USOO8183405B2

(12) United States Patent (10) Patent No.: US 8,183,405 B2 KWOn et al. (45) Date of Patent: May 22, 2012

(54) OBOVATOL DERIVATIVES OR FOREIGN PATENT DOCUMENTS PHARMACEUTICALLY ACCEPTABLE SALTS KR 10-0426450 4/2004 THEREOF, PREPARATION METHOD KR 10-0548743 2, 2006 THEREOF AND PHARMACEUTICAL KR 10-0799266 1, 2008 COMPOSITION FOR THE PREVENTION AND WO WO 2006/121258 A1 * 11/2006 TREATMENT OF CANCER CONTAINING OTHER PUBLICATIONS THE SAME ASAN ACTIVE INGREDIENT Ito et al., Obovatol and Obovatal, Novel Biphenyl Ether from (75) Inventors: Byoung-Mog Kwon, Daejeon (KR): the leaves of Magnolia obovata Thunb. Chem. Pharm. Bull., vol.30, Dong Cho Han, Daejeon (KR); No. 9, May 1982, pp. 3347-3353.* Su-Kyung Lee, Chungcheongbuk-do Denicourt, C., et al..."Targeting Apoptotic Pathways in Cancer Cells'. (KR); Hye-Nan Kim, Daejeon (KR): www.sciencemag.org, Science, vol. 305, pp. 141 1-1413. (Sep. 3, Young-Min Han, Daejeon (KR): 2004). Dae-Seop Shin, Chungcheongbuk-do Reed, J. C., “Apoptosis-Based Therapies', Natural Reviews/Drug Discovery, vol. 1, pp. 111-121, (Feb. 2002). (KR) Thomas Walle, "Methylation of Dietary Flavones Greatly Improves (73) Assignee: Korea Research Institute of Bioscience Their Hepatic Metabolic Stability and Intestinal Absorption'. and Biotechnology, Daejeon (KR) Molecular Pharmaceutics, vol. 4. No. 6, pp. 826-832. * cited by examiner (*) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 U.S.C. 154(b) by 496 days. Primary Examiner — Rosalynd Keys (74) Attorney, Agent, or Firm — The Nath Law Group; (21) Appl. No.: 12/219,726 Tanya E. Harkins; Mihsuhn Koh (22) Filed: Jul. 28, 2008 (57) ABSTRACT (65) Prior Publication Data Disclosed herein are novel obovatol derivatives represented by 1, and pharmaceutically acceptable US 2009/02399.55A1 Sep. 24, 2009 salts thereof. Having the ability to inhibit the growth of cancer cells and induce apoptosis in cancer cells, the derivatives or (30) Foreign Application Priority Data pharmaceutically acceptable salts thereof are useful in the Mar. 24, 2008 (KR) ...... 10 2008-00269.54 prevention and treatment of cancer and in the Suppression of cancer metastasis. Also, a method for preparing the deriva (51) Int. Cl. tives, and pharmaceutical compositions comprising the CD7C 69/66 (2006.01) derivatives as active ingredients are disclosed. CD7C 69/34 (2006.01) A61 K31/085 (2006.01) Chemical Formula 1 A6IP35/04 (2006.01) ORI CO7C 43/275 (2006.01) (52) U.S. Cl...... 560/180; 560/194 (58) Field of Classification Search ...... None See application file for complete search history. (56) References Cited R3 U.S. PATENT DOCUMENTS 5,135,746 A * 8, 1992 Matsuno et al...... 424,725 wherein R, R and Rare as defined in the specification. 2008/O194702 A1* 8, 2008 Kwon et al...... 514,719 2008/0312337 A1* 12/2008 Kwon et al...... 514,701 2010.0125103 A1* 5, 2010 Huh et al...... 514,548 2 Claims, 1 Drawing Sheet U.S. Patent May 22, 2012 US 8,183,405 B2

US 8,183,405 B2 1. 2 OBOVATOL DERVATIVES OR tumor Suppressor genes within cells as well as the expression PHARMACEUTICALLY ACCEPTABLE SALTS of oncogenes induce apoptosis. The destruction of damaged THEREOF, PREPARATION METHOD cells is disadvantageous to the cells themselves, but is very THEREOF AND PHARMACEUTICAL useful for the body in its entirety. The destruction of cells in COMPOSITION FOR THE PREVENTION AND 5 which oncogenic modification occurs functions to remove the TREATMENT OF CANCER CONTAINING potential risk of cancer. On rare occasions, if they do not THE SAME ASAN ACTIVE INGREDIENT experience cell death, genetically modified cells become can BACKGROUND OF THE INVENTION cerous (Andy Catherine Denicourt and Steven F. Dowdy, 10 Science, 305, 141 1-1413, 2004). 1. Field of the Invention Ongoing cancer cells have tactics for avoiding cell death. The present invention relates to a novel obovatol derivative The tumor Suppressor factor p53 has an anti-cancer mecha or a pharmaceutically acceptable salt thereof, a method for nism for inducing cells to destroy themselves. The cells in preparing the same, and a pharmaceutical composition for the which this protein is inactivated have weakened self-destruc prevention and treatment of cancer comprising the same as an 15 tion functions. Additionally, cancer cells produce the Bcl-2 active ingredient. protein in a large amount to avoid cell death. Recent studies 2. Description of the Related Art have disclosed that interfering with cell death not only causes With the development of civilization, the incidence of can the spread of tumors, but also induces the resistance of tumor cer has increased. The treatment of cancer is, for the most cells to chemicals, resulting in danger to cancer patients. Over part, dependent on Surgical therapy, radiation therapy and the years, it has been believed that radiotherapy and various chemical therapy, with 40 anticancer agents showing strong chemical therapies directly kill malignant cells by destroying cytotoxicity. However, because the application of these thera a wide range of their genes. Nonetheless, cancer cells have the pies is limited to cancer patients in early stages, or to certain ability to avoid cell death and become resistant to cancer types of cancers, cancer morbidity remains on an increasing drugs. These research results suggest that radiation and trend. 25 chemical therapies capable of inducing apoptosis, if devel In order to differentiate malignant tumors from benign oped, may be effective weapons against cancer (John C. ones, they should show malignant properties including cell Reed, Nature Review Drug Discovery 1, 111-121, 2002). growth promotion and invasion into adjacent tissues, and Apoptosis-inducing materials can be found in the prior art. Something that overcomes growth control signals from adja For example, Korean Patent Publication No. 2006-0000241 cent tissues is another criterion. Signaling pathways in nor 30 discloses a twin compound, synthesized through the pharma mal cells comprise only growth promoting signals, but also ceutical association of an anticancer drug with DMNO, which convey growth-controlling messages from the outer surface acts specifically on human cancer cell lines A549, HT-1080, deep into the nucleus. In cancer cells, however, signaling SK-OV-3 and U937 and can induce apoptosis in two p53 chains between cell growth inhibitory factors are loosened or mutated kinds of the human pulmonary epithelial cell line broken. In addition, cancer cells even neglect the signals 35 A549. induced from potential inhibitory factors present on the sur Korean Patent No. 426450 teaches an anti-cancer compo face thereof. In many cancer cells, a series of important sition comprising as active ingredients citric acid, albumin inhibitory factors induced by cancer Suppressor genes are lost and Zinc in pharmaceutically effective amounts in combina or inactivated (Bruce A. J. Ponder, Nature, 411, 336-341, tion with a pharmaceutically acceptable carrier or diluent. 2001). 40 Korean Patent No. 548743 describes a novel inhibitor F-3- Taken together, recent study results demonstrate that it is 2-5, isolated from a soil actinomycetes culture, capable of the molecular apparatus, known as the cell cycle clock, inducing apoptosis in cancer cells, a preparation method present in the nucleus that is ultimately affected by cell thereof, and its use in anti-cancer agents. growth factors and growth inhibitory factors. All cancer cells Korean Patent No. 799266 discloses a composition for the undergo cell division at very high rates without the ability to 45 prevention and treatment of cancer comprising widdrol, iso regulate the cell cycle. In normal cells, all of the signals lated from a Juniperus chinensis extract, as an active ingre transferred thereinto are collectively regulated to thus deter dient. mine the phase of the cell cycle to which each cell progresses. As such, a lot of effort has been made in organic synthesis If a decision is made to conduct cell division, the cell cycle and natural material fields to develop apoptosis-inducing starts to work (Gerard I. Evan and Karen H. Vousden. Nature, 50 materials. 411, 342-348, 2001). However, there is still a need for anticancer materials that Two mechanisms are described above for regulating the are harmless to the human body and highly effectively exert growth of normal cells to Suppress cancer generation. That is, growth regulation and apoptosis on cancer cells without side they are to suppress cell growth factors or activate cell growth effects. inhibitory factors. Despite the normal operation of the two 55 regulating mechanisms, cells may be activated by them SUMMARY OF THE INVENTION selves, or may replicate continuously regardless of external cell division stop signals so as to finally progress into cancer. Leading to the present invention, intensive and thorough Further, excessive genetic mutations within cells may incite research into the development of compounds which are harm cells to become cancerous in spite of all body defense func 60 less to the body and have excellent inhibitory and apoptotic tions. activity against cancer cells without side effects, conducted Each human cell has a self-destruction function which by the present inventors, resulted in the finding that obovatol starts to work when its fundamental constituents are damaged derivatives, chemically modified from obovatol, which is or its regulation system is not controlled. This phenomenon is obtainable from the herb-medicinally useful material Mag called apoptosis. For example, damage to nuclear genes may 65 nolia obovata Thunberg (Magnoliaceae), inhibit the growth induce programmed cell death. Further, many research of a variety of human cancer cell lines and induce apoptosis groups have recently reported that the functional loss of therein. US 8,183,405 B2 3 4 It is therefore an object of the present invention to provide 10) 1-(4-(E)-prop-1-enyl)phenoxy)-2,3-di-2-methoxy a novel obovatol derivative or a pharmaceutically acceptable acetyloxy-5-((E)-prop-1-enyl). salt thereof. The obovatol derivatives of the present invention, repre It is another object of the present invention to provide a sented by Chemical Formula 1, may be used in the form of method for preparing the novel obovatol derivative or the pharmaceutically acceptable salts. Useful are acid addition pharmaceutically acceptable salt thereof. salts formed of pharmaceutically acceptable free acids. The It is a further object of the present invention to provide a pharmaceutical composition for the prevention and treatment term “pharmaceutically acceptable salt as used herein refers of cancer comprising the novel obovatol derivative or the to any organic or inorganic salt of the base compounds of pharmaceutically acceptable salt as an active ingredient. 10 Chemical Formula 1, not exhibiting a side effect in which the beneficial activity of the base compounds of Chemical For BRIEF DESCRIPTION OF THE DRAWINGS mula 1 is degraded when it is present at a concentration causing no toxicity and harm in the body. The free acids may FIG. 1 is FACS plots showing the apoptotic effect of the obovatol on colorectal cancer cells as V-FITC stained cell 15 be inorganic or organic. Examples of useful inorganic free fractions ((a) negative control, (b) OuM of the obovatol acids include hydrochloric acid, bromic acid, nitric acid, Sul derivative of Example 1, (c)30 uM of the obovatol derivative furic acid, perchloric acid and phosphoric acid. As organic of Example 1, (d) 50 uM of the obovatol derivative of acids, citric acid, lactic acid, maleic acid, fumaric acid, glu Example 1). conic acid, methane Sulfonic acid, acetic acid, gluconic acid, DESCRIPTION OF THE PREFERRED Succinic acid, tartaric acid, 4-toluenesulfonic acid, galactur EMBODIMENTS onic acid, embonic acid, glutamic acid, aspartic acid, oxalic acid, (D)-or (L)-malic acid, ethane Sulfonic acid, p- The present invention pertains to obovatol derivatives rep Sulfonic acid, salicylic acid or malonic acid may be used. The resented by the following chemical formula 1 or pharmaceu 25 pharmaceutically acceptable salts may include alkali metal tically acceptable salts thereof. salts (sodium salt, potassium salt, etc.) and alkaline earth metal salts (calcium salt, magnesium salt, etc.). Acid addition salts useful in the present invention include, but are not lim Chemical Formula 1 30 ited to, acetate, aspartate, benzoate, besylate, bicarbonate/ ORI carbonate, bisulfatef sulfate, borate, camsylate, citrate, edisy late, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/ chloride, hydrobromide/, hydroiodide/iodide, 35 isethionate, lactate, malate, maleate, malonate, mesylate, R3 methylsulfate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, Saccharate, Stearate. Succi nate, tartrate, tosylate, trifluoroacetate, aluminum salt, argin 40 ine, benzathine, calcium, choline, diethylamine, diolamine, wherein, glycine, lysine, magnesium, meglumine, alamine, potassium RandR are independently or optionally hydrogen, C-C, salt, sodium salt, tromethamine, and Zinc salt, with hydro straight or branched alkyl, acetyl, C-C alkoxy or C-C, chloride or trifluoroacetate being preferred. alkoxyacetyl; and 45 Also, the obovatol derivatives of the present invention, R is hydrogen, C-C straight or branched alkyl, C-C, represented by Chemical Formula 1, may be in the form of alkenyl or allyl. conventionally producible salts, hydrates, and Solvates In Chemical Formula 1, preferably, RandR are indepen thereofas well as pharmaceutically acceptable salts. dently or optionally methyl, ethyl, acetyl, methoxy, methoxy Addition salts according to the present invention may be acetyl or ethoxyacetyl; and 50 prepared using a conventional method. For example, they R is hydrogen, propyl, propenyl or allyl. may be prepared by dissolving the compound of Chemical Concrete examples of the obovatol derivatives represented Formula 1 in a water-miscible organic solvent, such as by Chemical Formula 1 include: , , ethanol or acetonitrile, and adding an 1) 3-(4-propylphenoxy)-5-propylbenzene-1,2-diol; excess of organic acids oran excess of aqueous inorganic acid 2) 3-(4-allylphenoxy)-5-allyl-2-methoxyphenol; 55 Solutions So as to precipitate or crystallize salts. These addi 3) 1-(4-allylphenoxy)-5-allyl-2,3-diacetyloxybenzene: tion salts may be obtained by distilling the solvent or excess 4) 1-(4-allylphenoxy)-5-allyl-2,3-di-2-methoxyacety of acids from the Solution or by Suctioning and filtering the loxybenzene: precipitates. 5) 3-(4-propylphenoxy)-2-methoxy-5-propylphenol; Also, the present invention is concerned with a method for 6) 1-(4-propylphenoxy)-2,3-diacetyloxy-5-propylben 60 the preparation of the obovatol derivatives represented by Zene; Chemical Formula 1. 7) 1-(4-propylphenoxy)-2,3-di-2-methoxyacetyloxy-5- In accordance with an embodiment of the present inven propylbenzene: tion, the method for the preparation of the obovatol deriva 8) 3-(4-(E)-prop-1-enyl)phenoxy)-5-((E)-prop-1-enyl) tives, as illustrated by the following Reaction Scheme 1, benzene-1,2-diol; 65 comprises the reaction of an obovatol (2) with hydrogen in the 9) 1-(4-((E)-prop-1-enyl)phenoxy)-2,3-diacetyloxy-5- presence of a Pd-charcoal catalyst in a reaction solvent to ((E)-prop-1-enyl)benzene; and afford an obovatol derivative (1a). US 8,183,405 B2 5 6 In the method according to an embodiment of the present Reaction Scheme 1 invention, the reaction solvent where the reaction of an obo OH vatol (2) with hydrogen in the presence of a Pd-charcoal catalyst occurs may be ethylacetate, acetone, or acetonitrile. O OH In the method of the present invention, obovatol is prefer ably reacted with hydrogenata molar ratio of 1:10-1:50. This hydrogenation is preferably conducted at 20-25°C. for -C --- 10-12 hours. Reaction conditions beyond these limits may 10 result in a decrease in production yield. In accordance with another embodiment of the present invention, the method for the preparation of the obovatol 2 derivatives, as illustrated by the following Reaction Scheme 2, comprises the tautomerization of an obovatol (2) into an 15 obovatol derivative (1b) in the presence of a palladium chlo OH ride (PdCl) catalyst in an solvent.

Reaction Scheme 2 --O O OH OH

O OH

25 -C -- Ia

(wherein, compound 1a is a compound according to 30 2 Chemical Formula 1). The obovatol (2), serving as a starting material in Reaction Scheme 1, may be extracted from Magnolia obovata Thun berg (Magnoliaceae) according to the present invention or a 35 method known in the art, may be chemically synthesized, or OH may be commercially available. Preferably, obovatol is extracted from Magnolia obovata Thunberg (Magnoliaceae) O OH according to the present invention. In greater detail, obovatol may be prepared by 40 (a) soaking leaves of Magnolia obovata in an organic Sol -C vent to afford an extract; and (b) fractioning the extract in silica gel chromatography to N isolate pure obovatol. First, Step (a) is to dissolve obovatol present in leaves of 45 Magnolia obovata in an organic solvent. Ib No limitations are imposed with respect to the leaves of Magnolia obovata. They may be cultured or commercially (wherein, compound 1b is a compound according to procured. The leaves are washed until clean and dried. Then, Chemical Formula 1) they are sectioned into desired sizes and added to a suitable 50 volume of an organic solvent, preferably methanol. After the In the preparation method of obovatol derivatives accord leaf sections are left for 24-50 hours at room temperature, the ing to the present invention, the alcohol solvent may be organic solvent is filtered through a filter. The filtrate may be methanol or ethanol. then Subjected to an additional process such as concentration In this preparation method, the obovatol (2) is preferably or lyophilization. 55 reacted with palladium chloride at a molar ratio of 1:0.01-1: Next, step (b) is to purify obovatol from the filtered organic 0.02. Also, the tautomerization is preferably carried out at Solvent of step (a) using silica gel chromatography. 20-25°C. for 2-3 hours. Reaction conditions beyond these The organic layer thus formed is concentrated and the limits may result in a decrease in production yield. residue is purified by silica gel column chromatography using a mixture of and methanol, and is eluted with an 60 In accordance with a further embodiment of the present elution solvent of various ratios (9:1–6:4) of chloroform and invention, the method for the preparation of the obovatol methanol. derivatives, as illustrated by the following Reaction Scheme The compound may be isolated by silica gel chromatogra 3, comprises the reaction of a compound of Chemical For phy eluting with a mixture of ethyl acetate and hexane chlo mula 3 (compound 1a or 1b prepared in Reaction Scheme 1 or roform (90:10-80:20 V/v) as a mobile phase, with methylene 65 2, or obovatol) with a compound of Chemical Formula 4 in chloride serving as a solvent. The resulting eluate may be the presence of potassium carbonate (KCOs) in a solvent to further purified by Cs column chromatography. afford the obovatol derivative 1. US 8,183,405 B2 8 The term “prevention” as used herein with respect to can Reaction Scheme 3 cer, means any action inhibiting the formation of cancer or delaying the outbreak of cancer, including the administration OH of a pharmaceutical composition. By the term “treatment as O OH used herein with respect to diseases is meant any action by RIX which the symptoms of the diseases take a turn for the better 4 or are alleviated. R3 As used herein, the term “administration” means the pro vision of a therapeutically effective material for patients R3 10 using any Suitable method. As long as it allows the composi tion of the present invention to arrive at a target tissue, any ORI administration route may be taken in the present invention. In this regard, the composition of the present invention may be O OR2 administered orally or parenterally. The administration of the composition according to the present invention may be aided by a device which facilitates the delivery of the active ingre R3 O dient to target cells. R3 The pharmaceutical composition according to the present invention is usually formulated in combination with a diluent I or excipient, Such as a filler, a thickening agent, a binder, a wetting agent, a disintegrant, a surfactant, etc. (wherein, R is as defined in Reaction Scheme 1, R' is the same as R' except for hydrogen, R is hydrogen or R', and X Solid preparations intended for oral administration of the is a halogen element) compound of the present invention may take the form of In the method of preparing obovatol derivatives according tablets, pills, powders, granules, capsules, troches, and the to Reaction Scheme 3, the reaction solvent may be methyl 25 like. These solid preparations are formulated in combination acetate, acetone, or acetonitrile. with at least one excipient Such as starch, calcium carbonate, In this method, the compound of Chemical Formula 3 is Sucrose, lactose, or gelatine. In addition, a lubricant such as preferably reacted with the compound of Chemical Formula magnesium Stearate, talc, or the like may also be added. 4 at a molar ratio of 1:1.1-1:1.2. This reaction is preferably Liquid preparations intended for oral administration include carried out at 20-25°C. for 5-6 hours. Reaction conditions 30 Suspensions, internal use solutions, emulsion, syrups, and the beyond these limits may result in a decrease in production like. In addition to a simple diluent such as water or liquid yield. paraffin, various excipients, such as wetting agents, sweeten The compounds thus prepared can be isolated using silica ing agents, aromatics, preservatives, and the like may be gel column chromatography. They were identified as obova contained in the liquid preparations for the oral administra tolderivatives using UV and IR spectra, high resolution mass 35 tion of the compound of the present invention. spectrometry and NMR. Also, the compound of the present invention may be In addition, the present invention provides to a pharmaceu administered via a non-oral route. For this, Sterile aqueous tical composition for the prevention and treatment of cancer Solutions, non-aqueous solvents, Suspensions, emulsions, comprising an obovatol derivative represented by Chemical lyophilizates, Suppositories, and the like may be used. Inject Formula 1 or a pharmaceutically acceptable salt thereofas an 40 able propylene glycol, polyethylene glycol, vegetable oils active ingredient. Further, the present invention provides to a method for Such as olive oil, and esters such as ethyl oleate may be treating cancer, comprising administering the obovatol Suitable for non-aqueous solvents and Suspensions. The basic derivative of Chemical Formula 1 or a pharmaceutically materials of Suppositories include Witepsol. macrogol, acceptable salt thereofat a therapeutically effective dose to a Tween 61, cacao butter, laurin butter, glycerol, and gelatin. patent in need thereof. 45 The composition of the present invention is administered in The obovatol derivatives of Chemical Formula 1 according a therapeutically effective amount. The term “therapeutically to the present invention were found to effectively inhibit the effective amount’ as used herein means an amount of the growth of cancerous tissues as assayed in in vivo experiments composition that is sufficient to affect the anticancer activity in which the obovatol derivatives were administered to of the obovatol at a reasonable benefit/risk ratio applicable to immune-deficient mice implanted with a colorectal cancer 50 any medical treatment. The specific therapeutically effective cell line (see Table 3). Also, the obovatol derivatives of dose level for any particular patient will depend upon a vari Chemical Formula 1 according to the present invention were ety of factors including the disorder being treated and the proven to effectively induce cancer cells to undergo apoptosis severity of the disorder, the activity of the compound (FIG. 1). Therefore, the obovatol derivatives or pharmaceu employed, body sensitivity to the compound, the time of tically acceptable salts thereof in accordance with the present 55 administration, route of administration, rate of excretion, the invention are useful in the prevention and treatment of cancer. duration of the treatment, drugs used in combination or coin Examples of the cancerto which the obovatol derivatives or cidental therapy, and other factors known in the art. The pharmaceutically acceptable salts are therapeutically appli composition of the present invention may be administered cable include, but are not limited to, large intestine cancer, alone or in combination with other agents. In the latter case, stomach cancer, prostate cancer, breast cancer, renal cancer, 60 they may be administered sequentially or simultaneously. It is liver cancer, brain tumors, lung cancer, uterine cancer, col important to administer the compound at the minimal dose at orectal cancer, bladder cancer, pancreatic cancer, and blood which the maximal medicinal effect is obtained without side CaCC. effects, which can be readily determined by those skilled in The term “anti-cancer as used herein is intended to refer to the art. the inhibition or interruption of the formation or proliferation 65 For example, the effective dose of the compound according of cancer cells, thus leading to the prevention and treatment of to the present invention may vary depending on age, sex and CaCC. body weight. Typically, the compound according to the US 8,183,405 B2 10 present invention may be administered at a dose from 1 to 50 organic layer containing the active material was collected and mg per kg of body weight, and preferably at a dose from 1 to concentrated in a vacuum. The concentrate (1.2 g) was dis 10 mg per kg of body weight every day or every other day. The solved in methylene chloride (30 ml) and loaded onto a silica compound may be administered in a single dose or may be gel (Merck, Art No. 9385) so as to adsorb the active material divided into three doses per day according to the instructions thereonto. Silica gel column chromatography eluting with a of a physician orpharmacist. However, the dose does not limit gradient of a mixture of ethylacetate and hexane from 10:90 the present invention in any way because it may vary with to 20:80 afforded colorless 3-(4-allylphenoxy)-5-allyl-2- administration route, disease severity, sex, weight, age, etc. methoxyphenol (0.91 g, yield 90%). A better understanding of the present invention may be obtained through the following examples which are set forth 10 to illustrate, but are not to be construed as the limit of the EXAMPLE 3 present invention. EXAMPLE1 Synthesis of 15 1-(4-Allylphenoxy)-5-Allyl-2,3-Diacetyloxybenzene Synthesis of 3-(4-Propylphenoxy)-5-Propylbenzene-1,2-Diol

OAc

OH O OAc

25

30 The same procedure as in Example 2 was performed, with After being isolated from the leaves of Magnolia obovata the exception that potassium carbonate (K2CO) (1.2 g) and (growing naturally in the central region of Korea), obovatol(1 acetylchloride (500mg) were added to a solution of obovatol (1 g) in acetone (200 ml) and stirred at room temperature for g) was dissolved in acetone (200 ml) and added to Pd-char 5 hours, to afford colorless 1-(4-allylphenoxy)-5-allyl-2,3- coal (2 mg) at room temperature. Hydrogen was allowed to 35 flow into the catalyst. diacetyloxybenzene (1.2g, yield 90%) Subsequently, the organic layer containing an active mate rial was collected and concentrated in a vacuum. The concen EXAMPLE 4 trate (1.1 g) was dissolved in methylene chloride (100 ml) and loaded onto a silica gel (Merck, Art No. 9385) so as to adsorb 40 the active material thereonto. Silica gel column chromatog Synthesis of 1-(4-Allylphenoxy)-5-Allyl-2,3-di-2- raphy eluting with a gradient of a mixture of ethylacetate and Methoxyacetyloxybenzene hexane from 10:90 to 20:80 afforded colorless 3-(4-propy lphenoxy)-5-propylbenzene-1,2-diol (0.9 g, yield 90%). 45 EXAMPLE 2 Synthesis of 3-(4-Allylphenoxy)-5-Allyl-2-Methoxyphenol 50

55

60 The same procedure as in Example 2 was performed, with the except that potassium carbonate (KCO) (1.2 g) and To a solution of obovatol (1 g) in acetone (200 ml) were acetylchloride (1 mg) were added to a solution of obovatol (1 added potassium carbonate (KCO) (1.1 g) and methyl 65 g) in acetone (200 ml) and stirred at room temperature for 5 iodide (0.3 g), followed by stirring the solution at room tem hours, to afford colorless 1-(4-allylphenoxy)-5-allyl-2,3-di perature for 5 hours. After completion of the reaction, the 2-methoxyacetyloxybenzene (1.3 g, yield 90%) US 8,183,405 B2 11 12 EXAMPLE 5 EXAMPLE 7

Synthesis of Synthesis of 1-(4-Propylphenoxy)-2,3-di-2-Meth 3-(4-Propylphenoxy)-2-Methoxy-5-Propylphenol oxyacetyloxy-5-Propylbenzene

10

15

25 The same procedure as in Example 2 was performed, with The same procedure as in Example 2 was performed, with the exception that potassium carbonate (KCO) (1.1 g) and the exception that potassium carbonate (K2CO) (1.1 g) and methyl iodide (0.3 g) were added to a solution of tetrahy methoxyacetylchloride (500mg) were added to a solution of droobovatol (1 g), prepared in Example 1, in acetone (200 ml) tetrahydroobovatol (1 g), prepared in Example 1, in acetone and stirred at room temperature for 5 hours, to afford colorless 30 (200 ml) and stirred at room temperature for 5 hours, to afford 3-(4-propylphenoxy)-2-methoxy-5-propylphenol (1 g, yield colorless 1-(4-propylphenoxy)-2,3-di-2-methoxyacetyloxy 90%) 5-propylbenzene (1.2g, yield 90%)

EXAMPLE 6 35 EXAMPLE 8

Synthesis of 1-(4-Propylphenoxy)-2,3-Diacetyloxy Synthesis of 3-(4-((E)-Prop-1-enyl)Phenoxy)-5-((E)- 5-Propylbenzene Prop-1-enyl) Benzene-1,2-Diol 40

OH

45 OAc

O OAc

To a solution of obovatol (1 g) in methanol was added palladium chloride (PdCl) (10 mg), followed by stirring at room temperature for 5 hours. After completion of the reac tion, the organic layer containing the active material was 60 collected and concentrated in a vacuum. The concentrate (1.1 The same procedure as in Example 2 was performed, with g) was dissolved in methylene chloride (30 ml) and loaded the exception that potassium carbonate (KCO) (1.1 g) and onto a silica gel (Merck, Art No. 9385) so as to adsorb the acetyl chloride (500mg) were added to a solution of tetrahy active material thereonto. Purification through silica gel col droobovatol (1 g), prepared in Example 1, in acetone (200 ml) umn chromatography eluting with a gradient of a mixture of and stirred at room temperature for 5 hours, to afford colorless 65 ethylacetate and hexane from 10:90 to 20:80 afforded color 1-(4-propylphenoxy)-2,3-diacetyloxy-5-propylbenzene (1.1 less 3-(4-(E)-prop-1-enyl)phenoxy)-5-(E)-prop-1-enyl) g, yield 90%) benzene-1,2-diol (0.91 g, yield 90%)

US 8,183,405 B2 15 16 TABLE 2-continued ml culture tube, mixed with 5ul of annexin V-FITC and 10 ul of PI (propidium iodide), and incubated at room temperature NMR Data for 15 min in a light-tight condition. Then, 400 ul of 1xbind Cpd. NMR Data ing buffer was added to each tube, followed by FACS analysis for annexin V-FITC-stained cells (Moussa Alkhalaf, Abdulla Ex. 10 H-NMR (CDC1): 7.27 (H-3' and 5', d, J = 8.1 Hz), 6.95 (H-2' and H-6', d, J = 8.1 Hz), 6.77 (H-6, d, J = 1.5 Hz), El-Mowafy, Waleed Renno, Ousama Rachid, AhmedAli, and 6.17 (5H, m), 4.27 (2H, s), 4.14 (2H, s), Raja Al-Attyiah, Archives of Medical Research 39, 162-168, 3.525 (3H, s), 3.41 (3H, s), 1.88 (H-9, d, J = 6.6), 1.83 (H- 2006). 9', d, J = 6.6). The results are shown in FIG. 1. 10 In FACS plots of FIG. 1, the distribution of stained cells is shown. Cell migration to the second quadrant and the fourth EXPERIMENTAL, EXAMPLE1 quadrant refers to an increase in chromosome number, mean ing an increased number of cells in the G2/M phase or S Inhibitory Effect on Growth of Cancer Cell 15 phase. The third quadrant and the fourth quadrant of the FACS The compounds prepared in Examples 1, 2 and 3 were plot indicate cells expressing the early apoptosis marker evaluated for inhibitory activity against the growth of a annexin V. human cancer cell line using WST-1. Human cancer cell lines As seen in FIG. 1, the fraction of the cells migrating to the were incubated in 10% fetal bovine serum (FBS)-supple third quadrant increased from 14.12% at pre-treatment to mented media at 37°C. in a 5% CO atmosphere, followed by 59.58% after treatment with 50 uM of the obovatol derivative detaching the cells with 0.05% trypsin-EDTA. of the present invention, indicating that the obovatol deriva Cells were plated onto 90-well plates at a density of 4,000 tives of the present invention can increase the expression level cells (A549, MDA-MB-231), 5,000 cells (HEK293, NCI of the early apoptosis marker annexin V, resulting in apoptosis H23) or 6,000 cells (HCA-7, HCT116, SW620, DU145) per 25 induction. well, which were counted using a hematocytometer. Having the ability to induce apoptosis, therefore, the obo After incubation in 10% FBS-supplemented media at Vatol derivatives according to the present invention can be 37°C. for 24 hours in a 5% CO incubator, the media was applied to the prevention and treatment of cancer. changed with fresh media containing a control (0.1% DMSO) or the compound of Example 1 in an amount of 5, 10, 15, 20, 30 EXPERIMENTAL, EXAMPLE 3 25 or 30 g/ml (dissolved in DMSO and diluted in the medium). After 48 hours of treatment, WST-1 (Roche) was Assay for Acute Oral Toxicity in Rat added in an amount of 10 ul to each well and incubated for 2 hrs. Absorbance at 450 nm was read in an ELISA reader The obovatol derivatives of the present invention were (Bio-Rad). 35 assayed in Vivo for acute oral toxicity as follows. The results are given in Table 3, below. Specific pathogen free (SPF) SD rats 6 weeks old were used for this assay. The obovatol derivative prepared in TABLE 3 Example 1 or 2 was dissolved in distilled water, and the solution was orally administered at a dose of 500 mg/kg to Compounds OD 40 respective groups of two rats. Control O604 After the oral administration, the rats were observed for Example 1 (5 g/ml) O.S92 death, clinical symptoms, changes in body weight, and the Example 1 (10 g/ml) O.557 Example 1 (15 g/ml) O.474 like, and Subjected to hematological and Serobiochemical Example 1 (20 g/ml) O.247 tests. Autopsies were performed to check for abnormalities of Example 1 (25 g/ml) O.149 45 the thoracic and abdominal organs with the naked eye. Nei Example 1 (30 g/ml) O.141 ther particular clinical symptoms nor perished animals were observed. In addition, no acute toxicity was observed in body As seen in Table 3, the compound of Example 1, when weight change, haematological tests, serobiochemical tests, presentata concentration of 201g/ml or higher, was observed or autopsy examination. 50 These results demonstrate the compounds tested do not to inhibit the growth of cancer cells by 50% or more. induce toxicity up to a dose of 500 mg/kg in rats, and are EXPERIMENTAL, EXAMPLE 2 proven safe with an LDso of 500 mg/kg or more upon oral administration.

Apoptotic Effect on Cancer Cells FORMULATION EXAMPLE1 55 The obovatol derivatives according to the present invention were assayed for ability to induce programmed cell death as Preparation of Pharmaceutical Preparation follows. Apoptosis was analyzed using flow cytometry. First, col orectal cancer cells (SW620) were treated with 0.1% DMSO 60 (negative control), OuM, 30 uM or 50LM of the compound of <1-1> Preparation of Powder Example 1 for 48 hours and detached with 0.05% trypsin EDTA. After being harvested through centrifugation, the cells Obovatol derivative of Chemical Formula 1 were washed twice with PBS (phosphate-buffered saline) and Lactose suspended at a density of 1x10° cells/mlin a 1x binding buffer 65 (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, and 2.5 mM The above ingredients were mixed and loaded into an air CaCl). 100 ul of the cell suspension was transferred into a 5 tight sac to produce a powder agent. US 8,183,405 B2 17 18 Having the excellent ability to inhibit the growth of cancer cells and induce apoptosis in cancer cells, the obovatol deriva <1-2> Preparation of Tablet tives or pharmaceutically acceptable salts thereof in accor Obovatol derivative of Chemical Formula 1 100 mg dance with the present invention, as described above, are Corn Starch 100 mg useful in the prevention and treatment of cancer and in the Lactose 100 mg Suppression of cancer metastasis. MgStearate 2 mg Although the preferred embodiments of the present inven tion have been disclosed for illustrative purposes, those These ingredients were mixed and prepared into tablets skilled in the art will appreciate that various modifications, using a typical tabletting method. 10 additions and Substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims. What is claimed is: <1-3> Preparation of Capsule 1. A compound represented by the following Chemical Obovatol derivative of Chemical Formula 1 100 mg 15 Formula 1, or a pharmaceutically acceptable salt thereof Corn Starch 100 mg Lactose 100 mg MgStearate 2 mg Chemical Formula 1 These ingredients were mixed and loaded into gelatin cap Sules according to a typical method to produce capsules.

<1-4> Preparation of Injection 25 Obovatol derivative of Chemical Formula 1 10 g/ml DI. HC BP added to form pH 3.5 wherein, NaCl BP injection up to 1 ml R" and Rare independently or optionally methoxyacetyl or ethoxyacetyl; and The compound of the present invention was dissolved, 30 R is propyl, or propenyl orallyl. along with mannitol and Na HPO.12H2O, in distilled water, 2. The compound of Chemical Formula 1 or the pharma and the pH of the solution was adjusted to 7.4 beforesteril ceutically acceptable salt as set forth in claim 1, wherein the izing. An injection was prepared according to a typical pro compound is selected from the a group consisting of: cedure. 1-(4-allylphenoxy)-5-allyl-2,3-di-2-methoxyacetyloxy The obovatol derivative was dissolved in a suitable volume 35 ofan NaCl BP injection, and the solution was adjusted to a pH benzene: of 3.5 with diluted HC1 BP and to a desired volume with an 1-(4-propylphenoxy)-2,3-di-2-methoxyacetyloxy-5-pro NaCl BP injection, followed by sufficient mixing. The solu pylbenzene; and tion was loaded into transparent 5 ml type I ampules, which 1-(4-(E)-prop-1-enyl)phenoxy)-2,3-di-2-methoxyacety were hermetically sealed by melting, followed by autoclaving loxy-5-((E)-prop-1-enyl)benzene. at 120°C. for 15 minto prepare injections. k k k k k