AU2008200719B2 - 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: 822669 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 having 1,5-diene 15 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 geranic acid or 20 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 admission that 25 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 "comprises", is not intended to exclude other additives, components, integers or steps. 30 Disclosure of the Invention This invention provides geranyl compounds represented by the following formula (1-2): 3 NHCO R+COOHL (1.2) 5 in which R 2 stands for a residual group remaining after removing all carboxyl groups present in a carboxylic acid selected from the group consisting of malic acid, citric acid, succinic acid, fumaric acid, 2-oxoglutaric acid, pyruvic acid, p-pyruvoaminobenzoic acid, retinoic acid, tyrosine, cysteine, glutamic acid and serine, and where hydroxyl 10 or amino group(s) are present in the residual group, they may optionally be protected by acyl (e.g., lower alkanoyl) or benzyloxycarbonyl group(s), m is 1, 2 or 3, n is 0, 1 or 2, 15 m + n showing the number of -carhoxyhgroipswhch are present in said carboxylic acid. The geranylamide derivatives of above formula (1-2) include, for example, the following compounds. N,N'-digeranylmalic diamide 20 -HN HO-C-H OH -CN (6) CH2j-L NH 25 0 0-acetyl-N-geranylmalic monoamide 0 11 -OH 30 CH--0- -H (7) OOH-C-NH 0 35 4 0-acetyl-N,N'-digeranylmalic diamide 0 C-NH 5 CH-- --O--H 0 CH 2 -C-NH (8) 0 N,N',N''-trigeranylcitric triamide 0 10CH2-CNH HO-C- C-NH (9) 15 CH -C-NH 0 N-geranylsuccinic monoamide 20 HO 2

CCH

2

CH

2 C-NH (10) N,N'-digeranylsuccinic diamide 25

NH-C-CH

2

CH

2 C-NH || 11 o o N,N'-digeranylfumaric diamide 30 0 H C-NH C=C NHC H ll (12) 0 35 5 N-geranylfumaric monoamide 0 C=C/ (13) 5 HO2C H N,N'-digeranyl-2-oxoglutaric diamide 0

NH-C-CH

2

CH

2 C-C-NH 10 || {| o o (14) N-geranylpyruvamide CHi-C-C-NH (15) 0 0 N-geranyl-p-pyruvoaminobenzamide 20 CHg-C-C-NH 3l 111 -011 00 0 (16) 25 tyrosine geranylamide 0 HO - / CH 2 CH-C-NH

NH

2 (17) 30 N-acetyltyrosine geranylamide 6 0 It HO CH 2 CH-C-NH NH-C

CH

3 5 0 (18) Cysteine geranylamide 0 10 HSCH-CH-C-NH (19)

H

2 N Glutamic digeranyldiamide 0 15 11

NH-C-CH

2

CH

2 CH-C-NH 0

NH

2 (20) serine geranylamide 20

HOCH

2 CH-C-NH (21 1 (21)

H

2 N and 25 N-geranyl retinamide NH-C '. " 300 30 (22) 7 The geranylamide derivatives of the above formula (1-2) can be produced by, for example, subjecting geranylamine to an amidation reaction with reactive derivatives (e.g., mixed acid anhydride, active ester, halide or the like) of carboxylic acid represented by the formula 5 (I): R2 COOHLQ (II) in which R 2 , m and n have the earlier given significations, 10 in which hydroxyl or amino group(s) are protected with acyl (e.g., lower alkanoyl such as acetyl), benzyloxycarbonyl and the like groups. Said amidation reaction can be conducted following the conventional method of amidation reaction in the field of peptide chemistry, normally in an adequate inert organic solvent (e.g., 15 tetrahydrofuran, ether, dichloromethane, chloroform, N,N-dimethylformamide or the like) under cooling down to about 0*C or heating up to about 60 0 C, preferably at about 0*C to room temperature. The use ratio of geranylamine to a reactive derivative of 20 carboxylic acid of the formula (II) is variable depending on the number of geranyl group (m) to be introduced into the carboxylic acid, while it is normally preferred to use it within a range of 1 mole to (m+2) moles per mole of the reactive derivative. When the hydroxyl- or amino-protective groups are present 25 after the amidation reacetion, they are removed where necessary by a de-protection reaction such as hydrolysis to provide geranyl amide derivatives of the formula (1-2). Such geranylamide derivatives of the formula (1-2) produced in the above reactions can be isolated from the reaction 30 mixtures and purified by conventional means, for example, extraction, crystallization, chromatography or the like. Compounds of the formula (1-2) offered by the present invention possess excellent antitumor activity, as is clear from the following measurement results of antitumor effect. 35 8 Measurement of antitumor effect Carcinoma of HuH-7 cells (dendriform cell strain of human hepatoma) hypodermically implanted or subimplanted in the backs of 5-weeks old female nude mice (BALB/c, Ninox) was aseptically taken 5 out and crushed into 5 x 5 mm sized pieces in a phosphate buffer solution (PBS), a piece of which then being hypodermically implanted in the backs of the nude mice. Each test substance was dissolved in corn oil, and the solution was intraperitoneally administered to the nude mice consecutively 10 once per day at a rate of 250 pig/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 following equations. For the test six mice per group were used, and 15 the group administered with the solvent (corn oil) alone was made the control group: Antitumor effect (%) average tumor weight of a test group 20 -x 100 average tumor weight of the control group Weight loss (%) average body weight of a test group mice x 100 25 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 group values were held to be 100%. 30 Antitumor effect -: >100%, +/-: 100-75%, +: 75-50%, ++: 50-25%, +++: 25-0% 9 Weight loss - >110%, +/-: 110-100%, +: 100-95%, ++: 95-90%, +++: <90% Death rate (death rate during the test period) 5 -- none +/-: death occurred with high concentration administration (500 pg/mouse) +: 1-3 mice dead ++: 3-5 mice dead 10 ++: all 6 mice dead Synthetic evaluation -: weak antitumor effect and very strong toxicity against host mice 15 +/-: weak antitumor effect recognizable and toxicity against host mice also observable +: a fixed level of antitumor effect observable but toxicity against host mice also strong ++: strong antitumor effect observed and toxicity against host 20 mice week +++: strong antitumor effect observed and no toxicity against host mice The results are shown in the following Tables 1-3. 25 Table 1 Test Substance Antitumor '1bxicity Synthetic Effect Weight Death Evaluation loss rate 30 N-geranyl glucuronamide ++ rate ++ N-geranyl galacturonanmide +/ +/ N-galactosylgeranamide +++ + -+++ N-fucosegeranamide ++ +/- 35 10 Table 2 Antitumor Toxicity Synthetic 5Test Substance Effect Weight Death Evaluation loss rate N,N'-digeranylmalic diamide + + - ++ NN'-digeranylfumaric diamide ++ +/-

-

++ N-geranyl-4- ++ - -+++ pyruvoaminobenzamide 10 N-geranoyltyrosine ++ +/-

-

++ Tyrosine geranylamide + +/-

-

+ N-acetyltyrosine geranylamide + +/- - + Table 3 15 Antitumor Toxicity Synthetic 'lst Substance Effect Weight Death Evaluation loss rate N-glucosylmevalonamide ++ +/- 20 As is clear from the shown results, the compounds of the invention of the formula (1-2) 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 25 of various solid cancer represented by liver cancer. Where a compound of the present invention is used as a 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 30 a broad range depending on individual patients' symptoms, seriousness of the illness, body weight, age, and the doctor's diagnosis, 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. 35 11 Where a compound of the present invention is used as a medicine, an effective dose of the compound can be formulated with pharmaceutically acceptable carrier or diluent (e.g., excipient, solvent or other adjuvants) into a preparation form suitable for unit dose 5 administration, for example, tablet, powder, granule, capsule, enteric coated pill, troche, syrup, elixer, liquid, suspension, emulsion and the like. As carriers or diluents useful for the formulation, for example, excipients such as starch, lactose, sucrose, mannitol, carboxymethyl 10 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 starch, gelatin and the like; disintegrators such as potato starch, carboxymethyl cellulose and the like; and diluent solvents such as 15 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 may be incorporated where necessary. 20 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, referring to working Examples. 25 Examples 12 Synthesis Example 1: Synthesis of N,N'-digeranylfumaric diamide To a tetrahydrofuran (THF) (20 ml) solution containing fumaric acid (0.58 g, 5 mmlos), triethylamine (1.01 g, 10 mmols) was added and the solution was cooled to 0*C, into which a THF (5 ml) 5 solution containing isobutyl chloroformate (1.53 g, 10 mmols) was added dropwise. As the addition was continued, white precipitate started to form. After 30 minutes' stirring at 0*C, a THF (5 ml) solution containing geranylamine (1.53 g, 10 mmols) was added dropwise into the system, followed by an hour's stirring at 0*C and 10 further 4 hours' stirring at room temperature. After termination of the reaction, 50 ml of water was added to the reaction mixture which was then extracted with chloroform. The chloroform layer was washed with water and dried over magnesium sulfate. Filtering the magnesium sulfate off, the chloroform layer was condensed to provide 15 a white crystal. Recrystallizing the same from ethanol, 1.07 g of the title compound was obtained. The yield was 55%. IH NMR(CDCl 3 ) 6=1.60(6H, s), 1.62(6H, s), 1.68(6H, s), 2.01-2.10(8H, m), 3.95(4H, t, J=9.6 Hz), 5.04-5.09(2H, m), 20 5.20-5.25(2H, m), 5.94(2H, brs), 6.90(2H, s), 7.26(2H, s). Synthesis Example 2: Synthesis Example I was repeated except that the fumaric acid was replaced with corresponding carboxylic acid of the earlier 25 given formula (II) in each run, to provide the following compounds: N-geranylpyruvamide IH NMR(CDC1 3 ) 8=1.55(3H, s), 1.64(3H, s), 1.82(3H, s), 2.00(3H, s), 1.92-2.12(4H, m), 3.84(2H, d, J=7.2 Hz), 30 4.96-5.12(1H, m), 5.22-5.35(1H, m). N,N'-digeranylmalic diamide 'H NMR(CDCb3) 8=1.58(6H, s), 1.64(6H, s), 1.67(6H, s), 1.94-2.14(8H, m), 2.54(1H, dd, J=4.8, 14.8 Hz), 35 2.79(1H, dd, J=3.2, 14.4 Hz), 3.75-3.93(4H, m), 13 4.32-4.40(1H, m), 5.00-5.10(2H, -m), 5.10-5.22(2H, m). 0-acetyl-N-geranylmalic monoamide 'H NMR(CDC 3 ) 6-1.60(3H, s), 1.68(3H, s), 1.69(3H, s), 5 1.96-2.11(4H, m), 2.19(3H, s), 2.65(1H, dd, J=9.6, 22.8 Hz), 3.00(1H, dd, J=2.4, 22.8 Hz) 3.79-3.89(2H, m), 4.51-4.56(1H, m), 5.08(1H, t, J=7.2 Hz), 5.18(1H, t, J=6.0 Hz). 0-acetyl-NN'-digeranylmalic diamide, which was synthesized by a 10 similar method, using N-geranylmalic acid monoamide as the starting material. 'H NMR(CDC 3 ) 6=1.59(6H, s), 1.67(6H, s), 1.68(6H, s), 1.94-2.01(8H, m), 2.16(3H, s), 2.55(1H, dd, J=13.2, 22.8 Hz), 15 2.97(1H, dd, J=2.4, 22.8 Hz), 3.79-3.89(4H, m), 4.34-4.40(1H, m), 5.02-5.10(2H, m), 5.10-5.20(2H, m). N,N',N"-trigeranylcitric triamide 'H NMR(CDC 3 ) 6=1.60(9H, s), 1.66(9H, s), 1.68(9H, s), 20 1.98-2.08(12H, m), 3.76(6H, t, J=6.3 Hz), 4.26(4H, s), 5.07(6H, t, J=6.0 Hz), 5.20(6H, t, J=7.2 Hz). N-geranylsuccinic monoamide IH NMR(CDC 3 ) 8=1.60(3H, s), 1.70(3H, s), 1.72(3H, s), 25 1.92-2.15(4H, m), 2.52(2H, t, J=9.6 Hz), 2.70(2H, t, J=9.6 Hz), 3.80-3.90(2H, m), 5.08(1H, t, J=9.6 Hz), 5.18(1H, t, J=6.0 Hz), 5.61(1H, brs). N,N'-digeranylsuccinic diamide, which was synthesized by a similar 30 method, using N-geranylsuccenic monoamide as the starting material. IH NMR(CDC) 6=1.60(6H, s), 1.66(6H, s), 1.69(6H, s), 1.97-2.11(4H, m), 2.53(4H, s), 3.84(4H, t, J=5.5 Hz), 35 5.07(2H, t, J=4.9 Hz), 5.17(2H, t, J=5.5 Hz), 5.90(2H, brs).

14 N-geranylfumaric monoamide 'H NMR(CDC3) 6=1.59(3H, s), 1.67(3H, s), 1.70(3H, s), 1.94-2.16(4H, m), 3.88-4.04(2H, m), 5.06(1H, t, J=7.2 Hz), 5 5.21(1H, t, J=4.8 Hz), 6.30(1H, d, J=12.0 Hz), 6.46(1H, d, J=12.0 Hz). N,N'-digeranyl-2-oxoglutaric diamide 1H NMR(CDCL) 8=1.60(6H, s), 1.68(12H, s), 10 1.94-2.13(8H, m), 2.69(211, t, J=6.3 Hz), 3.26(2H, t, J=6.3 Hz), 3.81-4.04(4H, m), 5.02-5.10(2H, m), 5.15-5.22(2H, m). N-geranyl-p-pyruvoaminobenzamide 1H NMR(CDC) 6=1.60(3H, s), 1.68(3H, s), 1.70(3H, s), 16 2.03-2.11(4H, m), 2.17(3H, s), 3.95-4.04(2H, m), 4.83(lH, brs), 5.09(1H, t, J=6.6 Hz), 5.28(1H, t, J=6.9 Hz),5.94(1H, brs), 6.64(2H, d, J=8.7 Hz), 7.60(2H, d, J=8.7 Hz). N-geranylretinamide 20 1H NMR(CDCh) 6=1.03(6H, s), 1.12-1.63(6H, m), 1.60(3H, s), 1.66(3H,s), 1.68(3H, s), 1.72(3H, s), 1.87-1.93(4H, m), 2.01(3H, s), 2.37(3H, s), 3.82-3.92(2H, m3), 5.03-5.24(2H, m), 5.80(1H, s), 6.12-6.40(3H, m), 7.02(1H, d, J=12.0 Hz), 7.07(1H, d, J=12.0 Hz). 25 Synthesis Example 3: Synthesis of glutamic digeranyldiamide To a THF (20 ml) solution of N-benzyloxycarbonylglutamic acid (2.634 g, 9.4 mimols), triethylamine (1.899 g, 18.8 mmols) was added and cooled to 0*C. Into this mixture a THF (10 ml) solution of 30 isobutyl chloroformate (2.566 g, 18.8 mimols) was added dropwise, followed by 30 minutes' stirring at 0*C. Then a THF (10 ml) solution of geranylamine (2.880 g, 18.8 mmols) was added dropwise, followed by an hour's stirring at 00C and further 4 hours' stirring at room temperature. After termination of the reaction, 150 ml of chloroform 35 was added to the system, and the chloroform solution was washed 15 with water and dried over magnesium sulfate. The organic solvent was removed with an evaporator and the residue was separated on silica gel column chromatography. Thus 3.034 g of N-benzyloxycarbonylglutamic digeranyldiamide was obtained from 5 the hexane-acetone (2:1) distillate. The yield was 58.6%. Then the N-benzyloxycArbonylglutamic digeranyldiamide (3.034 g, 5.5 mmols) was dissolved in methanol (20 ml), and to the same solution 20 ml of 1N sodium hydroxide was added, followed by 5 hours' stirring at room temperature. The reaction mixture was 10 condensed with an evaporator, and the residue was separated on silica gel column chromatography to provide the object compound from the hexane-ethanol (3:1) distillate. Because the as-obtained product was viscous and amorphous, ether was added to the product for crystallization. Upon suction-filtering the system, 852 mg of the 15 object compound was obtained. The yield was 37.2%: IH NMR(CDCls)8=1.58(12H, s), 1.61(6H, s), 1.75-2.12(8H, m), 2.32-2.53(2H, m), 3.54-3.88(7H, m), 4.88-5.21(4H, m). 20 Synthesis Example 4: Repeating Synthesis Example 3 except that N-benzyloxycarbonylglutamic acid was replaced with tyrosine, N-acetyltyrosine, cysteine or serine, the following compounds, 25 respectively, were obtained. Where N-acetyltyrosine was used, the later deprotection operation was not conducted. Tyrosine geranylamide 'H NMR(CDCl)6=1.59(3H, s), 1.67(6H, s), 1.82-2.18(4H, m), 30 2.99-3.09(2H, m), 3.74-3.78(2H, m), 4.99-5.26(3H, m), 7.17-7.43(5H, m).

16 N-acetyltyrosine geranylamide 1H NMR(CDCla)6=1.60(3H, s), 1.68(3H, s), 1.98(3H, s), 2.00-2.11(4H, m), 2.18(3H, s), 2.90-3.00(2H, m), 3.69-3.79(2H, m), 4.59(lH, dd, J=15.6, 9.6 Hz), 5 5.00-5.10(2H, m), 6.70(2H, d, J=7.8 Hz), 7.01(2H, d, J=7.8 Hz), 7.27(1H, s). Cysteine geranylamide IH NMR(CDCIs)S=1.58(3H, s), 1.66(3H, s), 1.67(3H, s), 1.93-2.10(4H, m), 2.83-3.16(2H, m), 3.83-4.08(3H, m), 10 5.03-5.19(2H, m), 7.33(1H, s). Serine geranylamide 1H NMR(CDCl 3 )8=1.59(3H, s), 1.68(6H, s), 1.95-2.14(4H, m), 3.80-3.95(2H, m), 4.34-4.47(2H, m), 4.67(1H, t, J=10.8 Hz), 15 5.06(1H, t. J=6.0 Hz), 5.17(1H, t, J=6.0 Hz), 6.77(2H, brs). Formulation Example Two (2) g of N,N'-digeranylmalic diamide was dissolved in 1 liter of water for injection at ambient temperature, isotonized with 20 sodium chloride and sealed into ampoules. One (1) ml of this ~ njection contains 2 mg of the active ingredient. 25 30 35

Claims (7)

1. Geranyl compounds represented by the following formula (1-2): 5 NHCO R14COOHL (1-2) 10 in which R 2 stands for a residual group remaining after removing all carboxyl groups present in a carboxylic acid selected from the group consisting of malic acid, citric acid, succinic acid, fumaric acid, 2-oxoglutaric acid, pyruvic acid, p-pyruvoaminobenzoic acid, retinoic acid, tyrosine, cysteine, glutanic acid and seine, and where hydroxyl or amino group(s) are 15 present in the residual group, they may optionally be protected by acyl (e.g., lower alkanoyl) or benzyloxycarbonyl group(s), m is 1, 2, or3, n is 0, 1 or 2, m + n showing the number of carboxyl groups which are present in 20 said carboxylic acid.

2. An antitumor agent containing, as the active ingredient, a geranyl compound of the formula (1-2) as given in Claim 1. 25

3. A pharmaceutical composition comprising an activity-effective amount of geranyl compound of the formula (1-2) as given in Claim 1, and a pharmaceutically acceptable carrier or diluent.

4. Method of treating cancer comprising administration of antitumorically 30 effective amount of a geranyl compound of the formula (1-2) as given in Claim 1 to a patient. 18

5. Use of a geranyl compound of formula (1-2) as given in Claim 1 for treatments against cancer.

6. Use of a geranyl compound of the formula (I-2) as given in Claim 1 for 5 production of pharmaceutical preparations.

7. A geranyl compound according to claim 1 substantially as herein described with reference to anyone of the Examples. 10