WO2010150927A1

WO2010150927A1 - Pharmaceutical composition for prevention and treatment of cancer diseases comprising benzamide derivatives

Info

Publication number: WO2010150927A1
Application number: PCT/KR2009/003420
Authority: WO
Inventors: Mi-Jung Lim; Hyun-Jung Kwak; Man-Young Cha; Sang-Rak Choi; Sun-Gwan Hwang; Kyung-Chul Cho
Original assignee: Sk Holdings Co., Ltd.
Priority date: 2009-06-25
Filing date: 2009-06-25
Publication date: 2010-12-29

Abstract

There is provided a pharmaceutical composition for prevention and treatment of cancer diseases comprising benzamide derivatives. The pharmaceutical composition comprises benzamide derivatives represented by the following formula 1 as indicated in the specification, or pharmaceutically available salts thereof. Therefore, the pharmaceutical composition may be useful to prevent or treat a variety of cancer diseases such as colorectal cancer (CRC) since the pharmaceutical composition is associated with the cell growth inhibition and the tumor growth retardation, including the suppression of the Wnt signaling pathway.

Description

PHARMACEUTICAL COMPOSITION FOR PREVENTION AND TREATMENT OF CANCER DISEASES COMPRISING BENZAMIDE DERIVATIVES

The present invention relates to a pharmaceutical composition for prevention and treatment of cancer diseases, and more particularly, to a pharmaceutical composition for prevention and treatment of cancer diseases such as colorectal cancer comprising benzamide derivatives or pharmaceutically available salts thereof as active components.

Studies on biological activities of benzamide derivatives have been conducted for several years. As a result, medicines having various structures have been known in the art. Among the compounds, some low-molecular compounds have been developed and commercially available as anticancer drugs (Phieng Siliphaivanh and Paul Harrington, 2007; Neeru Khanna and H.J. Jayaram, 2004).

According to the present invention, previously known compounds containing benzamide group were selected to find a group of biologically active compounds by conducting experiments on intracellular signaling pathways whose much research has been recently conducted on mechanisms in which the compounds show anticancer effects.

Cancer is a disease that is defined as uncontrolled proliferation and abnormal cell spreading, and is an incurable disease that takes the second place next to unexpected accidents. In the year 2006, about 1,399,790 Americans were diagnosed as cancer, and about 564,830 patients were died of cancer in the same year (more than 1,500 a day) (American cancer society, 2006). The cancer diseases have appeared as social and economic problems due to continuous increases in cancer incidence and early cancer outbreaks. The outbreak causes of cancer are mainly divided into extrinsic factors (smoking, chemical drugs, radiations, etc.) and intrinsic factors (genetic mutations, hormones, immune conditions, etc.). These factors have been known to act together, or act sequentially to initiate or promote carcinogenesis. Many genes (oncogenes or tumor suppressor genes) affecting the induction of cancer have been known, and the cancer induced by radical accumulation of genetic mutants induces abnormal cell growth. Finally, the proliferated cancer cells move to other human tissues or organs.

Single nucleotide polymorphism (SNP) may cause errors in signaling pathways since the SNP affects the signaling pathways that require normal roles of a target gene. For example, one representative example of the signaling pathways includes a Wnt/β-catenin signaling pathway. The Wnt signaling pathway is directly associated with the anticancer activities, and is recognized as the most general characteristics of human malignancy. Wnt is a group of highly conserved proteins that are associated with the differentiation, growth, maturation and movement of cells. It was known that these proteins maintain homeostasis in adult tissues during a germinating period of cells, and regulate the growth, morphology and motility of cells (Mark L. Johnson et al., 2006; Janssens N. et al., 2006). Typical Wnt signal transduction stabilizes β-catenin proteins by the control of protein kinases, thus to induces the migration into the cell nucleus and the transcriptional activities. It was reported that these transcriptional activities are induced by transcription factors including a group of Lef1/Tcf (Moon RT et al., 2002; Reya T and Clevers H, 2005; Wodarz A and Nusse R, 1998).

When there is no Wnt signal, glycogen-synthase kinase-3β (GSK-3β forms a complex together with adenomatous polyposis coli (APC) and Axin proteins (a scaffolding protein complex) to induce the phosphorylation of a main regulator β-catenin. The phosphorylated β-catenin is decomposed by its ubiquitination, and reduced in volume in cytoplasm, thereby suppressing the transcriptional activities that are mediated by Lef1/Tcf (Hart M et al., 1999; Winston JT et al., 1999).

Meanwhile, when there are Wnt signals, Wnt activates a transducer protein, Dvl (Dishevelled), to suppress the activities of GSK-3β. The suppression of the GSK activities results in the accumulation of β-catenin in the cytoplasm, and the accumulated β-catenin migrates into the nucleus and binds to a variety of transcription factors to induce the expression of target genes. Representative target genes associated with the Wnt signal transduction include a large number of oncogenes such as matrix metalloproteinases (MMP, for example, MMP2, MMP3, MMP7 and MMP9), cyclin D1, Cox-2, c-myc, c-jun, Fra-1 and VEGFR, which are all associated with the outbreak and invasion of cancer. In addition of the presence of the Wnt proteins, the mutations of various regulators (for example, APC, Axin, β-catenin, etc.) continue to activate the Wnt signaling pathway. Actually, the mutations of APC or β-catenin have been found in a large number of cancer diseases (liver cancer, prostate cancer, ovarian cancer and stomach cancer). In particular, it was reported that the Wnt signaling pathway is abnormally activated in more than 90% of patients suffering from the colorectal cancer (Willert, K., and Nusse, R., 1998, Luu H.H., et al., 2004,).

According to the recent research results, the in vitro and in vivo experiments using monoclonal antibody or Wnt signal inhibitors found that the suppression of the activation of the Wnt signal transduction suppresses the cell growth or cell invasion, and induces the apoptosis of cancer cells (He, B. et al., 2004; You, L.et al., 2004).

Most of cytotoxic drugs have been used are used as the therapeutic agents for treating cancer disease to treat cancer. However, these cytotoxic drugs adversely affect the quality of patients lives due to their sever side effects, and have their limits such as the unexpectedly low treatment effects on advanced tumor such as recurring or metastatic cancer diseases. Therefore, there is an urgent demand for development of new drugs that show beneficial effects on progressive cancers and may relieve side effects by improvement of the cancer cell selectivity and specificity. In order to satisfy the demand, a molecular target drug, EGFR inhibitor, or a VEGFR inhibitor, Avastin, which is recently on the market, has been used along with the cytotoxic drugs.

In developing drugs that may treat cancer-related diseases such as breast cancer, stomach cancer liver cancer and colorectal cancer on the basis of the importance of the previously proposed Wnt signaling pathway, Wnt signaling molecules have been recognized to be a good target to screen drugs. Accordingly, there are many attempts to find compounds (activators or inhibitors) that can control the Wnt signaling molecules, and to develop new drugs.

Accordingly, the present inventors have made ardent attempts to solve the above-mentioned problems, and found that benzamide derivatives and their pharmaceutically available salts, which act as antagonists that have the effect of suppressing the Wnt/β-catenin signaling pathway, show an anticancer effect. Therefore, the present invention was completed on the basis of the above facts.

The present invention is designed to solve some of the problems of the prior art, and therefore it is an object of the present invention to provide a pharmaceutical composition having the effect of prevention and treatment of cancer diseases since the pharmaceutical composition comprises benzamide derivatives or their pharmaceutically available salts as active components.

Also, it is another object of the present invention to provide a method for prevention or treatment of cancer using the benzamide derivatives or their pharmaceutically available salts as the active components, wherein the benzamide derivatives or their pharmaceutically available salts show the anticancer activity since they have the effect of suppressing the Wnt/β-catenin signaling pathway.

According to an aspect of the present invention, there is provided a pharmaceutical composition for prevention and treatment of cancer including benzamide derivatives represented by the following formula 1, or pharmaceutically available salts thereof as active components:

Formula 1

wherein, A is -(CH₂)₀- or -(NR₄)-, B is -(CH₂)₀- or -(NH)n(NHR₅)-, n is an integer of 0 and 1, wherein R₄ and R₅ are same or different, and each of the R₄ and R₅ is a group represented by hydrogen or alkyl,

R₁ and R₂ are same or different, and each of R₁ and R₂ is selected from the group consisting of hydrogen; alkyl group; alkyl group substituted with alkynyl alkoxy, cycloalkyl or aryl; and aryl group substituted with alkyl, alkynyl alkoxy, cycloalkyl or aryl, or

R₁ and R₂ form a pyrrolidine, morpoline, piperidine or piperazine ring together with adjacent N, wherein

at least one hydrogen in the pyrrolidine, morpoline, piperidine or piperazine ring may be substituted with alkyl group; alkynyl- or alkoxy-substituted alkyl group; aryl group; aryl group substituted with alkyl, alkynyl or alkoxy; carboxy group; or carbamoyl group, or may be fused with other aryl group, and

R₃ is selected from the group consisting of hydrogen, alkyl, aryl-substituted alkyl group, aryl group, aryl group substituted with halo, alkoxy, carboalkoxy, carbamoyl, cyano, hydroxyl, nitro or thio, heteroaryl group, and heteroaryl group substituted with alkyl, halo, alkoxy, carboalkoxy or aryl group.

In this case, the benzamide derivatives may be presented by the following formula 2:

Formula 2

wherein, n = 1,

R₁, R₂, R₃ and R₅ are defined in the same manner as described above.

Also, the benzamide derivatives may be represented by the following formula 3:

Formula 3

wherein, n = 1, and m = 0 or 1,

X is CH₂-, O, S, NR₇R₈,

R₃ and R₅ are defined in the same manner as described above,

R₆ is selected from the group consisting of hydrogen, alkyl group, alkynyl- or alkoxy-substituted alkyl group, phenyl group, and substituted phenyl group, or forms a fused ring together with the other substituted ring,

R₇ and R₈ are same or different, and each of R₇ and R₈ is selected from the group consisting of hydrogen, alkyl group, alkynyl-, hydroxyl- or alkoxy-substituted alkyl group, hydroxyl, carboxyalkoxy and carbamoyl group.

In addition, the benzamide derivatives may be represented by the following formula 4:

Formula 4

wherein, n = 0, and

R₁, R₂, R_3,and R₅ are defined in the same manner as described above.

The residue R₃ may be heteroaryl selected from the group consisting of thiazole; oxadiazole; benzothiazole; and rings thereof in which at least one hydrogen substituted with alkyl, halo, alkoxy, carboalkoxy, aryl or heteroaryl.

Additionally, the benzamide derivatives may be represented by the following formula 5:

Furthermore, the benzamide derivatives may be represented by the following formula 5:

Formula 5

wherein, R_1, R₂, and R₄ are defined in the same manner as described above, and

R₃ is selected from the group consisting of aryl group, alkyl-substituted alkyl group, aryl group substituted with aryl, halo, alkyl, alkoxy, carboalkoxy, carbamoyl, cyano, hydroxyl, nitro or thio, heteroaryl group, and heteroaryl group substituted with alkyl, halo, alkoxy, carboalkoxy or aryl group.

As described above, the pharmaceutical composition according to one exemplary embodiment of the present invention may be useful to prevent or treat a variety of cancer diseases such as colorectal cancer (CRC) since the pharmaceutical composition is associated with the cell growth inhibition and the tumor growth retardation, including the suppression of the Wnt signaling pathway.

FIG. 1 shows an effect of a pharmaceutical composition according to one exemplary embodiment of the present invention on Tcf-4 activities.

FIG. 2 shows the body weight change after the administration of the pharmaceutical composition according to one exemplary embodiment of the present invention.

FIG. 3 shows the tumor volume changes after the administration of the pharmaceutical composition according to one exemplary embodiment of the present invention.

The terms used in this specification are defined as follows, prior to detailed description of the present invention.

a) Alkyl group:

Alkyl group has 1 to 10 carbon atoms and may be composed of linear or branched saturated or unsaturated hydrocarbon. Here, at least one hydrogen may be substituted to the maximum extent with at least one substituent selected from the group consisting of acyl, amino, carboalkoxy, carboxy, carboxyamino, -O-carbamoyl (-O-(C=O)-NH₂), cyano, halo, hydroxyl, nitro, thio, alkyl, cycloalkyl, aryl, alkoxy, aryloxy, sulfoxy, and guanido, regardless of the order and kinds of the substituents.

b) Cycloalkyl group:

Cycloalkyl group has a 3 to 12-membered ring structure comprising saturated or partially unsaturated hydrocarbon, and may include 0 to 5 heteroatoms such as oxygens, sulfur, nitrogen and the like. Here, the ring structure is a 3 to 12-membered single ring or fused ring compound. Here, at least one hydrogen may be substituted to the maximum extent with at least one substituent selected from the group consisting of acyl, amino, carboalkoxy, carboxy, carboxyamino, -O-carbamoyl (-O-(C=O)-NH₂), cyano, halo, hydroxyl, nitro, thio, alkyl, cycloalkyl, aryl, alkoxy, aryloxy, sulfoxy, and guanido, regardless of the order and kinds of the substituents.

Specific examples of the cycloalkyl group include, but are not particularly limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohehexyl, cycloheptyl, cyclooctyl, morpholinyl, homomorpholinyl, thiomorpholinyl, homothiomorpholinyl, thiomorpholinyl S-oxide, thiomorpholinyl S,S-dioxide, piperidinyl, homopiperidinyl, piperazinyl, homopiperazinyl, pyrrolidiny, pyrrolinyl, tetrahydropyranyl, tetrahydrofuranyl, tetrahydrothienyl, oxazolidinonyl, dihydropyrazolyl, dihydropyrrolyl, dihydropyrazinyl, dihydropyridinyl, dihydropyrimidinyl, dihydrofuryl, dihydropyranyl, and the like.

c) Aryl group:

Aryl group include both aromatic group composed of 5 to 15-membered single-ring or fused-ring unsaturated hydrocarbons and heteroaromatic group containing 1 to 5 heteroatoms such as oxygen, sulfur, nitrogen and the like.

Here, at least one hydrogen may be substituted to the maximum extent with at least one substituent selected from the group consisting of acyl, amino, carboalkoxy, carboxy, carboxyamino, -O-carbamoyl (-O-(C=O)-NH₂), cyano, halo, hydroxyl, nitro, thio, alkyl, cycloalkyl, aryl, alkoxy, aryloxy, sulfoxy, and guanido, regardless of the order and kinds of the substituents.

Specific examples of the aryl group include, but are not particularly limited to, phenyl, 1-naphtyl, 2-naphtyl, pyridinyl, pyrimidinyl, quinolinyl, benzothienyl, indolyl, pyrazinyl, isoindolyl, isoquinolyl, qunazolinyl, quinoxalinyl, phthalazinyl, imidazolinyl, isoxazolyl, pyrazolyl, oxazolyl, thiazolyl, indolizinyl, indazolyl, benzothiazolyl, benzimidazolyl, benzofuranyl, thienyl, pyrrolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, oxazolopyridinyl, imidazopyridinyl, isothiazolyl, cinnolinyl, carbazolyl, isochromanyl, chromanyl, tetrahydroisoquinolinyl, isoindolinyl, isobenzotetrahydrofuranyl, isobenzotetrahydrothienyl, isobenzothienyl, benzoxazolyl, pyridopyridinyl, benzotetrahydrofuranyl, benzotetrahydrothienyl, purinyl, benzodioxolyl, triazinyl, phenoxazinyl, phenothiazinyl, pteridinyl, benzothiazolyl, imidazopyridinyl, imidazothiazolyl, dihydrobenzisoxazinyl, benzisoxazinyl, benzoxazinyl, dihydrobenzisothiopyranyl, benzopyranyl, benzothiopyranyl, coumarinyl, isocoumarinyl, chromonyl, chromanonyl, pyridinyl-N-oxide, tetrahydroquinolinyl-N-oxide, dihydroquinolinyl, dihydroquinolinonyl, dihydroisoquinolinonyl, dihydrocoumarinyl, dihydroisocoumarinyl, isoindolinonyl, benzodioxanyl, benzoxazolinonyl, pyrrolyl-N-oxide, pyrimidinyl-N-oxide, pyrazinyl-N-oxide, quinolinyl-N-oxide, indolyl-N-oxide, indolinyl-N-oxide, pyrazinyl-N-oxide, isoquinolyl-N-oxide, qunazolinyl-N-oxide, quinoxalinyl-N-oxide, phthalazinyl-N-oxide, imidazolinyl-N-oxide, isoxazolyl-N-oxide, oxazolyl-N-oxide, thiazolyl-N-oxide, indolizinyl-N-oxide, indazolyl-N-oxide, benzothiazolyl-N-oxide, benzimidazolyl-N-oxide, pyrrolyl-N-oxide, oxadiazolyl-N-oxide, thiadiazolyl-N-oxide, triazolyl-N-oxide, tetrazolyl-N-oxide) and the like.

d) Halo:

Halo is the general term for fluoro, chloro, bromo and iodo.

Hereinafter, exemplary embodiments of the present invention will be described in more detail.

In addition to the compounds represented by the Formulas 1 to 5, the benzamide derivatives according to one exemplary embodiment of the present invention pharmaceutically available acids or base addition salts, and stereochemical isomers of the compounds of the Formulas 1 to 5. Here, the base addition salts include any salts that maintain the activities of parent compounds and does not induce undesirable effects in objects to be administered, but the present invention is not particularly limited thereto. These salts comprise inorganic salts and organic salts, and preferably include the following acids. More particularly, the acids include, but are not particularly limited to, acetic acid, nitric acid, aspartic acid, sulfonic acid, sulfuric acid, maleic acid, glutamic acid, formic acid, succinic acid, phosphoric acid, phthalic acid, tannic acid, tartaric acid, hydrobromic acid, propionic acid, benzenesulfonic acid, benzoic acid, stearic acid, esyl acid, lactic acid, bicarbonic acid, busulfuric acid, bitartaric acid, oxalic acid, butyric acid, calcium edentate acid, camsylic acid, carbonic acid, chlorobenzoic acid, citric acid, edetic acid, toluenesulfonic acid, edisylic acid, esylic acid, fumaric acid, gluceptic acid, pamoic acid, gluconic acid, glycollylarsanilic acid, methylnitric acid, hydrochloric acid, hydroiodic acid, hydroxynaphthoic acid, isethionic acid, lactobionic acid, madelic acid, estolic acid, methylsulfuric acid, mucic acid, muconic acid, p-nitromethanesulfonic acid, hexamic acid, pantothenic acid, monohydrogen phosphoric acid, dihydrogen phosphoric acid, salicylic acid, sullfamic acid, sulfanilic acid, methanesulfonic acid, teoclic acid) and the like.

Also, the basic slats include, for example, ammonium salts, alkaline metal or alkaline earth metal salts, particularly lithium, sodium, potassium, magnesium and calcium salts, salts containing an organic base, for exemple, bezatin, N-methyl-D-glucamine, hydrabamin salts, and salts containing an amino acid, for example, arginine, lysine and the like.

On the contrary, the salt form may be converted into free salt form by treatment with suitable bases or acids.

According to one exemplary embodiment, the preferred salt of the benzamide derivatives of the present invention is benzo[1,3]-dioxole-5-carboxylic acid[4-(4-methoxy-phenylsulfamoyl)-phenyl]-amide.

Also, the present invention provides a method for prevention or treatment of cancer using as the active components the benzamide derivatives or their pharmaceutically available salts represented by Formulas 1 to 5, wherein the benzamide derivatives or their pharmaceutically available salts show the anticancer activity since they have the effect of suppressing the Wnt/β-catenin signaling pathway.

The pharmaceutical composition according to one exemplary embodiment of the present invention may be useful to prevent and treat a variety of cancer diseases. Here, the cancer diseases may include, but are not particularly limited to, colorectal cancer, breast cancer, liver cancer, prostate cancer, ovarian cancer, stomach cancer, etc. Preferably, the cancer disease is colorectal cancer.

Preparation of compounds

The compounds of the present invention may be prepared by a variety of synthesis or para-synthesis technologies. Suitable starting materials are known in the art, or may be prepared by the known synthesis methods or be commercially purchased. Therefore, it should be understood that other equivalents and modifications of the synthetic pathway may be made by those skilled in the art in order to use other starting materials or selective reagents, and reaction conditions (for example, temperature, solvents and the like) may be suitably changed in order to realize the desired equivalents and modifications. Additionally, it may be recognized by those skilled in the art that protective groups may be required to prepare any of the compounds, and the reaction conditions suitable for the selected protective groups are also widely known. Therefore, it should be understood that the methods and exemplas proposed herein are just preferable examples for the purpose of illustrations only, not intended to limit the scope of the invention.

According to one exemplary embodiment, the method for preparing benzamide derivatives represented by the Formula 4 is described in more detail with reference to the following Scheme 1.

Scheme 1

Intermediate (1) Intermediate (2) Formula 4

Wherein, n is an integer of 0 or 1,

R₃ is selected from the group consisting of hydrogen, alkyl, aryl-substituted alkyl group, aryl group, aryl group substituted with halo, alkoxy, carboalkoxy, carbamoyl, cyano, hydroxyl, nitro or thio, heteroaryl group, and heteroaryl group substituted with alkyl, halo, alkoxy, carboalkoxy or aryl group,

R₅ is a group represented by hydrogen or alkyl, and

X is a group activated by halo, methansulfonyl, phenylsulfonyl, alkoxy and the like.

The Scheme 1 is a coupling reaction used to prepare the compound represented by the Formula 4. More particularly, the intermediate (1) activated by alkoxy may be, for example, refluxed with stirring at the presence of the intermediate (2) and an ethanol solvent to obtain benzamide derivatives of Formula 4.

Also, the coupling reaction such as Scheme 1 may be carried out to obtain benzamide derivatives of Formulas 1 to 3.

In addition, the method for preparing benzamide derivatives represented by the Formula 5 is described in more detail with reference to the following Scheme 2.

Scheme 2

Intermediate (3) Intermediate (4) Formula 5

wherein, R₁ is defined in the same manner as described above,

R₄ is a group represented by hydrogen or alkyl,

R₃ is selected from the group consisting of hydrogen, alkyl, aryl-substituted alkyl group, aryl group, aryl group substituted with halo, alkoxy, carboalkoxy, carbamoyl, cyano, hydroxyl, nitro or thio, heteroaryl group, and heteroaryl group substituted with alkyl, halo, alkoxy, carboalkoxy or aryl group, and

The Scheme 2 is a coupling reaction used to prepare the compound represented by the Formula 5. More particularly, the activated intermediate (4) may be, for example, refluxed with stirring at the presence of the amine intermediate (3) and an acetonitrile solvent to obtain benzamide derivatives of Formula 5.

Pharmaceutical composition and administrations

According to another exemplary embodiment of the present invention, a pharmaceutical composition suitable for pharmaceutical use comprising at least one compound of the present invention and pharmaceutically available carrier, excipients or diluents is provided.

The term "composition," as used herein, is intended to comprise any products that are directly or indirectly obtained from a combination of certain amounts of certain components, as well as products comprising (when necessary, certain amounts of) the certain components. The term "pharmaceutically available" means that the carrier or excipient is compatible with the other components of the formulations, and is not toxic to its receptors.

For the preparation of the pharmaceutical composition, carriers may be selected according to the kinds of the formulations to be prepared, and the pharmaceutical composition may be formulated by mixing the carriers with the active components, benzamide derivatives, at a suitable ratio.

The pharmaceutical composition of the present invention may be parenterally, locally, orally or topically administered for therapeutic treatment. For example, various aqueous vehicles such as water, buffer, 0.04% saline and 0.3% glycol may be used for the pharmaceutical composition, and the pharmaceutical composition may also comprise other proteins, such as albumin, lipoproteins globulins, which reinforce the stability of the pharmaceutical composition. As a result, the pharmaceutical composition may be sterilized by widely known sterilization techniques. Solutions may be packaged for the future use, and filtered and dry-frozen under an aseptic condition. In this case, the dry-frozen products are mixed with a sterile solution prior to the administration.

In general, oral compositions include an inert diluent or an edible vehicle. The compositions may be sealed in a gelatin capsule or be compressed into tablets. For the purpose of the oral therapeutic administration, the active compound is mixed with an excipient, and may be used in the form of a tablet, a troche or a capsule.

Conventional pharmaceutical carriers may be used to prepare the oral composition. For example, water, glycol, oil, alcohol and the like may be used as the carrier in the case of the oral liquid formulations such as suspensions, syrups, elixirs and solutions, and starch, sugar, kaoline, a lubricating agent, a bonding agent, a disintegrating agent may be used in the case of the solid formulations such as powders, pills, capsules and tablets. Considering the ease of administration, the tablets and capsules may be delivered in the most convenient manner, and the tablets and pills are more preferably formulated into enteric agents.

In the case of the parenteral formulation, conventional sterile water may be used as the carrier, and include other components such as a solution adjuvant.

Injectable formulations, for example, sterile injectable aqueous or oily suspensions may be prepared with a suitable component such as powders, a wetting agent or a suspending agent according to the known technologies. The solvent that may be used herein includes water, a Ringer's solution and an isotonic NaCl solution, and a sterile fixed oil may also be generally used as the solvent or the suspension medium. Any non-pungent fixed oils comprising mono or di-glycerides may be use to this purpose, and fatty acids such as oleic acid may also be used for preparation of the injectable formulations.

A therapeutically effective amount of the pharmaceutical composition according to the present invention may be determined by experience by subjecting a target compound to a known in vivo and in vitro model system for diseases to be treated. The term "therapeutically effective amount" means an amount of an active component that is suitable to relieve or lower symptoms of diseases in need of treatment, or to reduce or delay clinical markers or symptoms of diseases in need of prevention.

When one of the active components of the composition according to the present invention, more particularly the benzamide derivatives represented by Formulas 1 to 5 is administered for the clinical purpose, it may be administered to patients in a single dose or divided doses. Here, a daily dose of the active component is preferably in a range of 0.1 to 100 ㎎ per 1㎏ of body weight. However, a specific dose level of the active component for certain patients may be varied according to certain compounds used, the weight, sex, health, diet of patients, the time and routes of administration of drugs, the methods of administration, the excretion rate, the admixture of drugs and the severity of diseases.

When necessary, the benzamide derivatives may be used to formulate effective pharmaceutical compositions into the form of their prodrugs.

Also, the composition according to the present invention may further comprise other components that do not suppress the functions of an active component or assist the functions of the active component, and may be formulated into the other various forms known in the art. Preferably, the composition according to the present invention may further comprise anticancer drugs known in the art.

Hereinafter, the description of the present invention will be described in more detail with reference to the following exemplary embodiments. However, it should be understood that the description proposed herein is just a preferable example for the purpose of illustrations only, not intended to limit the scope of the invention.

Compound 1: Benzoic acid, 4-[[bis(2-methoxyethyl)amino]sulfonyl]-, 2-(4-methoxy-7-methyl-2-benzothiazolyl)hydrazide

The compound used herein was purchased from Chemdiv (Cat No. C502-0586).

Compound 2: Benzoic acid, 4-(1-pyrrolidylsulfonyl)-, 2-(4,7-dimethoxy-2-benzothiazolyl)hydrazide

The compound used herein was purchased from Chemdiv (Cat No. C502-0643).

Compound 3: Benzoic acid, 4-(1-pyrrolidylsulfonyl)-, 2-(4-methoxy-7-methyl-2-benzothiazolyl)hydrazide

The compound used herein was purchased from Chemdiv (Cat No. C502-0559).

Compound 4: Benzoic acid, 4-(1-piperidinylsulfonyl)-, 2-(5,7-dimethyl-2-benzothiazolyl)hydrazide

The compound used herein was purchased from Chemdiv (Cat No. C502-0515).

Compound 5: Benzoic acid, 4-(1-piperidinylsulfonyl)-, 2-(6-methoxy-2-benzothiazolyl)hydrazide

The compound used herein was purchased from Chemdiv (Cat No. C502-0290).

Compound 6: Benzoic acid, 4-[(3,4-dihydro-2(1H)-isoquinolinyl)sulfonyl]-, 2-(4-methoxy-2-benzothiazolyl)hydrazide

The compound used herein was purchased from the Chemdiv (Cat No. C502-0066).

Compound 7: Benzoic acid, 4-[(2,6-dimethyl-4-morpholinyl)sulfonyl]-, 2-(4-methoxy--2-benzothiazolyl)hydrazide

The compound used herein was purchased from Life Chemical (Cat No. F0642-0594).

Compound 8: 1-piperazinecarboxylic acid, 4-[[4-[[2-(4-methoxy-7-methyl-2-benzothiazolyl)-hydrazinyl]carbonyl]phenyl)sulfonyl]-, ethyl ester

The compound used herein was purchased from Chemdiv (Cat No. C502-0571).

Compound 9: Benzamide, 4-[(2,6-dimethyl-morpholinyl)sulfonyl]-N-[5-(4-fluorophenyl)-1,3,4oxadiazol-2-yl]

The compound used herein was purchased from Chemdiv (Cat No. C079-0047).

Compound 10: 1-piperazinecarboxylic acid, 4-[[4-[[[5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-yl]amino]carbonyl]sulfonyl]-, ethyl ester

The compound used herein was purchased from Chemdiv (Cat No. C079-0139).

Compound 11: Benzamide, 4-[(2,6-dimethyl-4-morpholinyl)sulfonyl]-N-(5-methoxy-2-benzothiazolyl)-

The compound used herein was purchased from Chemdiv (Cat No. C059-0001).

Compound 12: Benzamide, N-[4-(1,3-benzodioxol-5-yl)-2-thiazolyl]-4-[3,5-dimethyl-1-piperidinyl]sulfonyl]-

The compound used herein was purchased from Chemdiv (Cat No. 3331-4726).

Compound 13: 1,3-benzodioxole-5-carboxyl amide, N[4-[[(4-methoxyphenyl)amino]sulfonyl]phenyl]

The compound used herein was purchased from Chemdiv (Cat No. 2884-3651).

Hereinafter, the above-mentioned compounds were tested for anticancer effect using the following method.

Experimental example 1 : Wnt/β-catenin signaling activity test using cell lines

1) IC₅₀ Measurement

a) Method

In order to determine Wnt/β-catenin signaling activity in an HEK293 cell, a plasmid, which comprises a binding site of a Tcf/Lef transcription regulator protein to which 5 β-catenins bind, and a marker protein, firefly luciferase, expressed under the control of the Tcf/Lef transcription regulator protein, was introduced into cells to prepare a transformed cell line (PCT/KR2006/005837). One day before the treatment of the compound, 2 x 10⁴ transformed cells were plated on a 96 well plate, and then treated with the compound (50.0, 16.7, 5.6, 1.9, 0.6 and 0.2 uM), which was serially diluted with a mixture obtained by mixing a Wnt-conditioning medium and a generic culture medium at a ratio of 1:4. Then, the transformed cells were incubated for 18 hours, and an expression rate of a marker gene (luciferase) was examined using a Steady-Glo Luciferase kit (Promega). A Wnt-conditioning medium is a culture solution obtained by incubating a cell line CRL2647 (ATCC) expressing Wnt3a for 2 days, and collecting and filtering the cell-cultured medium. The negative control used herein used a cell line treated only with a normal culture solution, and the positive control used a cell line treated only with a Wnt-conditioned medium. An inhibitory rate of the Wnt signal transduction is calculated as represented by the following Equation 1.

Equation 1

Inhibitory rate = (Ct-Cn)/(Cp-Cn)*100

(Ct: Experimental group, Cn: Control (medium-treated group), Cp: Wnt-conditioned medium-treated group)

b) Experimental results

Table 1

The activities of the Wnt signaling pathway were measured against the positive control in which the compound was treated only with the Wnt-conditioned medium. As a result, it was revealed that most of the compounds show the reduction in the expression rate of luciferase by more than 50% when the compounds were present in an amount of 10 uM or less.

2) TOP-Flash/FOP-Flash reporter assay

a) Method

In order to examine the effect of selectively suppressing the Wnt signaling pathway, 0.4 μg of a reporter plasmid (TOP-Flash) containing a Tcf-4 binding site or a reporter plasmid (FOP-Flash) (Korinek V. et al, 1997) containing a mutant Tcf-4 binding site and 0.1 μg of Renilla Luciferase reporter plasmid pRLTK (promega) for correcting the transfection were introduced into a HCT116 cell line in which the Wnt signaling pathway was activated, by using Lipofectamin 2000 (Invitrogen), and incubated for 18 hours. Then, the transfected HCT116 cells were treated with the compounds for 18 hours, and the changes in expression of a marker gene by the Tcf-4 promoter were than measured using a Dual-Glo Luciferase kit (Promega).

b) Experimental results

The experimental results were shown in FIG. 1. As shown in FIG. 1, the two plasmids were instantly co-expressed in the HCT116 cells, and their expression rates were compared to that of the TOP-Flash that was not treated with the compounds. Here, the expression rate of the TOP-Flash was set to 100 %. As a result, it was revealed that the expression rate of the TOP-Flash was reduced in a dose-dependent manner by the treatment with

Compounds

3 and 4, but there is no effect of reducing the expression of FOP-Flash plasmid by the treatment with the compounds when the FOP-Flash plasmid was introduced into the HCT116 cells.

Experimental example 2 : Test of Cell growth inhibition

1. Method

In order to determine the effect on cell growth suppression of the HCT116 cells and SW480 cells, each cell line was divided at a concentration of 1 10⁴into a 96 well plate, and incubated for 24 hours. The cell lines were treated, respectively, with 6 decreasing concentrations (50.0, 16.7, 5.6, 1.9, 0.6 and 0.2 uM) of the serially diluted compounds. 48 hours after the treatment with the compounds, a GI₅₀ concentration where a concentration of a drug is required to reduce growth by 50% was determined using a CellTiter-Glo Cell Viability Assay kit (Promega). A GI₅₀ value was determined by the following Equation 2.

Equation 2

GI₅₀=[(Ti-Tz)]/(C-Tz)]*100 (if, Ti=Tz)

GI₅₀=[(Ti-Tz)]/Tz*100 (if, Ti<Tz)

Time zero: Tz, Control: C, Experimental group: Ti

2. Experimental results

Table 2

As listed in Table 2, it was revealed that most of the compounds show the effect on the 50% cell growth inhibition when the HCT116 cells are treated with less than 10 uM concentration of the compounds, and the Compounds 1, 2, 14 and 15 show the effect on the 50% cell growth inhibition when the HCT116 cells are treated with more than 10 uM concentration of the compounds. On the contrary, it was revealed that the 50% cell growth inhibition by the Compound 13 is not observed until the compound is present in a concentration of 40 uM.

Experimental example 3 : Western analysis

1. Method

An HCT116 cell line was incubated in a DMEM culture solution under a standard culture condition (5% CO₂, 37℃, 100% relative moisture). Here, the DMEM culture solution was supplemented with penicillin-streptomycin (100 Units/mL) and 10% fetal bovine serum inactivated by thermal treatment. A test compound was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 10 μM. Cells (3x10⁷) were incubated with/without the test compound for 24 hours. In order to selectively separate only cytoplasm from the incubated cells, the incubated cells were lysed by treatment with a high concentration of salt. Then, the resultant cell suspension was centrifuged at 12,000 rpm for 10 minutes to collect a supernatant.

The collected cytoplasm solution was electrophoresized in a 10% PAGE gel, and probed with cyclin D1 and survivin antibodies (Cell signaling). The probing procedure was performed using a chemiluminescence system (ECL, Amersham). For comparison with an equivalent amount of the protein, an actin protein was used as the control. The results are listed in the following Table 3.

2. Experimental results

Table 3

It has been known that the cyclin D1 and survivin are used as a cell growth inhibitor and a cell death inhibitor, respectively, and play an important role in the proliferation of cancer cells. As listed in Table 3, it was revealed that when the HCT116 cells were treated with the compounds, the expression of the cyclin D1 and survivin known as the target gene of β-catenin is reduced to a high extent. That is, the expression of the cyclin D1 gene was reduced by the minimum of 19.8% (Compound 10) to the maximum of 64.9% (Compound 7), and the expression of the survivin gene was reduced by the minimum of 30.5% (Compound 10) to the maximum of 98.1% (Compound 3).

Experimental example 4 : Tumor regression assay

1. Method

Balb/c nude mice (15-16 g, 6 weeks old, female) were prepared and adapted in a clean room for more than 1 week, and then used for assays. 0.3 ml of an HCT 116 cancer cell line per mouse was subcutaneously injected via an axilla region between the right shoulder and walls of the chest to induce the tumorigenesis. From a time point that a mean tumor volume was in a range of approximately 50 to 60 mm³, the test compounds were peritoneally administered once a day at a dose of 50mg/10ml/kg by a day before the closing day (day 20). Irinotecan used as the positive control was repeatedly administered peritoneally once a week at a dose of 50mg/10ml/kg. The changes in body weight of the mice were measured three times a week during the test period to determine the toxicity of drugs such as reduction of mouse body weight caused by the administration of the drugs. The tumor volume was measured in 3 directions using a verier caliper, and then represented by the following Equation 3.

Equation 3

Tumor volume = (length × width × height)/2

2. Experimental results

In order to determine the toxicity of a compound when the compound was repeatedly administered peritoneally at a dose of 50 mg/10ml/kg into the HCT 116 cancer cell-engrafted nude mice for 20 days (one a day), General symptoms and body weight changes of the nude mice were observed during the administration period. The general symptoms and the statistically significant body weight changes caused by the administration of drugs were not observed in the nude mice during the administration period. In the case of the positive control in which Irinotecan was administered at a dose of 50 mg/kg, the statistically significant reduction of mouse body weight by 16% (p<0.005) were observed in the nude mice on 19^th day after the administration of drugs (three mice of the solvent control died on the 19^th closing day after the administration), compared to the solvent control (Table 4, FIG. 2).

Table 4

The changes in mean tumor volume after the engrafting of the HCT 116 cancer cell line were listed in Table 3 and shown in FIG. 3. It was revealed that the tumor regression by 32% to 18% (p<0.5 vs. Control) was observed in the nude mice of the positive control on 19^th day after the administration of drugs (three mice of the solvent control died on the 19^th closing day after the administration), compared to the solvent control, and the statistically significant tumor regression of 75% (p<0.005 vs. Control) was observed in the positive control (Irinotecan-administered group) (Table 3, FIG. 3).

When the compound was repeatedly administered peritoneally into the human colorectal cancer cell line (HCT-116)-engrafted nude mice at a dose of 50 mg/kg, the specific general symptoms and the statistically significant body weight changes were not observed during the administration period, and the died mice were observed but the death of the mice was considered to be derived from the growth of tumor rather than the administration of drugs.

Also, it was confirmed that

Compounds

3, 5, 6 and 8 have the effect of effectively suppressing the growth of tumor in the anticancer animal model without the changes in the body weight of the mice.

Claims

A pharmaceutical composition for prevention and treatment of cancer, comprising benzamide derivatives represented by the following formula 1, or pharmaceutically available salts thereof as active components:

Formula 1

wherein, A is -(CH₂)₀- or -(NR₄)-, B is -(CH₂)₀- or -(NH)n(NHR₅)-, n is an integer of 0 and 1, wherein R₄ and R₅ are same or different, and each of the R₄ and R₅ is a group represented by hydrogen or alkyl,

R₁ and R₂ are same or different, and each of R₁ and R₂ is selected from the group consisting of hydrogen; alkyl group; alkyl group substituted with alkynyl alkoxy, cycloalkyl or aryl; and aryl group substituted with alkyl, alkynyl alkoxy, cycloalkyl or aryl, or

R₁ and R₂ form a pyrrolidine, morpoline, piperidine or piperazine ring together with adjacent N, wherein at least one hydrogen in the pyrrolidine, morpoline, piperidine or piperazine ring may be substituted with alkyl group; alkynyl- or alkoxy-substituted alkyl group; aryl group; aryl group substituted with alkyl, alkynyl or alkoxy; carboxy group; or carbamoyl group, or may be fused with other aryl group, and

R₃ is selected from the group consisting of hydrogen, alkyl, aryl-substituted alkyl group, aryl group, aryl group substituted with halo, alkoxy, carboalkoxy, carbamoyl, cyano, hydroxyl, nitro or thio, heteroaryl group, and heteroaryl group substituted with alkyl, halo, alkoxy, carboalkoxy or aryl group.
The pharmaceutical composition of claim 1, wherein the benzamide derivatives are presented by the following formula 2:

Formula 2

wherein, n = 1,

R₁, R₂, R₃ and R₅ are defined in the same manner as in claim 1
The pharmaceutical composition of claim 1, wherein the benzamide derivatives are represented by the following formula 3:

Formula 3

wherein, n = 1, and m = 0 or 1,

X is CH₂-, O, S, NR₇R₈,

R₃ and R₅ are defined in the same manner as in claim 1,

R₆ is selected from the group consisting of hydrogen, alkyl group, alkynyl- or alkoxy-substituted alkyl group, phenyl group, and substituted phenyl group, or forms a fused ring together with the other substituted ring,

R₇ and R₈ are same or different, and each of R₇ and R₈ is selected from the group consisting of hydrogen, alkyl group, alkynyl-, hydroxyl- or alkoxy-substituted alkyl group, hydroxyl, carboxyalkoxy and carbamoyl group.
The pharmaceutical composition of claim 1, wherein the benzamide derivatives are represented by the following formula 4:

Formula 4

wherein, n = 0, and

R₁, R₂, R_3,and R₅ are defined in the same manner as in claim 1.
The pharmaceutical composition of claim 4, wherein the residue R₃ is heteroaryl selected from the group consisting of thiazole; oxadiazole; benzothiazole; and rings thereof in which at least one hydrogen substituted with alkyl, halo, alkoxy, carboalkoxy, aryl or heteroaryl.
The pharmaceutical composition of claim 1, wherein the benzamide derivatives are represented by the following formula 5:

Formula 5

wherein, R_1, R₂, and R₄ are defined in the same manner as in claim 1, and

R₃ is selected from the group consisting of aryl group, alkyl-substituted alkyl group, aryl group substituted with aryl, halo, alkyl, alkoxy, carboalkoxy, carbamoyl, cyano, hydroxyl, nitro or thio, heteroaryl group, and heteroaryl group substituted with alkyl, halo, alkoxy, carboalkoxy or aryl group.
The pharmaceutical composition of claim 6, wherein the residue R₃ is heteroaryl selected from the group consisting of thiazole; oxadiazole; benzothiazole; and rings thereof in which at least one hydrogen substituted with alkyl, halo, alkoxy, carboalkoxy, aryl or heteroaryl.
The pharmaceutical composition of any one of claims 1 to 7, wherein the pharmaceutical composition is used to prevent and treat a cancer disease selected from the group consisting of colorectal cancer, breast cancer, liver cancer, prostate cancer, ovarian cancer and stomach cancer.
The pharmaceutical composition of any one of claims 1 to 7, comprising a pharmaceutically available carrier that is suitable for being formulated into an oral formulation, a parenteral formulation, an injectable formulation or a transdermal formulation.
A method for prevention or treatment of cancer, comprising: administering the pharmaceutical composition defined in any one of claims 1 to 7 to patients to suppress a Wnt/β-catenin signaling pathway in order to show anticancer activities.