED DESCRIPTION

[0077] Hereinafter, the disclosure will be described in more detail by using the following examples. However, these examples are only intended to illustrate the present disclosure, and the scope of the present disclosure is not limited thereto.

Example 1. Isolation of Lenziamide and Identification of Structure Thereof

[0078] 1-1. Isolation of Lenziamide-Producing Strain

[0079] A Lenziamide-producing Streptomyces sp. PC5 strain was isolated from an integument of Onthophagus lenzii. PC5 strain was isolated from Actinomycete Isolation solid medium (2 g of sodium caseinate, 0.1 g of asparagine, 4 g of sodium propionate, 0.5 g of dipotassium phosphate, 0.1 g of magnesium sulfate, 0.001 g of ferrous sulfate and 15 g of agar powder per 1 L of sterile water) (FIG. 1). Based on 16S rDNA sequence analysis results, the strain was identified to be a Streptomyces species. The strain was named Streptomyces sp. PC5 strain, and the strain was deposited to Korea Research Institute of Bioscience and Biotechnology on Jan. 29, 2020 (accession number KCTC1411BP).

[0080] 1-2. Culture of Streptomyces sp. PC5 Strain

[0081] A Streptomyces sp. PC5 strain was plated on sterilized yeast extract-malt extract (YEME) solid medium (4 g of yeast extract, 10 g of malt extract, 4 g of glucose, and 18 g of agar per 1 L of distilled water) and cultured for about 4 weeks at about 30° C.

[0082] The spores of the cultured PC5 strain were inoculated to a 50 mL volume of YEME liquid medium (4 g of yeast extract, 10 g of malt extract, and 4 g of glucose per 1 L of distilled water), and cultured for about 8 days at 30° C. with shaking at 200 rpm. 5 mL of the culture fluid was inoculated to a 200 mL volume of modified YEME liquid medium (4 g of yeast extract, 10 g of malt extract, 4 g of glucose, and 1 g of humic acid per 1 L of distilled water) and cultured for about 5 days at 30° C. with shaking at 170 rpm.

[0083] 1-3. Isolation and Purification of Lenziamide

[0084] Culture fluid of PC5 strain was obtained from Example 1-2.

[0085] 1 L of PC5 strain culture fluid obtained at the end of the culturing and about 1.5 L volume of ethyl acetate (EtOAc, Daejung Hwageum Co., Ltd.) were poured into a separatory funnel which was mounted on a stand, and the funnel was closed with a stopper and shook for 3 minutes up and down and right and left to conduct a primary extraction. Afterwards, the separatory funnel was again mounted on a stand for complete separation of a water layer and an EtOAc layer, and then the valve of the separator funnel was opened to remove the water layer. New EtOAc was added to the water layer, and iterative extractions were carried out in the same manner. The EtOAc layers were stored separately in a clean flask, and anhydrous sodium sulfate (Daejung Hwageum Co., Ltd.) was added thereto to remove the remaining water from the culture fluid, and the solution was filtered through several layers of clean gauzes to remove impurities. The dehydrated EtOAc layer was transferred to a 3 L round bottom flask and dried under a reduced pressure in a vacuum dry oven. A total of 150 L of culture fluid was used to obtain 5 g of crude extract by extraction.

[0086] A reverse-phase fractionation was first carried out by using an open column to purify lenziamide from the PC5 strain extract. After stabilizing the open column filled with C.sub.18 resin with 20% (v/v) methanol (MeOH)/80% (v/v) H.sub.2O, the extract adsorbed on Celite 545 was filled thereon. The extract was fractionated by each of 20% (v/v), 40% (v/v), 60% (v/v), 80% (v/v), 100% (v/v) methanol solvent. Fractions of 80% (v/v) and 100% (v/v) methanol solvent where lenziamide A was distributed were transferred to a round bottom flask and dried under a reduced pressure.

[0087] In order to obtain pure lenziamide A from dried 80% (v/v) and 100% (v/v) methanol fractions, high-performance liquid chromatography (HPLC) was performed, followed by further purification in three steps below.

[0088] (1) Fractionation was conducted with use of a reverse-phase column (C.sub.18 YMC-Pack ODS-A S-5 μm 250×10.0 mm) and elution with concentration gradient from 50% (v/v) to 80% (v/v) acetonitrile (ACN) to which 0.1% (v/v) of formic acid is added/aqueous solution (flow rate: 2 mL/min, detection: UV 210 nm) for about 50 minutes. Lenziamide A, B, C, D, and E were respectively identified in fractions obtained at about 31-minute, about 26-minute, about 28-minute, and about 23-minute.

[0089] (A-2) The 31-minute fraction dried under a reduced pressure was further fractionated by using a reverse-phase column (C.sub.18 YMC-Pack ODS-A S-5 μm 250×10.0 mm) under the isocratic condition of 76% (v/v) methanol (MeOH) to which 0.1% (v/v) of formic acid is added/aqueous solution (flow rate: 2 mL/min, detection: UV 230 nm). Lenziamide A was identified in a fraction at about 28-minute.

[0090] (A-3) The 28-minute fraction dried under a reduced pressure was further fractionated under the same condition as in (A-2) to obtain pure lenziamide A. By conducting iterative experiments, about 100 mg of lenziamide A was obtained from about 5 g of crude extract.

[0091] (B-2) The 26-minute fraction dried under a reduced pressure was further fractionated for 40 minutes by using a cyano column (CN YMC-Pack S-5 μm 250×4.6 mm) with a concentration gradient of 40% (v/v) to 70% (v/v) methanol (MeOH) to which 0.1% (v/v) of formic acid is added/aqueous solution (flow rate: 0.8 mL/min, detection: UV 230 nm). Pure lenziamide B was obtained in a fraction at about 25-minute.

[0092] (C-2) The 28-minute fraction dried under a reduced pressure was further fractionated for about 40 minutes by using a cyano column (CN YMC-Pack S-5 μm 250×4.6 mm) with a concentration gradient of 40% (v/v) to 70% (v/v) methanol (MeOH) to which 0.1% (v/v) of formic acid is added/aqueous solution (flow rate: 0.8 mL/min, detection: UV 230 nm). Pure lenziamide C was obtained in a fraction at about 27-minute.

[0093] (D-2) The about 23-minute fraction dried under a reduced pressure was further fractionated for about 40 minutes by using a cyano column (CN YMC-Pack S-5 μm 250×4.6 mm) with a concentration gradient of 40% (v/v) to 70% (v/v) methanol (MeOH) to which 0.1% (v/v) of formic acid is added/aqueous solution (flow rate: 0.8 mL/min, detection: UV 230 nm). Pure lenziamide D was obtained from a fraction at about 23-minute, and lenziamide E was identified mixed with impurities from a fraction at about 21-minute.

[0094] (E-3) The about 21-minute fraction obtained in the course of obtaining pure lenziamide D and dried under a reduced pressure was further fractionated by using a reverse-phase column (C.sub.18 (2) Luna 5 μm 250×4.6 mm) with the isocratic condition of 73% (v/v) methanol (MeOH) to which 0.1% (v/v) of formic acid is added/aqueous solution (flow rate: 2 mL/min, detection: UV 230 nm). Lenziamide E was obtained in a fraction at about 21-minute.

[0095] 1-4. Identification of Chemical Structure of Lenziamide

[0096] The structure of lenziamide A was identified based on 1D and 2D nuclear magnetic resonance(NMR) spectrum. For the nuclear magnetic resonance spectrum (.sup.1H NMR, .sup.13C NMR), Bruker's 500 MHz NMR was used, and acetone-d.sub.6 was used as a solvent.

[0097] The positioning of the structure of lenziamide A by the nuclear magnetic resonance spectrum is shown in Table 1.

[0098] [Lenziamide A]

[0099] (1) Molecular formula: C.sub.57H.sub.87N.sub.11O.sub.11.

[0100] (2) Molecular weight: 1101

[0101] (3) Color: Transparent

[0102] (4) .sup.1H-NMR (Acetone-d.sub.6, 850 MHz): Refer to Table 1

[0103] (4) .sup.13C-NMR (Acetone-d.sub.6, 212.5 MHz): Refer to Table 1

TABLE-US-00001 TABLE 1 Amino acid Position δ.sub.C Type δ.sub.H mult. (J in Hz) Ile  1 174.5 C  2 53.9 CH 4.78 dd (11.0, 8.5)  2-NH 7.73 d (8.5)  3 35.5 CH 2.36 m  4 25.5 CH.sub.2 1.43 m 1.18 m  5 9.6 CH.sub.3 0.86 t (7.5)  6 14.8 CH.sub.3 1.01 d (6.5) N-Me-Phe  7 169.6 C  8 63.3 CH 4.50 dd (11.5, 1.5)  9a 34.7 CH.sub.2 2.85 m  9b 2.52 d (14.0) 10 139.0 C 11 130.1 CH 6.41 d (5.5) 12 129.4 CH 7.10 m 13 127.3 CH 7.11 m 14 129.4 CH 7.10 m 15 130.1 CH 6.41 d (5.5) 16 28.5 CH.sub.3 2.77 s Pip 17 172.3 C 18 46.4 CH 4.35 dd (5.5, 3.5) 19a 23.3 CH.sub.2 0.49 m 19b 0.06 m 20a 19.4 CH.sub.2 1.59 m 20b 0.90 m 21a 47.0 CH.sub.2 2.96 m 21b 2.67 m 21-NH 4.77 dd (13.0, 2.0) N-Me-Ser 22 171.1 C 23 69.9 CH 4.41 m 24a 62.1 CH.sub.2 4.21 ddd (12.0, 12.0, 7.0) 24b 3.77 m 24-OH 4.91 dd (11.5, 2.0) 25 39.1 CH.sub.3 2.96 s Leu 26 174.6 C 27 48.4 CH 4.96 ddd (10.0, 10.0, 4.0) 27-NH 8.79 d (9.5) 28a 41.4 CH.sub.2 1.67 ddd (14.5, 10.5, 4.0) 28b 1.38 m 29 24.8 CH 1.57 m 30 24.2 CH.sub.3 0.90 d (7.0) 31 22.5 CH.sub.3 0.91 d (7.0) N-Me-Phe 32 171.3 C 33 59.0 CH 6.00 dd (12.5, 3.0) 34a 37.2 CH.sub.2 3.58 dd (14.5, 3.5) 34b 3.04 dd (14.5, 12.5) 35 139.9 C 36 129.6 CH 7.29 d (7.5) 37 129.4 CH 7.33 dd (7.5, 7.5) 38 127.6 CH 7.23 d (7.5) 39 129.4 CH 7.33 dd (7.5, 7.5) 40 129.6 CH 7.29 d (7.5) 41 32.4 CH.sub.3 3.33 s Ala 42 176.3 C 43 45.0 CH 4.84 qd (9.0, 7.0) 43-NH 6.74 d (8.5) 44 18.0 CH.sub.3 0.69 d (7.0) N-Me-Val 45 170.1 C 46 62.4 CH 4.58 d (10.0) 47 25.1 CH 2.22 m 48 19.9 CH.sub.3 0.91 d (6.5) 49 18.6 CH.sub.3 0.78 d (6.5) 50 30.0 CH.sub.3 2.74 s -Ala 51 173.6 C 52a 33.6 CH.sub.2 2.64 ddd (16.5, 5.5, 2.0) 52b 2.42 ddd (16.5, 10.5, 2.5) 53a53b 35.1 CH.sub.2 3.57 m 3.37 m 53-NH 7.23 m N-Me-Ala 54 172.2 C 55 52.7 CH 5.62 q (7.0) 56 16.5 CH.sub.3 1.39 d (7.5) 57 31.9 CH.sub.3 3.42 s

[0104] The chemical structure of lenziamide A analyzed from the nuclear magnetic resonance spectrum data and the stereo-structure analysis result is shown below.

##STR00004##

[0105] On the other hand, the chemical structures of lenziamides B to E are shown below.

##STR00005## ##STR00006##

Example 2. Identification of Anticancer Activity of Lenziamide

[0106] 2-1. Evaluation of Growth Inhibitory Activity in Cancer Cells

[0107] 6 types of cancer cell lines of Lung adenocarcinoma cell A549, colorectal cancer cell HCT116, breast cancer cell MDA-MB-231, liver adenocarcinoma cell SK-HEP-1, stomach adenocarcinoma cell SNU638, and chronic myeloid leukemia cell K562, and a normal cell line of pulmonary fibroblast MRC-5 were purchased from Korean Cell Line Bank (Seoul, Korea).

[0108] With Lenziamide compound prepared as in Example 1 added, the cell lines were cultured for about 72 hours. Thereafter, the resulting cell cultures were stained with sulforhodamine (SRB) that only stains live cells and washed to remove non-binding dye, and the stained cells were suspended in 10 mM of Tris (pH 10.0). The absorbance was measured at 515 nm and the cell proliferation was measured. Using TableCurve 2D v5.01 software (Systant Software Inc., Richmond, Calif., USA), half maximal inhibitory concentration (IC.sub.50) value was calculated by nonlinear regression analysis method. All reagents were purchased from Sigma-Aldrich. The calculated IC.sub.50 values (μM) are shown in Table 2. As a positive control, etoposide was used.

TABLE-US-00002 TABLE 2 MDA- SK- IC.sub.50(μM) A549 HCT116 MB-231 HEP-1 SNU638 K562 MRC-5 Lenziamide A 0.80 0.37 0.62 0.39 0.44 0.77 >20 Lenziamide B 0.43 0.20 0.23 0.22 0.25 0.36 >20 Lenziamide C 0.76 0.52 0.96 0.53 0.43 0.65 >20 Lenziamide E 1.78 1.01 1.59 0.96 1.32 1.21 >20 Etoposide 0.59 1.86 3.36 0.45 0.48 0.76 11.57

[0109] As shown in Table 2, lenziamide compounds were found to effectively inhibit growth of cancer cells.

[0110] 2-2. Cell Cycle Control Efficacy of Lenziamide A in Colorectal Cancer Cells.

[0111] In order to identify the action mechanism of lenziamide A showing an excellent cell proliferation inhibiting efficacy, cells of colorectal cancer cell line HCT116 were treated with lenziamide A at a concentration of 0 μM, 1.25 μM, 2.5 μM, or 5 μM. The cell cycle control effect of lenziamide A was confirmed by using flow cytometry method, and the results are shown in FIGS. 2A and 2B.

[0112] As shown in FIGS. 2A and 2B, G2/M cell cycle arrest was increased until 24 hours after treatment of lenziamide A, followed by sub G1 cell accumulation, which is cell death in the presence of a high concentration of lenziamide. That is, it was found that lenziamide A shows inhibitory efficacy of cancer cell proliferation through cell death by inducing G2/M cell cycle arrest.

[0113] 2-3. Apoptosis Induction Efficacy of Lenziamide A in Colorectal Cancer Cells

[0114] In order to confirm that sub G1 cell accumulation due to G2/M arrest effect of lenziamide A leads to cell apoptosis, HCT116 cells were treated with different concentrations of lenziamide A. Thereafter, cell death induction efficacy of lenziamide A was evaluated by using Annexin V/propidium iodide dual-stain analysis method which can identify cell deaths. The results of Annexin V/propidium iodide dual-stain analysis were shown in FIG. 3A, and stages of cells according to the concentration of lenziamide A were shown in FIG. 3B. The ratios (%) of live cells, early apoptosis, late apoptosis, necrosis, and total cell death were calculated.

[0115] As shown in FIGS. 3A and 3B, apoptosis increased dependent on the concentration of lenziamide A. Particularly, when 5 μM of lenziamide A was treated, a population of about 20.37% of early apoptosis and 51.40% of late apoptosis cells was shown. Thus, lenziamide A was confirmed to inhibit growth of cancer cells by inducing cell apoptosis.

[0116] 2-4. In Vivo Growth Inhibitory Efficacy of Lenziamide A in Colorectal Cancer Cells

[0117] In order to evaluate antitumor efficacy of lenziamide A in an animal model, xenograft animal models were prepared by grafting colorectal cancer cells.

[0118] Cells of HCT116, a human-derived colorectal cancer cell line, were diluted to a concentration of 5×10.sup.6 units/mL in RPMI (Roswell Park Memorial Institute) medium, and 0.2 mL of the diluted cells was injected subcutaneously to right flank of BALB/c mice (5-week old, male). When the size of the tumor reached about 100 mm.sup.3, groups were randomly divided based on the size of the tumor.

[0119] Lenziamide A was administered by intravenous injection at a dose of 10 mg/body weight kg or 30 mg/body weight kg to the prepared xenograft animal models. A mixture mixed at a ratio of dimethyl sulfoxide:Cremophor:saline=0.5:0.5:9 was used as a negative control, and irinotecan was used as a positive control. Volume of the tumor (mm.sup.3), body weight of the xenograft animal model (g), and final weight of the tumor (g) were measured from the first day of administration, and the results are shown in FIGS. 4A to 4C.

[0120] As shown in FIGS. 4A to 4C, a concentration-dependent tumor growth inhibitory effect was found at the two concentrations (10, 30 mg/kg) of lenziamide A. On the other hand, toxicity caused by lenziamide A was evaluated with body weight (g) during the administration period, and no significant weight decrease was observed.

[0121] 2-5. Growth Inhibitory Efficacy of Lenziamide A in a 5-Fluorouracil Resistant-Colorectal Cancer Cell Line

[0122] In order to evaluate efficacy of lenziamide A in resistant cell lines, HCT116-5-FU, a colorectal cancer cell line resistant to 5-fluorouracil (5-Fu), which is used as the primary therapeutic agent for colorectal cancer, was prepared.

[0123] In order to prepare HCT116-5-FU resistant cell line, 5-fluorouracil was treated to HCT116 cells 3 times a week, and only surviving cells were subcultured with treatment of increasing concentrations of 5-fluorouracil for 3 months. HCT116-5-FU resistant cell line was established by finally collecting and culturing only the surviving cells.

[0124] 5-Fu, irinotecan, etoposide, and lenziamide A were added to HCT116 cell line without resistance and HCT116-5-FU resistant cell line, and half maximal inhibitory concentration (IC.sub.50) values were calculated as described in Example 2-1. IC.sub.50 values (μM) according to the compound, and fold change of IC.sub.50 value of HCT116-5-FU with respect to IC.sub.50 value of HCT116 were calculated, and the results are shown in Table 3.

TABLE-US-00003 TABLE 3 Fold change (HCT116-5-FU/ IC.sub.50(μM) HCT116 HCT116-5-FU HCT116) 5-fluorouracil 4.22 36.70 8.70 Irinotecan 1.52 5.65 3.72 Etoposide 0.76 1.92 2.53 Lenziamide A 0.36 0.42 1.16

[0125] As shown in Table 3, HCT116-5-FU resistant cell line was found resistant to other drugs used for treating colorectal cancer as well as 5-FU, but lenziamide A effectively inhibited growth of HCT116-5-FU resistant cell line. Thus, lenziamide A was confirmed to have an effective growth inhibition effect on resistant cancer cells.

[0126] 2-6. In Vivo Growth Inhibitory Efficacy of Lenziamide A in Resistant Tumor Cells and Efficacy of Lenziamide A in Combination with 5-FU.

[0127] In order to verify efficacy of lenziamide A at an animal experiment level, xenograft animal models were developed using a resistant cancer cell line.

[0128] HCT116-5-FU resistant cell line prepared as described in Example 2-5 was diluted to a concentration of 5×10.sup.6 units/mL in RPMI medium, and 0.2 mL of the diluted cell line was injected subcutaneously to right flanks of BALB/c mice (5-week old, male). When the size of the tumor reached about 100 mm.sup.3, groups were randomly divided based on the size of the tumor.

[0129] Lenziamide A (10 mg/body weight kg), 5-FU (30 mg/body weight kg), or a combination thereof (10 mg/body weight kg of lenziamide A+30 mg/body weight kg of 5-FU) was administered by intravenous injection to resistant cancer cell line injected xenograft animal models. A mixture mixed at a ratio of dimethyl sulfoxide:Cremophor:saline=0.5:0.5:9 was used as a negative control. Volumes of tumor (mm.sup.3) and body weights of the xenograft animal models (g) were measured from the first day of administration, and the results are shown in FIGS. 5A and 5B.

[0130] As shown in FIGS. 5A and 5B, when lenziamide A is administered at a dose of 10 mg/kg, lenziamide A effectively inhibited growth of resistant tumor without significant weight loss. Further, in the case of a combined administration of lenziamide

[0131] A and 5-FU, a synergistic effect was observed compared to administration of 5-FU alone. Thus, lenziamide A was confirmed to have an in vivo growth inhibitory effect for resistant cells, and show a synergistic effect when administered in combination with other anti-cancer agents such as 5-FU.

[0132] 2-7. Anti-Metastasis Efficacy of Lenziamide A

[0133] (1) Identification of In Vitro Anti-Metastasis Efficacy

[0134] In general, metastatic cancer is known to have faster growth rate that that of non-metastatic cancer, thereby making it very difficult to treat it. A colorectal cancer cell line (C-1) was injected into appendix of laboratory mice and left for 6 weeks to artificially induce growth and metastasis of tumor, and colorectal cancer cells metastasized to the liver (L-2) were isolated and cultured. After administering 0 μM to 4 μM of lenziamide A to colorectal cancer cell line (C-1) and metastatic colorectal cancer cell line (L-2) obtained above, expression of biomarkers related to metastasis of cancer was identified by reverse transcription polymerase chain reaction (RT-PCR), and the results are shown in FIG. 6A.

[0135] As shown in FIG. 6A, metastatic colorectal cancer cells (L-2) had a reduced expression of E-cadherin, an epithelial biomarker, compared to a colorectal cancer cell line (C-1), but had an increased expression of N-Cadherin and vimentin, which are biomarkers related to metastasis of cancer. When lenziamide A was treated to colorectal cancer cells metastasized to liver (L-2), expression of E-cadherin increased, while expression of N-Cadherin and vimentin decreased. Thus, lenziamide A was confirmed to have anti-metastasis efficacy in vitro.

[0136] (2) Identification of In Vivo Anti-Metastasis Efficacy

[0137] As described in Example 2-7 (1), a metastatic colorectal cancer cell line (L-2) was re-injected into appendix of laboratory mice to form tumor, and lenziamide A was administered at a dose of 5 mg/body weight kg or 15 mg/body weight kg. A mixture mixed at a ratio of dimethyl sulfoxide:Cremophor:saline=0.5:0.5:9 was used as a negative control. 10 mg/body weight kg of irinotecan was administered as a positive control. Volumes of tumor (mm.sup.3) and body weights of the xenograft animal model (g) were measured from the first day of administration, and the results are shown in FIGS. 6B and 6C.

[0138] As shown in FIGS. 6B and 6C, in groups to which 5 mg/kg or 15 mg/kg of lenziamide A was administered, metastasis of the tumor to other organs was effectively inhibited, and growth of tumor in the appendix was also inhibited. Thus, lenziamide A was confirmed to have excellent anti-metastasis efficacy in vivo.

[0139] <Accession Number>

[0140] Name of Depositary Authority: Korea Research Institute of Bioscience and Biotechnology

[0141] Accession number: KCTC14117BP

[0142] Date of Receipt: 20200129