AU2008200720B2 - Geranyl compounds - Google Patents

Description

AUSTRALIA Patents Act COMPLETE SPECIFICATION (ORIGINAL) Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: Name of Applicant: Ohgen Research Laboratories Ltd. Actual Inventor(s): Kiyoshi Akiyama Address for Service and Correspondence: PHILLIPS ORMONDE & FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: GERANYL COMPOUNDS Our Ref: 822675 POF Code: 1349/160761 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): -1 - 2 SPECIFICATION GERANYL COMPOUNDS 5 Technical Field This invention relates to novel geranyl compounds and to their utilization as antitumor agents. 10 Background Art Many geranyl compounds having 1,5-diene structure are present in vivo, and are known as in vivo precursors of substances having polyene structure and exhibiting various physiological activities. These substances 15 having 1,5-diene structure and polyenes derived therefrom invariably start from mevalonic acid and biosynthesized. I noticed, as such geranyl compounds having 1,5-diene structure, geranic acid or geranylamine, and furthermore mevalonic acid which is the base for biosynthesis of polyenes, synthesized various derivatives of 20 geranic acid or geranylamine and mevalonic acid and investigated their physiological activities, in particular, antitumor activity and toxicity, and have come to complete the present invention. The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an 25 admission that any of the material referred to was published, known or part of the common general knowledge in Australia as at the priority date of any of the claims. Throughout the description and claims of the specification the word "comprise" and variations of the word, such as "comprising" and 30 "comprises", is not intended to exclude other additives, components, integers or steps.

3 Disclosure of the Invention This invention provides geranyl compounds represented by the following formula (1-3): 5 R' CH 2 CHCOOH (I-3) NH 0 in which R 3 stands for p-hydroxyphenyl or mercapto group. 10 The geranylamide derivatives of above formula (1-3) include the following two compounds: N-geranoyltyrosine 15 HO

-

/

CH

2 CHCO 2 H( 3 15 (23) NH 0 and N-geranoylcysteine 20 HSCHg-HC-CO 2 H NH (24) 25 Those geranylamide derivatives of the formula (1-3) can be prepared by, for example, subjecting reactive derivatives of geranic acid (e.g., mixed acid anhydride, active ester, halide and the like) to an amidation reaction with tyrosine or cysteine. 30 This amidation reaction can also be conducted following conventional method of amidation reaction in the field of peptide chemistry, normally in an adequate inert organic solvent (e.g., 4 tetrahydrofuran, ether, dichloromethane, chloroform, N,N-dimethylformamide or the like) or in water, under cooling down to about 00C or heating up to about 60 0 C, preferably at about 0*C to room temperature. 5 The use ratio of tyrosine or cysteine to a reactive derivative of geranic acid is not strictly limited, but it is normally preferred to use either of them within a range of 1-2 moles per mole of the reactive derivative. Such geranylamide derivatives of the formula (1-3) produced 10 in the above reactions can be isolated from the reaction mixtures and purified by conventional means, for example, extraction, crystallization, chromatography or the like. Compounds of the formula (1-3) offered by the present invention possess excellent antitumor activity, as is clear from the 15 following measurement results of antitumor effect. Measurement of antitumor effect Carcinoma of HuH-7 cells (dendriform cell strain of human hepatoma) hypodermically implanted or subimplanted in the backs of 20 5-weeks old female nude mice (BALB/c, Ninox) was aseptically taken out and crushed into 5 x 5 mm sized pieces in a phosphate buffer solution (PBS), a piece ofwhich then being hypodermically implanted in the backs of the nude mice. Each test substance was dissolved in corn oil, and the solution 25 was intraperitoneally administered to the nude mice consecutively once per day at a rate of 250 pg/mouse for 3 weeks, starting from a week after the implantation. After termination of the administration, the carcinomas were taken out and weighed to calculate the antitumor effect and the mice' weight loss by the 30 following equations. For the test six mice per group were used, and the group administered with the solvent (corn oil) alone was made the 5 control group: Antitumor effect (%) average tumor weight of a test group 5 -X 100 average tumor weight of the control group Weight loss (%) average body weight of a test group mice 10 X 100 average body weight of the control group mice Evaluations of antitumor effect and weight loss were conducted according to the following standard, where the control 15 group values were held to be 100%. Antitumor effect -: >100%, +/-: 100-75%, +: 75-50%, ++: 50-25%, +++: 25-0% 20 Weight loss -: >110%, +/-: 110-100%, +: 100-95%, ++: 95-90%, +++: <90% Death rate (death rate during the test period) none 25 +/-: death occurred with high concentration administration (500 gg/mouse) +: 1-3 mice dead ++: 3-5 mice dead +++: all 6 mice dead 30 Synthetic evaluation -: weak antitumor effect and very strong toxicity against host mice +/-: weak antitumor effect recognizable and toxicity against host 35 mice also observable +: a fixed level of antitumor effect observable but toxicity 6 against host mice also strong ++: strong antitumor effect observed and toxicity against host mice week +++: strong antitumor effect observed and no toxicity against 5 host mice The results are shown in the following Tables 1-3. Table 1 10 est Substance Antitumor ThUct Synthetic Effect Weight Death Evaluation loss rate N-geranyl glucuronamide ++ +/- - ++ N-geranyl galacturonamide +/- +/ N-galactosylgeranamide ++++ N-fucosegeranamide ++ +- -+++ Table 2 Tst Substance Antitumor Ibxicity Synthetic Effect Weight Death Evaluation loss rate N,N'-digeranylmalic diamide + + - ++ N,N'-digeranylfumaric diamide ++ +/- - ++ N-geranyl-4 pyruvoaminobenzamide + N-geranoyltyrosine ++ +/- - ++ Tyrosine geranylamide + +/- - + N-acetyltyrosine geranylamide + +/- - + 7 Table 3 Ihst Substance Antitumor TOxicity Synthetic Effect Weight Death Evaluation loss rate N-glucosylmevalonamide ++ +/- + As is clear from the shown results, the compounds of the invention 5 of the formula (1-3) possess excellent antitumor effect against HuH-7 cells and furthermore almost no toxicity, and are expected to be useful as antitumor agents for treatment and therapy of various solid cancer represented by liver cancer. Where a compound of the present invention is used as a 10 medicine such as an antitumor agent, it can be administered orally or parenterally (e.g., intravenous injection, intramuscular injection, hypodermic injection, or the like). The effective dose is variable over a broad range depending on individual patients'symptoms, seriousness of the illness, body weight, age, and the doctor's diagnosis, 15 etc. Normally, however, taking a case of administration by injection, the dose can be about 1 - about 50 mg/kg/day, which may be administered at a single time or at plural times dividedly in a day. Where a compound of the present invention is used as a medicine, an effective dose of the compound can be formulated with 20 pharmaceutically acceptable carrier or diluent (e.g., excipient, solvent or other adjuvants) into a preparation form suitable for unit dose .administration, for example, tablet, powder, granule, capsule, enteric coated pill, troche, syrup, elixer, liquid, suspension, emulsion and the like. 25 As carriers or diluents useful for the formulation, for example, excipients such as starch, lactose, sucrose, mannitol, carboxymethyl cellulose and the like; lubricants such as magnesium stearate, sodium laurylsulfate, tale and the like; binders such as dextrin, microcrystalline cellulose, polyvinylpyrrolidone, gum Arabic, corn 30 starch, gelatin and the like; disintegrators such as potato starch, carboxymethyl cellulose and the like; and diluent solvents such as 8 water for injection, physiological saline, aqueous dextrose solution, vegetable oil for injection, propylene glycol, polyethylene glycol and the like can be named. Furthermore, taste- or odor-correcting agent, colorant, isotonicity, stabilizer, antiseptic, soothing agent and the like 5 may be incorporated where necessary. In the pharmaceutical preparations according to the invention, moreover, other pharmacologically active substance(s) may be incorporated where necessary. Hereinafter the invention is explained still more specifically, 10 -eferring to working Examples. Examples Synthesis Example 1: Synthesis of N-geranoylcysteine 15 To a THF (20 ml) solution containing geranic acid (1.68 g, 10 mmols), triethylamine (1.01 g, 10 mmols) was added and cooled to 0*C, into which a THF (5 ml) solution of isobutyl chloroformate (1.37 g, 10 mmols) was added dropwise, followed by 30 minutes' stirring at 00C. To the reaction mixture a solution of cysteine (1.35 g, 10 mmols) as 20 dissolved in 1N sodium hydroxide (10 ml) was added, followed by an hour's stirring at 0*C and further 4 hours' stirring at room temperature. After termination of the reaction, 10 ml of 1N hydrochloric acid was added to the reaction mixture and stirred for 10 minutes at room temperature. Then the reaction mixture was 25 condensed with a rotary evaporator. To the residue ethanol was added, and whereupon precipitated sodium chloride was removed by filtration. The ethanol solution was once again condensed under reduced pressure with the evaporator, and the residue was separated on silica gel column chromatography. Thus 0.556 g of the title 30 compound was obtained from the hexane-acetone (2:1) distillate. The yield was 19.5%: 9 1H NMR(CDCls)8=1.59(6H, s), 1.68(3H, s), 2.00-2.24(4H, m), 2.60-2.77(1H, m), 3.00-3.30(2H, m), 4.48-4.58(1H, m), 5.00-5.13(1H, m), 8.96(1H,s). Synthesis Example 2: N-geranoyltyrosine Synthesis Example 1 was repeated except that the cysteine was replaced with tyrosine, to provide the title compound: 1 H NMR(CDCl 3 )8=1.55(6H, s), 1.64(3H, a), 1.96-2.00(4H, m), 10 2.90-3.17(2H, m), 4.81-5.06(3H, m), 6.40-7.21(4H, m), 7.25(1H, s).

Claims (7)

1. Geranyl compounds represented by the following formula (1-3): R- CH 2 CHCOOH 54H (1-3) NH 0 in which RW stands for p-hydroxyphenyl or mercapto group. 10

2. An antitumor agent containing, as the active ingredient, a geranyl compound of the formula (1-3) as given in Claim 1.

3. A pharmaceutical composition comprising an activity-effective 15 amount of geranyl compound of the formula (1-3) as given in Claim 1, and a pharmaceutically acceptable carrier or diluent.

4. Method of treating cancer comprising administration of antitumorically effective amount of a geranyl compound of the formula (I 20 3) as given in Claim 1 to a patient.

5. Use of a geranyl compound of formula (1-3) as given in Claim 1 for treatments against cancer. 25

6. Use of a geranyl compound of the formula (1-3) as given in Claim 1 for production of pharmaceutical preparations.

7. A geranyl compound according to claim 1 substantially as herein described with reference to any one of the Examples. 30