[2] Background Art [3] Cancer is the second leading cause of disease-induced death, next to cardiovascular diseases, world-wide, regardless of men and women. Cancer is generally known to be the result of abnormal cell growth and proliferation due to various reasons. Inherent gene abnormality and gene alterations by exposures to the chemical carcinogens or by infection with viruses are examples of those reasons.

[4] In normal cells, cell proliferation is controlled properly by balanced functions of proto-oncogenes, tumor suppressor genes and apoptosis-regulating genes. Proto- oncogene is basically essential for cell growth and differentiation by stimulating protein synthesis and intracellular signal transduction. However, once mutated, it induces over-proliferation of a cell, leading cells more prone to tumorigenesis. In the meantime, tumor suppressor inhibits excessive cell growth by regulating cell cycles and repairing genetic defects. Nevertheless, cancer can be developed by loss- of-function mutation of tumor suppressor genes or gain-of-function mutation of proto- oncogenes. Cells with mutated proto-oncogenes or tumor suppressor genes generally go through apoptosis process by apoptosis-regulating genes. In addition to oncogene, tumor suppressor gene and apoptosis-regulating gene, there are many other protective mechanisms with functions of gene repair, signal transmission, etc. , to keep cells healthy. Despite those double or triple protective mechanisms in cells, cancer can be developed when cells are continuously and excessively exposed to carcinogens and genetic defects occur to the genes.

[5] Therefore, various techniques and materials have been developed to overcome cancer, i. e. , surgical operations, radiotherapy, chemotherapy, immunotherapy and gene-therapy [6] Chemotherapy has been popular after choriocarcinoma was completely cured by using methotrexate, and more than 50 chemotherapeutic agents are now in use. In particular, cancers such as choriocarcinoma, leukemia, Wilms'tuDor, Ewing's sarcoma, rhabdona, retinoblastoma, lymphom and testis tumors are generally well-treated with chemotherapeutic agents.

[7] Nbst chemtherapeutic agents inhibit the synthesis of nucleic acid or poly- merization of nucleic acids. However, those chemotherapeutic agents not only work for cancer cells but also damage normal cells, especially actively proliferating cells, resulting in serious side effects such as dysfunction of bone marrow, injury of gas- trointestinal mucosa, depilation, etc. So, the biggest problem of chemotherapeutic agents is non-specificity, i. e. , they damage normal cells showing active cell division, for example, bone marrow cells, gastrointestinal epithelial cells, hair follicle cells, etc.

As a result, most of patients under chemotherapy suffer side effects such as dysfunction of bone marrow, gastrointestinal disorder, depilation, etc. The only difference in the effect of chemotherapy between normal cells and cancer cells is not in quality but in quantity. The only reason why cancer cells are destroyed more than normal cells is because they are more sensitive than normal cells and regeneration of normal cells are faster.

[8] One of the popular anticancer-approaches is apoptosis induction by finding or improving a prominent candidate which is able to induce apoptosis of cancer cells.

Apoptosis is a kind of active mechanism causing cell death, which functions not only in normal physiological circumstances such as development and differentiation but also in pathological environments such as cell defect or infection with a mi- croorganism. External apoptosis-inducing factors are UV, g-ray irradiation, heat shock, ceramide, anticancer drugs, reactive oxygen species, virus infection and elimination of a growth factor, etc, and those are divided into two groups according to their action mechanism; 1) factors acting through a death receptor and 2) apoptosis-inducers working other pathways. Apoptosis-inducers working through a death receptor usually induce the expression of death receptors, i. e., Fas, tudor necrotizing factor receptor 1 (TNFR1), TNF-related apoptosis-inducing ligand (TRAIL), TNF-receptor-related apoptosis-mediated protein (TRAMP), nerve growth factor (NGF), etc. Apoptosis- inducers that do not use the death receptors generally induce the expression of bax, a pro-apoptotic protein of bcl-2 family inducing apoptosis, leading to caspase activation, and a breakdown of mitochondrial membrane potential. Currently, studies are actively undergoing on the mechanism of g-ray irradiation-, heat shock-and anticancer drug- induced apoptosis and those works are used for estimating the proper combinations of various cancer therapies and elucidating reasons for the cases where conventional therapies did not work. Thus, finding a protein playing an important role in apoptosis is very important and encouraging, along with elucidating the mechanism of apoptosis in connection with various anticancer drugs.

[9] The primary goal of most anticancer agents is to induce apoptosis of cancer cells.

The conventional anticancer agents induce apoptosis, but cannot control specific apoptosis-regulating genes. One of apoptosis-targeted anticancer agents under de- velopment now, is Aptosyn developed by a company (Cell pathway, Horsham, PA, USA). According to them, Aptosyn induces apoptosis of abnormal cells specifically by inhibiting cyclic GMP phosphodiesterase. In the meantime, G-3139, developed by another company (Genta, Lexington, MA, USA), is a substance lowering the amount of bcl-2 protein in a cancer cell by inhibiting the synthesis of mRNA of bcl-2 gene.

The possibility seems to be real that G-3139 might be used in conjunction with other anticancer agents such as docetaxel, irinotecan or paclitaxel.

[10] Lck expression is generally restricted to T-lymphocytes and NK cells. However, over-expression of Lck was also reported in a blood cancer patient, in particular B cell neoplasia. B cell neoplasia is exemplified by Burkitt lymphom (Cheung RK & Dosch M. 1991, J. Biol. Chem. 266: 8667-8670; Jucker M et al. 1991. Leukemia 5: 528-530; Knethen AV et al. 1997. Leukemia & Lymphom 26: 551-562), which is a B- transformed cell line, non-Hodgkin B-cell lymphom (Rouer et al. 1993. Leukemia 7: 246-250), B-lymphoblastoid cells, chronic B-lymphocytic leukemia (Knethen AV et al. 1997. Leukemia & Lymphom 26: 551-562; Majolini MB et al. 1998. Blood 91: 3390-3396), etc. In addition, abnormal expression of Lck was also observed in a lung cancer, colon cancer cell line (Veillette et al. 1987. Oncogene Res. 1: 357-374), colorectal cancer tissue (Nakamura K et al. 1996. Eur. J. Cancer 32A 1401-1407) and in breast cancer tissues (Koster A et al. 1991. Anticancer Res. 11: 193-201).

[11] Leukemia, a malignant tumor generated in bone marrow and blood, is resulted from abnormal proliferation of blood cells. Leukemia is classified into four groups; acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, and chronic lymphoblastic leukemia. Myelogenous leukemia is caused by abnormality of myeloid cells, erythrocytes or megakaryocytes in bone marrow, and Lymphoblastic leukemia is caused by abnormality of lymphoblastic cells such as T-cells or B-cells.

[12] Radiotherapy, bone marrow transplantation and chemotherapy are used for the treatment of leukemia. Although bone marrow transplantation is the most fundamental treatment for leukemia among many methods, it is limited in use because of difficulty in securing a donor. Thus, chemotherapy is taking the place of it. Generally, more than two anticancer agents are combinatorially used for leukemia. In fact, about 40 anticancer agents are administered either singly or combinatorially. M) st chemotherapeutic agents are DNA synthesis inhibitors or cell growth inhibitors, and since they target actively-proliferating cells no matter it is normal or cancerous, side effects are inevitable. Moreover, after all those treatments, leukemia patients are not completely recovered and, in some patients, chemotherapy merely extend the survival period.

[13] Rosmarinic acid has been known to have various activities in vivo. In particular, it has anti-viral, anti-bacterial, anti-oxidant activities and inhibitory activities on the synthesis of tumor necrosis factor. Rosmarinic acid also has anti-5-lipoxygenase, anti- cyclooxygenase, anti-complement and anti-inflammatory activities.

[14] The present inventors have eagerly studied to find a substance being able to kill cancer cells only without damaging normal cells. At last, the present inventors have completed this invention by confirming that apoptotic effect of hydroxylphenyl derivatives of rosmarinic acid is mostly restricted to cancer cells only.

[15] Disclosure of Invention Technical Solution [16] It is an object of this invention to provide a pharmaceutical composition for anticancer containing hydroxylphenyl derivatives of rosmarinic acid as an effective ingredient.

[17] Brief Description of the Drawings [18] The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein: [19] [20] FIG. 1 is a graph showing the apoptotic effect of hydroxylphenyl derivatives of rosmarinic acid on various leukemia cell lines, [21] [22] FIG. 2 is a graph showing the apoptotic effect of hydroxylphenyl derivatives of rosmarinic acid on various leukemia cell lines according to the concentration of the same, [23] [24] FIG. 3 is a graph showing the apoptotic effect of hydroxylphenyl derivatives of rosmarinic acid on peripheral blood mononuclear cells of a healthy donor, [25] [26] FIG. 4 is a graph showing the apoptotic effect hydroxylphenyl derivatives of rosmarinic acid on peripheral blood mononuclear cells of a patient with B-cell chronic lymphoblastic leukemia.

[27] Best Mode for Carrying Out the Invention [28] The present invention provides a pharmaceutical composition for anticancer containing hydroxylphenyl derivatives of rosmarinic acid represented by the following <formula 1> as an effective ingredient.

[29] <Formula 1> [30] [31] Wherein, [32] R, R, R, R and R are independent each other, at least one of them is hydroxy, 1 2 3 4 S and the others are selected from a group consisting of H, halogen atom, C-C alkoxy, 1 3 aldehyde, carboxyl, amino, trifluormethyl, and nitro; [33] R, R, R, R and R are independent each other, at least one of them is hydroxy, 6 7 8 9 10 and the others are selected from a group consisting of H, halogen atom, C-C alkoxy, 1 3 aldehyde, carboxyl, amino, trifluormethyl, and nitro; [34] X is O, S, -NH-,-N (CH)-,-N (CH CH)-, or-NHNH; 1 3 2 3 [35] X is-CH-,-C (=O)-,-C (=S)-, or-C (=O)-NH-; z z [36] X is selected from a group consisting of , and- (CH)- ; provided that A is H, C ~C straight or branched alkyl, thiol, phenyl, 2 m 1 1 4 cyano, or C-C alkoxycarbonyl; A is H, or CI-C 4 straight or branched alkyl ; n is 0,1 1 3 2 1 4 or 2; m is 0,1 or 2 ; [37] Y is selected from a group consisting of H,-CH-,-C (=O)-,-C (=S) -, amine substituted with C-C straight or branched alkyl or aryl, 1 4 [38] Y is not existed or-NZ Z,-O-Z, or-S-Z ; 2 11 12 2 2 [39] Z11 or Z12 is independent each other, H, amine substituted with t-butoxycarbonyl or not, C1#C12 straight or branched alkyl, aryl, cycloalkyl, or heteroalkyl; [40] Z is H, C #C straight or branched alkyl, aryl, cycloalkyl, or heteroalkyl; 2 1 12 [41] B is H or alkyl ; [42] * represents a chiral carbon.

[43] [44] The compounds of <Formula 1> are optical isomers of R or S. In the present invention, all the optical isomers and racemic mixtures thereof are included.

[45] Preferably, R, R and R are H; R and R are hydroxy; R, R and R are H; R and 4 5 2 3 6 7 10 8 R are hydroxy; X is O, S, -NH-or-N (CH)-; X is -CH -, -C(=O)- or -C(=S)-; X is- 9 1 3 2 2 3 CH=CH- ; Y is O or not; Y is C-C alkoxy, -NH or hydroxy; B is Q 1 2 1 4 2 [46] [47] Preferably, the compounds of hydroxylphenyl derivatives of rosmarinic acid comprise : [48] 1) 3- (3, 4-dihydroxy-phenyl)- (R)-2- [3-trans- (3, 4-dihydroxy-phenyl)-acryloylamino] - propionic acid methyl ester; [49] 2) 3-(3,4-dihydroxy-phenyl)-(R)-2-[3-trans-(3,4-dihydroxy-pheny l)-acryloylamino] - propionic acid; [50] 3) 3-(3,4-dihydroxy-phenyl)-(R)-2-[3-trans-(3,4-dihydroxy-pheny l)-acryloylamino] - propionic acid ethyl ester; [51] 4) 3-(3,4-dihydroxy-phenyl)-(R)-2-[3-trans-(3,4-dihydroxy-pheny l)-acryloylamino] - propionic acid propyl ester; [52] 5) 3- (3, 4-dihydroxy-phenyl)- (R)-2- [3-trans- (3, 4-dihydroxy-phenyl)-acryloylamino] - propionic acid isopropyl ester; [53] 6) 3- (3, 4-dihydroxy-phenyl)- (R)-2- [3-trans- (3, 4-dihydroxy-phenyl)-acryloylamino] - propionic acid tert-butyl ester; [54] 7) 3- (3, 4-dihydroxy-phenyl)- (R)-2- [3-trans- (3, 4-dihydroxy-phenyl)-acryloylamino] - propionamide ; [55] 8) 3- (3, 4-dihydroxy-phenyl)- (S)-2- [3-trans- (3, 4-dihydroxy-phenyl)-acryloylamino]- propionic acid methyl ester; [56] 9) 3- (3, 4-dihydroxy-phenyl)- (S)-2- [3-trans- (3, 4-dihydroxy-phenyl)-acryloylamino]- propionic acid; [57] 10) 3- (3, 4-dihydroxy-phenyl)- (S)-2- [3-trans- (3, 4-dihydroxy-phenyl)-acryloylamino ]-propionic acid ethyl ester; [58] 11) 3- (3, 4-dihydroxy-phenyl)- (S)-2- [3-trans- (3, 4-dihydroxy-phenyl)-acryloylamino ] -propionic acid propyl ester; [59] 12) 3- (3, 4-dihydroxy-phenyl)- (S)-2- [3-trans- (3, 4-dihydroxy-phenyl)-acryloylamino ]-propionic acid isopropyl ester; [60] 13) 3- (3, 4-dihydroxy-phenyl)- (S)-2- [3-trans- (3, 4-dihydroxy-phenyl)-acryloylamino ]-propionic acid tert-butyl ester; [61] 14) 3- (3, 4-dihydroxy-phenyl)- (S)-2- [3-trans- (3, 4-dihydroxy-phenyl)-acryloylamino ]-propionamide ; [62] 15) 3- (3, 4-dihydroxy-phenyl)- (R)-2- [3-trans- (3, 4-dihydroxy-phenyl)-thioacryloylamino]-p ropionic acid methyl ester; [63] 16) 3- (3, 4-dihydroxy-phenyl)- (R)-2- [3-trans- (3, 4-dihydroxy-phenyl)-thioacryloylamino]-p ropionic acid; [64] 17) 3- (3, 4-dihydroxy- phenyl)- (R)-2- [3-trans- (3, 4-dihydroxy-phenyl)-thioacryloylamino]-propionic acid ethyl ester; [65] 18) 3- (3, 4-dihydroxy-phenyl)- (R)-2- [3-trans- (3, 4-dihydroxy-phenyl)-thioacryloylamino]-p ropionic acid propyl ester; [66] 19) 3- (3, 4-dihydroxy-phenyl)- (R)-2- [3-trans- (3, 4-dihydroxy-phenyl)-thioacryloylamino]-p ropionic acid isopropyl ester; [67] 20) 3- (3, 4-dihydroxy-phenyl)- (R)-2- [3-trans- (3, 4-dihydroxy-phenyl)-thioacryloylamino]-p ropionic acid tert-butyl ester; [68] 21) 3- (3, 4-dihydroxy-phenyl)- (R)-2- [3-trans- (3, 4-dihydroxy-phenyl)-thioacryloylamino]-p ropionamide ; [69] 22) 3- (3, 4-dihydroxy-phenyl)- (S)-2- [3-trans- (3, 4-dihydroxy-phenyl)-thioacryloylamino]-p ropionic acid methyl ester; [70] 23) 3- (3, 4-dihydroxy-phenyl)- (S)-2- [3-trans- (3, 4-dihydroxy-phenyl)-thioacryloylamino]-p ropionic acid; [71] 24) 3- (3, 4-dihydroxy-phenyl)- (S)-2- [3-trans- (3, 4-dihydroxy- phenyl)-thioacryloylamino]-propionic acid ethyl ester; [72] 25) 3- (3, 4-dihydroxy-phenyl)- (S)-2- [3-trans- (3, 4-dihydroxy-phenyl)-thioacryloylamino]-p ropionic acid propyl ester; [73] 26) 3- (3, 4-dihydroxy-phenyl)- (S)-2- [3-trans- (3, 4-dihydroxy-phenyl)-thioacryloylamino]-p ropionic acid isopropyl ester; [74] 27) 3- (3, 4-dihydroxy-phenyl)- (S)-2- [3-trans- (3, 4-dihydroxy-phenyl)-thioacryloylamino]-p ropionic acid tert-butyl ester; [75] 28) 3- (3, 4-dihydroxy-phenyl)- (S)-2- [3-trans- (3, 4-dihydroxy-phenyl)-thioacryloylamino]-p ropionamide ; [76] 29) 3-(3, 4-dihydroxy-phenyl)-(R)-2-{[3-trans-(3, 4-dihydroxy-phenyl)-acryloyl]- methyl-amino}-propionic acid methyl ester; [77] 30) 3-(3, 4-dihydroxy-phenyl)-(R)-2-{[3-trans-(3, 4-dihydroxy-phenyl)-acryloyl]- methyl-amino}-propionic acid; [78] 31) 3-(3,4-dihydroxy-phenyl)-(R)-2-{[3-trans-(3,4-dihydroxy-phen yl)-acryloyl] - methyl-amino}-propionic acid ethyl ester; [79] 32) 3-(3, 4-dihydroxy-phenyl)-(R)-2-{[3-trans-(3, 4-dihydroxy-phenyl)-acryloyl]- methyl-amino}-propionic acid propyl ester; [80] 33) 3-(3, 4-dihydroxy-phenyl)-(R)-2-{[3-trans-(3, 4-dihydroxy-phenyl)-acryloyl]- methyl-amino}-propionic acid isopropyl ester; [81] 34) 3-(3,4-dihydroxy-phenyl)-(R)-2-{[3-trans-(3,4-dihydroxy-phen yl)-acryloyl] - methyl-amino}-propionic acid tert-butyl ester; [82] 35) 3- (3, 4-dihydroxy-phenyl)- (R)-2- [3-trans- (3, 4-dihydroxy-phenyl)-acryloyl]- methyl-amino]-propionamide ; [83] 36) 3-(3, 4-dihydroxy-phenyl)-(S)-2-{[3-trans-(3, 4-dihydroxy-phenyl)-acryloyl]- methyl-amino}-propionic acid methyl ester; [84] 37) 3-(3, 4-dihydroxy-phenyl)-(S)-2-{[3-trans-(3, 4-dihydroxy-phenyl)-acryloyl]- methyl-amino}-propionic acid; [85] 38) 3-(3,4-dihydroxy-phenyl)-(S)-2-{[3-trans-(3,4-dihydroxy-phen yl)-acryloyl]-me thyl-amino}-propionic acid ethyl ester; [86] 39) 3-(3, 4-dihydroxy-phenyl)-(S)-2-{[3-trans-(3, 4-dihydroxy-phenyl)-acryloyl]- methyl-amino}-propionic acid propyl ester; [87] 40) 3-(3,4-dihydroxy-phenyl)-(S)-2-{[3-trans-(3,4-dihydroxy-phen yl)-acryloyl] - methyl-amino}-propionic acid isopropyl ester; [88] 41) 3-(3,4-dihydroxy-phenyl)-(S)-2-{[3-trans-(3,4-dihydroxy-phen yl)-acryloyl] - methyl-amino}-propionic acid tert-butyl ester; [89] 42) 3- (3, 4-dihydroxy-phenyl)- (S)-2- [3-trans- (3, 4-dihydroxy-phenyl)-acryloyl]- methyl-amino]-propionamide ; [90] 43) 3- (3, 4-dihydroxy-phenyl)-N- [2-trans- (3, 4-dihydroxy-phenyl)-ethyl]- acrylamide ; [91] 44) 3- (3, 4-dihydroxy-phenyl)-N- [2-trans- (3, 4-dihydroxy-phenyl)-ethyl]- Nmethyl-acrylamide ; [92] 45) (R)-2- [trans-3- (3, 4-dihydroxy-phenyl)-acryloylamino]- 3- (4-hydroxy-phenyl)-propionic acid methyl ester; [93] 46) (R)-2- [trans-3- (3, 4-dihydroxy-phenyl)-acryloylamino]- 3- (4-hydroxy-phenyl)-propionic acid; [94] 47) (S)-2- [trans-3- (3, 4-dihydroxy-phenyl)-acryloylamino]- 3- (4-hydroxy-phenyl)-propionic acid methyl ester; [95] 48) (S)-2- [trans-3- (3, 4-dihydroxy-phenyl)-acryloylamino]- 3- (4-hydroxy-phenyl)-propionic acid; [96] 49) (S)-2- [3- (3, 4-dihydroxy-benzyl)-ureido]-3- (3, 4-dihydroxy-phenyl)-propionic acid methyl ester; [97] 50) 3- (3, 4-dihydroxy-phenyl)-2- [2- (3, 4-dihydroxy-phenyl)-acethylamino]- propionic acid methyl ester; [98] 51) 2- (3, 4-dihydroxy-benzoylamino)-3- (3, 4-dihydroxy-phenyl)-propionic acid methyl ester; [99] 52) 3- (3, 4-dihydroxy-phenyl)-2- [3- (3, 4-dihydroxy-phenyl)-propyonylamino]- propionic acid methyl ester; [100] 53) 3- (3, 4-dihydroxy-phenyl)-2- [3- (3, 4-dihydroxy-phenyl)-arylamino]-propionic acid methyl ester; [101] 54) (R)-3- (3, 4-dihydroxy-phenyl)-acrylic acid- 2-(3, 4-dihydroxy-phenyl)-lmethoxycarbonyl ethyl ester; [102] 55) (R)-3- (3, 4-dihydroxy-phenyl)-acrylic acid- 2- (3, 4-dihydroxy-phenyl)-1-propoxycarbonyl ethyl ester; [103] 56) (R)-3- (3, 4-dihydroxy-phenyl)-acrylic acid-2- (3, 4-dihydro xy- phenyl)-l-t-butoxycarbonyl ethyl ester; [104] 57) (R)-3- (3, 4-dihydroxy-phenyl)-acrylic acid- 2- (3, 4-dihydroxy-phenyl)-l-carbamoyl ethyl ester; and [105] 58) (R)-3- (3, 4-dihydroxy-phenyl)-acrylic acid- 2- (3, 4-dihydroxy-phenyl)-l-isopropylcarbamoyl ethyl ester.

[106] [107] The hydroxylphenyl derivatives of rosmarinic acid of the present invention can be prepared by the preparation method disclosed in WD 03/089405.

[108] Hydroxylphenyl derivatives of rosmarinic acid of the present invention induce apoptosis of various leukemia cell lines. Particularly, Jurkat and LBRM-33, derived from T-cells expressing Lck, and BCL-1, derived from B-cells not expressing Lck until tunorigenesis, are killed by the derivatives effectively. On the other hand, J. CaMl. 6, Raji and mononuclear (THP-1, U937) cell lines, not expressing Lck, are not affected by the hydroxylphenyl derivatives of rosmarinic acid. However, when the concentration of hydroxylphenyl derivatives of rosmarinic acid is over 50 uM, even cancer cells not expressing Lck might be killed, indicating that apoptotic effect of hy- droxylphenyl derivatives of rosmarinic acid is not limited to cancer cell lines expressing Lck.

[109] Hydroxylphenyl derivatives of rosmarinic acid of the present invention loses apoptotic effect when hydroxyl groups in the phenyl groups located in both ends of the derivatives are either eliminated or masked. Thus, hydroxyl groups of phenyl groups located in both ends of the derivatives play very important role in apoptosis.

[110] Hydroxylphenyl derivatives of rosmarinic acid of the present invention do not destroy peripheral blood mononuclear cells of a healthy donor but kill peripheral blood mononuclear cells of a leukemia patient, more particularly, B-cell leukemia cells taking 80% of mononuclear lymphocytes of a leukemia patient.

[111] Therefore, hydroxylphenyl derivatives of rosmarinic acid of the present invention can be effectively used for preventing or treating cancers by inducing apoptosis in cancer cells only without damaging normal cells.

[112] The composition of the present invention can be prepared by comprising one or more pharmaceutically acceptable carrier additionally in addition to abovementioned effective ingredients for administration. A pharmaceutically acceptable carrier can use by mixing saline, sterilized aqueous solution, linger solution, buffer saline, dextrose solution, malto-dextrin solution, glycerol, ethanol and one or more ingredient of them, and as occasion demands, it can use by adding other conventional additives such as an- tioxidant, buffer solution, fungistats etc. It can prepared to parenteral formulation such as aqueous solution, suspension, emulsion, or pellet, capsule, granule, or tablets by addition diluent, dispersion agent, surfactant, binding agent and lubricant additionally.

The composition can further be prepared in suitable forms for each diseases or according to ingredients by following a method represented in Remington's Phar- maceutical Science (the newest edition), Mack Publishing Company, Easton PA [113] The composition of the present invention can be administered orally or parenterally by objective method (for example, intravenous, hypodermic, local or peritoneal injection). The effective dosage of the composition can be determined according to weight, age, gender, health condition, diet, administration frequency, administration method, excretion and severity of a disease. The dosage of hydroxylphenyl derivatives of rosmarinic acid is 0. 05-500 mg/kg per day when they are administered by in- tramuscular injection and 1-5 g/kg per day when they are orally administered, and ad- ministration frequency is once a day or preferably a few times a day.

[114] Estimated LD50 value of the hydroxylphenyl derivatives of rosmarinic acid of the present invention is much greater than 1 g/kg, which is confirmed by acute toxicity assay with mice tested via oral administration.

[115] A composition of the present invention can be used singly or together with other treatments such as surgical operation, hormone treatment, pharmacologic treatment, biological regulators, etc. , for preventing and treating cancers.

[116] Anticancer agents available for co-use with the composition of the present invention are cell growth inhibitors such as zinostatin, pirarubicin, idarubicin, el- liptiniun acetate, streptozocin and zinostatin stimalamer, antibodies such as lentinan, procodazol, TheraCys, OncoVAX-CL, ukrain, BCG vaccine and sizofilan, hormones such as aminoglutethimide, fadrozole, formestane and trilostane, and others such as razoxane, etoposide phosphate, vindesine, nitracrine, tretinoin, amsacrine, vinorelbine and sobuzoxane. Cytarabine, doxorubicin, daunorubicin, mitoxantrone, thioguanine, mercaptopurine, prednisone, etoposide, asparaginase, vincristin, cyclophosphamide, 5-FU and paclitaxel can be also included in the above drug category.

[117] Mode for the Invention [118] EXAMPLES [119] Practical and presently preferred embodiments of the present invention are il- lustrative as shown in the following Examples.

[120] However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

[121] [122] Example 1: Apoptotic effect of hvdroxvlphenvl derivatives of rosmarinic acid on various leukemia cell lines [123] Following experiments were performed to investigate apoptotic effect of hy- droxylphenyl derivatives of rosmarinic acid of the present invention on various leukemia cell lines.

[124] Cell lines derived from T lymphocytes, B-lymphocytes and monocytes such as Jurkat (hunan acute T-cell leukemia cell line), J. CaMl. 6 (hunan acute T-cell leukemia cell line not expressing Lck), LBRM-33 (mouse T-cell lymphom cell line), BCL-1 (mouse B-cell leukemia cell line), Raji (human Burkitt's lymphom cell line), THP-1 (hunan monocyte cell line), U937 (hunan rmonocyte cell line), etc were used.

All the cell lines were purchased from ATCC (American Type Culture Collection).

[125] Each cell line was maintained in RPMI 1640 mediun containing 10% FBS, 2mM L-glutamine, 100 U/m of penicillin and 100, ug/mQ of streptomycin.

[126] The cell density of each cell line was adjusted to lx10 cells/mQ. 10 uM of hy- droxylphenyl derivative of rosmarinic acid [3- (3, 4-dihydroxy-phenyl)- (R)-2- [3-trans- (3, 4-dihydroxy-phenyl)-acryloylamino]-prop ionic acid methyl ester] was added and cultured for 16-40 hours. Then, the cells were stained with annexin V-FITC to measure the apoptotic effect of hydroxylphenyl derivatives of rosmarinic acid by FACS.

[127] The results were shown in FIG. 1.

[128] As shown in FIG. 1, hydroxylphenyl derivative of rosmarinic acid [3- (3, 4-dihydroxy-phenyl)- (R)-2- [3-trans- (3, 4-dihydroxy-phenyl)-acryloylamino]-prop ionic acid methyl ester] destroyed 40-50% Jurkat, LBRM-33 (both are T-cell derived leukemia cell lines expressing Lck) and BCL-1 (B-cell leukemia cell line) cells for 16 hours and 100% of them all after 40 hours, indicating that apoptotic effect of the hy- droxylphenyl derivative of rosmarinic acid was time-dependent. On the contrary, apoptotic effect of the derivative was not detected in those cell lines not expressing Lck such as Jurkat T-lymphocyte derived J. CaMl. 6, B-cell derived Raji, monocyte cell line THP-1, U937, etc.

[129] [130] Apoptosis effects of other hydroxylphenyl derivatives of rosmarinic acid were further investigated in Lck over-expressing BCL-1 cell line.

[131] The cell density of BCL-1 for culture was lx 10 cells/mQ. Hydroxylphenyl de rivatives of rosmarinic acid (compounds 1-58) were added at the concentrations of 100,50, 25,12. 5,6. 25,3. 125 uM each and cultured for 40 hours. The cells were stained with annexin V-FITC to measure apoptotic effects of them by FACS.

[132] The results are shown in Table 1.

[133] Table 1 Derivative Apoptoticactivity ( [E Derivative Apoptoticactivity ( [E D50(uM)] D50(uM)] 1 12 30 30.8 2 30 31 14 3 15.8 32 12.5 4 12.4 33 12.6 5 11.8 34 12.6 6 12 35 13.1 7 12.8 36 11.9 8 12.1 37 31 9 25 38 12.8 10 15.1 39 11.2 11 12.5 40 11.9 12 11.9 41 11.9 13 12.5 42 12.5 14 13 43 50 15 7.3 44 52 16 20 45 47.1 17 10.5 46 52.1 18 6.9 47 49.5 19 7.5 48 64.2 20 7.9 49 40 21 8.1 50 50 22 6.9 51 51.2 23 21 52 75.1 24 9.5 53 44.7 25 6.8 54 13.1 26 7.7 55 11 27 8.1 56 12.8 28 8.0 57 16 29 12.1 58 11.9 [134] [135] As shown in Table 1, hydroxylphenyl derivatives of rosmarinic acid showed apoptotic effects with ED of 5-20 uM, regardless of the change of X, or X, unless SO 12 the basic carbon structure became shorter. Precisely, apoptotic effects were all observed in rosmarinic acid derivatives when X was-NH-,-O-,-NCH-and when X 1 3 2 was-C (=O)- or-C (=S). The highest apoptotic effect was seen when X was . But, when carbon-carbon double bond was substituted with single bond or the number of carbon decreased less than two, apoptotic effect was lost or dropped rapidly.

[136] When a compound is cleaved into two compounds at the position X, the compound loses apoptotic activity rapidly. Therefore a whole molecular structure ought to be maintained to keep apoptotic effect.

[137] In the meantime, when Y and Y formed ester or amide, apoptotic effect was not 1 2 changed. But, when Y1 and Y2 were removed, the apoptotic activity disappeared rapidly. When Y was a hydrophobic group such as -OCH, -OCH CH, -O(CH) CH, 2 3 2 3 2 2 3 - OCH (CH), or-OC (CH), apoptotic activity increased greatly. In this invention, a 3 2 3 3 hydrophobic group was not limited to the above.

[138] The apoptotic activities of hydroxylphenyl derivatives of rosmarinic acid decreased remarkably when hydroxyl groups of phenyl groups positioned at both ends of the derivatives were eliminated or masked, indicating that hydroxyl groups of phenyl groups in both ends play a very important role in inducing apoptosis.

[139] Therefore, R1#R5 of hydroxylphenyl derivatives of rosmarinic acid, represented by Formula 1, should be independent each other and at least one of them must be a hydroxyl group. And, R-R of the derivatives should be independent each other and 6 10 at least one of them must be a hydroxyl group. However, in this invention, R1#R10 of the derivatives are not necessarily hydroxyl groups but can be substituted with other groups having similar characteristics to hydroxyl groups.

[140] [141] Example 2: Apoptotic effect of hvdroxvlphenvl derivatives of rosmarinic acid on various leukemia cell lines [142] The present inventors have performed following experiments to investigate apoptotic effect of hydroxylphenyl derivatives of rosmarinic acid of the present invention on various leukemia cell lines.

[143] Cell lines derived from T lymphocytes, B-lymphocytes and monocytes such as Jurkat (hunan acute T-cell leukemia cell line), H9 (human T cell leukemia cell line), CCRF-CEM (human acute T cell leukemia cell line), BCL-1 (mouse B-cell leukemia cell line expressing Lck), J. CaMl. 6 (hunan acute T-cell leukemia cell line not expressing Lck), EL4 (mouse T cell leukemia cell line), LBRM-33 (mouse T-cell lymphom cell line), WEHI-3 (mouse leukemia cell line), K-562 (mouse myelogenous leukemia cell line), etc were used. All the cell lines were purchased from ATCC (American Type Culture Collection).

[144] Each cell line was maintained in a mediun according to the instructions provided by ATCC. Cell density was lx10 cells/mQ and hydroxylphenyl derivatives of rosmarinic acid were added at the concentrations of 5,10, 20,30 and 50 uM, re- spectively. Culture was continued for 24-48 hours. Then, the cells were stained with annexin V-FITC to measure apoptotic effects of them by FACS.

[145] The results are shown in FIG. 2.

[146] As shown in FIG. 2, hydroxylphenyl derivatives of rosmarinic acid effectively destroyed T-cell leukemia cell lines (Jurkat, H9, CCRF-CEM) and B-cell leukemia cell line, BCL-1, expressing Lck abnormally, showing ED of 12 uM. su [147] Hydroxylphenyl derivatives of rosmarinic acid also killed other leukemia cell lines not expressing Lck (J. CaMl. 6, EL-4, WEHI, K-562, etc), which was, though, not significant (showing ED at 25-40 uM). so [148] Hydroxylphenyl derivatives of rosmarinic acid of the present invention can destroy cancer cell lines not expressing Lck with the concentrations over 50 uM, meaning that their apoptotic effects were not limited to cancer cell lines expressing Lck.

[149] Therefore, hydroxylphenyl derivatives of the present invention are expected to induce apoptosis of leukemia cell lines even originated from B-lymphocytes showing abnormal Lck expression, for example blood cancer cell lines, lung cancer cell lines, large intestine cancer cell lines, colon carcinoma cell lines and breast cancer cell lines.

[150] [151] Example 3: Apoptotic effect of hydroxylphenyl derivatives of rosmarinic acid on peripheral blood mononuclear cells of healthy donors [152] The present inventors have performed following experiments to investigate apoptotic effect of hydroxylphenyl derivatives of rosmarinic acid on peripheral blood mononuclear cells of a healthy donor.

[153] Blood was taken from healthy donors and mononuclear cells (Hunan Peripheral Blood nonuclear Cells; hPBMC) were isolated therefrom by using Ficoll-paque.

The separated mononuclear cells were maintained in RPMI mediun containing 10% FBS, 2 mM L-glutamine, 100 U/m of penicillin and 100, ug/mQ of streptomycin.

[154] Nbnonuclear cells (lx10 cells/mQ) were treated with various concentrations (12. 5- 50 uM) of hydroxyl phenyl derivatives of rosmarinic acid in the absence of any stimulators for 24-48 hours. Then, the mononuclear cells were stained with annexin V-FITC to measure apoptotic effect by FACS.

[155] The results are shown in FIG. 3.

[156] As shown in FIG. 3, hydroxylphenyl derivatives of rosmarinic acid did not induce apoptosis of mononuclear cells from healthy donors.

[157] It is implicated from the experiments that hydroxylphenyl derivatives of rosmarinic acid of the present invention induces apoptosis of cancer cells only, but not resting peripheral blood mononuclear cells from healthy donors.

[158] [159] Example 4: Apoptotic effect of hvdroxvlphenvl derivatives of rosmarinic acid on peripheral blood mononuclear cells from B-cell chronic Ivmphocvtic leukemia patients [160] The present inventors performed following experiments to investigate apoptotic effect of hydroxylphenyl derivatives of rosmarinic acid of the present invention on mononuclear cells from leukemia patients.

[161] Bloods were taken from B-cell chronic lymphocytic leukemia patients and mononuclear cells were isolated therefrom by using Ficoll-paque. The isolated mononuclear cells were maintained in RPMI medium containing 10% FBS, 2 mM L- glutamin, 100 U/m of penicillin and 100, ug/mQ of streptomycin.

[162] In order to observe the subset populations of the isolated mononuclear cells, mononuclear cells were stained with anti-CD3-Cy-chrome and anti-CD19-PE, followed by FACS analysis. Around 80% of mononuclear cells were CD19-positive (B cell marker) in B-cell chronic lymphocytic leukemia patients, which were greatly increased percentages of B cells compared to a normal range of B cell percentage (more or less than 5%).

[163] Nbnonuclear cells from B-cell chronic lymphocytic leukemia patients were treated with different concentrations of hydroxylphenyl derivatives of rosmarinic acid [3- (3, 4-dihydroxy-phenyl)- (R)-2- [3-trans- (3, 4-dihydroxy-phenyl)-acryloylamino]-prop ionic acid methyl ester] at the concentrations of 100,50, 25 and 12.5 uM each and cultured for 2 days. Then, apoptotic effect was measured by FACS.

[164] The results were shown in FIG. 4.

[165] As shown in FIG. 4, hydroxylphenyl derivative of rosmarinic acid induced apoptosis more than 50% of mononuclear cells from B-cell chronic lymphocytic leukemia patients at the concentration of 12.5 uM. Peripheral blood mononuclear cells from leukemia patients are almost homogeneous to B-lymphocyte populations because leukemic B-lymphocytes take more than 80% of total peripheral blood mononuclear cells from B-cell leukemia patients.

[166] Therefore, hydroxylphenyl derivatives of rosmarinic acid were confirmed to induce apoptosis of peripheral mononuclear cells from chronic lymphocytic leukemia patients, more precisely, leukemic B-lymphocytes of chronic lymphocytic leukemia patients.

[167] [168] Experimental Example : Acute oral toxicitv test in rats [169] The following experiments were performed to see if the compounds of hy- droxylphenyl derivatives of rosmarinic acid of the present invention had acute toxicity in rats.

[170] Six-week old SPF SD line rats were used in the tests for acute toxicity test. The compounds of hydroxylphenyl derivatives of rosmarinic acid were suspended in 0.5% methyl cellulose solution and orally administered to 4 rats per group with the dosage of lg/kg/15m. Death, clinical symptoms, and weight change in rats were observed, hematological tests and biochemical tests of blood were performed, and any abnormal signs in the gastrointestinal organs of chest and abdomen were checked during autopsy. The results showed that the test compounds did not cause any specific clinical symptoms, weight change, or death in rats. No change was observed in hematological tests, biochemical tests of blood, and autopsy.

[171] The compounds used in this experiment were evaluated to be safe substances since they did not cause any toxic change in rats up to the level of 500mg/kg and their estimated LD values were much greater than 1 g/kg in rats. so [172] [173] Manufacturing Example 1: Preparation of injectable solutions [174] The present inventors prepared injectable solutions containing 10mg of hy- droxylphenyl derivatives of rosmarinic acid as an effective ingredient as follows.

[175] lg of hydroxylphenyl derivatives of rosmarinic acid, 0.6 g of sodiun chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 m of solution. The solution was put in a bottle and heated at 20°C for 30 minutes for sterilization.

[176] The constituents of the injectable solutions are as follows.

[177] [178] Hydroxylphenyl derivatives of rosmarinic acid 1 g [179] Sodiun chloride 0.6 g [180] Ascorbic acid 0. 1 g [181] Distilled water Proper amount [182] [183] Manufacturing Example 2: Preparation of syrups [184] Syrups containing hydroxylphenyl derivatives of rosmarinic acid by 2% (weight/volune) as an effective ingredient were prepared as follows.

[185] Hydroxylphenyl derivatives of rosmarinic acid, saccharin and glucose were dissolved in 80 g of warm water. The mixture was cooled down, to which a mixture of glycerin, saccharin, flavors, ethanol, sorbic acid and distilled water was added. Water was added to the mixture, making a total volune of 100 m.

[186] The constituents of the syrups are as follows.

[187] [188] Hydroxylphenyl derivatives of rosmarinic acid 2 g [189] Saccharin 0.8 g [190] Glucose 25.4 g [191] Glycerin 8.0 g [192] Flavor 0. 04 g [193] Ethanol 4. 0 g [194] Sorbic acid 0.4 g [195] Distilled water Proper amount [196] [197] Manufacturing Example 3: Preparation of tablets [198] Tablets containing 15 mg of hydroxylphenyl derivatives of rosmarinic acid as an effective ingredient were prepared as follows.

[199] 250 g of hydroxylphenyl derivatives of rosmarinic acid, 175.9 g of lactose, 180 g of potato-starch and 32 g of colloidal silicic acid were all mixed together. 10% gelatin solution was added to the mixture, which was then pulverized and filtered with a 14mesh sieve. The pulverized mixture was dried, to which 160 g of potato-starch, 50 g of talc and 5 g of magnesium stearate were added to produce tablets.

[200] The constituents of the tablets are as follows.

[201] [202] Hydroxylphenyl derivatives of rosmarinic acid 250 g [203] Lactose 175.9 g [204] Potato starch 180 g [205] Colloidal silicic acid 32 g [206] 10% gelatin solution [207] Potato starch 160 g [208] Talc 50 g [209] Magnesiun stearate 5 g [210] Industrial Applicability [211] The hydroxylphenyl derivatives of rosmarinic acid of the present invention have excellent apoptotic effect on both T-cell originated leukemia cells and B-cell originated leukemia cells expressing Lck abnormally. On the other hand, hy- droxylphenyl derivatives of rosmarinic acid of the present invention do not induce apoptosis of B-cells and monocytes, which do not express Lck.

[212] Hydroxylphenyl derivatives of the present invention do not induce apoptosis of peripheral blood mononuclear cells from healthy donors, but induce apoptosis of peripheral blood mononuclear cells from leukemia patients.

[213] Therefore, hydroxylphenyl derivatives of rosmarinic acid of the present invention can be effectively used for preventing and treating cancers since they induce apoptosis of cancer cells but not normal cells.