CN1237176A - Tricyclic compounds useful as FPT inhibitors - Google Patents

Abstract

Novel tricyclic compounds and pharmaceutical compositions are disclosed which are inhibitors of the enzyme farnesyl protein transferase. Also disclosed are methods of inhibiting Ras function and thus inhibiting abnormal growth of cells. The method comprises administering a novel tricyclic compound to a biological system. In particular, the method inhibits the abnormal growth of cells in a mammal, such as a human.

Description

Tricyclic compounds useful as FPT inhibitors

background

Patent application WO 95/00497 (January 5, 1995) published under the Patent Cooperation Treaty (PCT) describes the inhibition of the enzyme, i.e. farnesyl-protein transferase (FPT or FTase), and thus the oncogene protein Ras Nesylated compounds. Oncogenes frequently encode protein components of signal transduction pathways leading to stimulation of cell growth and mitogenesis. Expression of oncogenes in cultured cells leads to transformation of cells characterized by the ability of cells to grow in soft agar and dense focal growth of cells lacking the contact inhibition exhibited by non-transformed cells. Mutation and/or overexpression of some oncogenes are often associated with cancer in humans.

To gain transforming ability, the precursor of the Ras oncoprotein must undergo farnesylation of a cysteine residue located in the carboxy-terminal tetrapeptide. Thus, inhibitors of the enzyme that catalyzes this change (ie, farnesyl protein transferase) have been shown to be anticancer agents for certain tumors (ie, tumors in which Ras contributes to transformation). Mutated, oncogenic forms of Ras are frequently found in many human cancers, most notably in more than 50% of colon and pancreatic cancers (Kohl et al., Science, Vol. 260, pp. 1834-1837, 1993).

Given the relevance of farnesyl protein transferase inhibitors, a valuable contribution to the art would be additional compounds useful as farnesyl protein transferase inhibitors. The present invention provides such a contribution.

SUMMARY OF THE INVENTION

Inhibition of farnesyl protein transferase by the tricyclic compounds of the present invention has not been reported before. Accordingly, the present invention provides methods of inhibiting farnesyl protein transferase using tricyclic compounds of the invention which: (i) effectively inhibit farnesyl protein transferase in vitro, but do not inhibit geranylgeranyl Protein transferase I; (ii) Blocking the phenotypic changes induced by the transformed Ras form of the farnesyl receptor, but not the engineered transformed Ras form of the geranylgeranyl receptor (iii) block intracellular processing of Ras to farnesyl receptors, but not engineered Ras to geranylgeranyl receptors; and (iv) block processing of Ras produced by transformed Ras Induced abnormal growth of cells in culture. Several compounds of the invention have demonstrated antitumor activity in animal models.

The present invention provides methods of inhibiting abnormal growth of cells, including transformed cells, by administering an effective amount of a compound of the present invention. Abnormal growth of cells refers to cell growth independent of normal regulatory mechanisms such as loss of contact inhibition. This includes the abnormal growth of: (1) tumor cells (tumors) that express an activated Ras oncogene; (2) tumor cells in which the Ras protein is activated by an oncogenic mutation in another gene; and (3) in which Aberrant Ras activation in benign and malignant cells of other proliferative diseases.

The tricyclic compounds of the present invention include the following compounds or pharmaceutically acceptable salts or solvates thereof:

4-[8-Chloro-3,7-dibromo-5,6-dihydro-11H-benzo[5,6]arhepta[1,2-b]pyridin-11-yl]-1 -(4-thiomorpholinoacetyl)piperidine

4-[8-Chloro-3,7-dibromo-6,11-dihydro-5H-benzo[5,6]arhepta[1,2-b]pyridin-11-yl]-1 -(4-thiomorpholinoacetyl)piperidine S-oxide

(+,-)-1-(3-bromo-8,10-dichloro-5-ethyl-6,11-dihydro-5H-benzo[5,6]arhepta[1,2 -b]pyridin-11-yl)-4-(4-pyridylacetyl)piperidine N4-oxide

(+,-)-4-(3-Bromo-8,10-dichloro-6,11-dihydro-5H-benzo[5,6]arhepta[1,2-b]pyridine- 11-yl)-1-[2-(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)-1-oxoethyl]piperidine; and

(+,-)-4-(3,10-Dibromo-8-chloro-6,11-dihydro-5H-benzo[5,6]arepta[1,2-b]pyridine- 11(R)-yl)-1-[(1-oxopropyl-4-piperidinyl)acetyl]piperidine. In another embodiment, the present invention relates to a pharmaceutical composition for inhibiting abnormal cell growth, which comprises an effective amount of a tricyclic compound and a pharmaceutically acceptable carrier.

In another embodiment, the invention is directed to a method of inhibiting abnormal growth of cells, including transformed cells, comprising administering to a mammal (eg, a human) in need of such treatment an effective amount of a tricyclic compound. Abnormal growth of cells refers to cell growth independent of normal regulatory mechanisms such as loss of contact inhibition. This includes abnormal growth of: (1) tumor cells (tumors) that express an activated Ras oncogene; (2) tumor cells in which the Ras protein is activated by an oncogenic mutation in another gene; (3) in which abnormal Ras-activated benign and malignant cells of other proliferative diseases, and (4) benign or malignant cells activated by mechanisms other than Ras proteins. Without wishing to be bound by theory, it is believed that these compounds may work by inhibiting the function of G-proteins such as ras p21 by blocking G-protein prenylation, thereby making them useful in the treatment of proliferative diseases such as tumor growth and cancer, or by inhibiting ras farnesyl protein transferase, thereby targeting their antiproliferative activity in ras-transformed cells.

The suppressed cells may be tumor cells expressing an activated ras oncogene. For example, cell types that can be inhibited include pancreatic tumor cells, lung cancer cells, myelogenous leukemia tumor cells, thyroid follicular tumor cells, myelodysplasia tumor cells, epidermal cancer tumor cells, bladder cancer tumor cells, or colon tumor cells. Inhibition of farnesyl protein transferase by ras treatment with tricyclic compounds can inhibit abnormal cell growth. It also has an inhibitory effect on tumor cells in which the Ras protein is activated by oncogenic mutations in genes other than Ras genes. In addition, tricyclic compounds can also inhibit tumor cells activated by proteins other than Ras proteins.

The invention also provides a method of inhibiting tumor growth by administering an effective amount of a tricyclic compound to a mammal (eg, a human) in need of such treatment. Specifically, the present invention provides methods for inhibiting the growth of tumors expressing activated Ras oncogenes by administering an effective amount of the above-mentioned compounds. Examples of tumors that can be suppressed include (but are not limited to) lung cancer (such as lung adenocarcinoma), pancreatic cancer (such as pancreatic cancer like exocrine pancreatic cancer), colon cancer (such as colorectal cancer like colon adenocarcinoma and colon cancer) , myeloid leukemia (such as acute myelogenous leukemia (AML), follicular carcinoma of the thyroid, myelodysplastic syndrome (MDS), bladder cancer, and epidermal cancer.

The present invention is also believed to provide methods of inhibiting certain benign and malignant proliferative diseases in which the Ras protein is activated by other The Ras gene is abnormally activated by an oncogenic mutation of the gene, that is, the Ras gene itself is not activated by the mutation into an oncogenic form. For example, the benign proliferative disorder neurofibromatosis or in which Ras is activated by mutation or overexpression of tyrosine kinase oncogenes (such as neu, src, abl, lck, and fyn) can be inhibited by the tricyclic compounds described herein. tumor.

In another embodiment, the present invention relates to a method of inhibiting farnesyl protein transferase of ras and farnesylation of the oncoprotein Ras by administering to a mammal, especially a human, an effective amount of a tricyclic compound. Administration of compounds of the present invention to patients to inhibit farnesyl protein transferase is useful in the treatment of the above-mentioned cancers.

Detailed description of the invention

As used herein, unless otherwise specified, the following terms used are defined as follows:

M ⁺ - represents the molecular ion of the molecule in the mass spectrum;

MH ⁺ - represents the molecular ion hydrogenation of the molecule in the mass spectrum;

Bu- represents butyl;

Et-represents ethyl;

Me-represents methyl;

Ph-represents phenyl;

The following solvents and reagents are abbreviated: Tetrahydrofuran (THF); Ethanol (EtOH); Methanol (MeOH); Acetic acid (HOAc or AcOH); Ethyl acetate (EtOAc); Trifluoroacetic acid (TFA); trifluoroacetic anhydride (TFAA); 1-hydroxybenzotriazole (HOBT); m-chloroperbenzoic acid (MCPBA); triethylamine (Et ₃ N); diethyl ether (Et ₂ O); ethyl chloroformate (ClCO ₂ Et) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (DEC).

The positions of the substituents R ¹ , R ² , R ³ and R ⁴ are referred to based on the following numbered ring structures:

Some of the compounds of the present invention may exist as different isomers (eg, enantiomers or diastereomers). The present invention is intended to include all such stereoisomers in pure form and in mixtures, including racemic mixtures. For example, the carbon atom at position C-11 can be in S or R configuration.

Some tricyclic compounds are acidic, such as those with carboxyl or phenolic hydroxyl groups. These compounds can form pharmaceutically acceptable salts. Examples of such salts include sodium, potassium, calcium, aluminum, gold and silver salts. Also included are salts formed with pharmaceutically acceptable amines, such as ammonia, alkylamines, hydroxyalkylamines, N-methylglucamine, and the like.

Partially basic tricyclic compounds may also form pharmaceutically acceptable salts, such as acid addition salts. For example, a pyridine-nitrogen atom can form salts with strong acids, while compounds with basic substituents such as amino groups can also form salts with weak acids. Examples of acids suitable for forming salts are hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, citric acid, oxalic acid, malonic acid, salicylic acid, malic acid, fumaric acid, succinic acid, ascorbic acid, maleic acid, methanesulfonic acid and others Inorganic and carboxylic acids are well known in the art. Said salts are prepared by contacting the free base form with a sufficient amount of the desired acid to produce the salt by conventional and convenient procedures. The free base form can be regenerated by treating the salt with an appropriate dilute aqueous base, such as dilute aqueous sodium hydroxide, potassium carbonate, ammonia and sodium bicarbonate. These free base forms differ somewhat from their respective salt forms in certain physical properties, such as solubility in polar solvents, but for the purposes of the present invention, the acid and base salts are the same as their respective free bases. The base form is however equivalent.

All such acid and base salts are useful as pharmaceutically acceptable salts within the scope of the invention and all acid and base salts are considered equivalent to the free forms of the corresponding compounds for purposes of the invention.

The tricyclic compound of formula 1.0 can be isolated from the reaction mixture by conventional methods such as extraction of the reaction mixture from water with an organic solvent, evaporation of the organic solvent, followed by chromatography on silica gel or other suitable chromatographic medium.

The compounds of the invention and the starting materials for their preparation are illustrated by the following examples, which should not be construed as limiting the scope of the disclosure.

Example 1

4-[8-Chloro-3,7-dibromo-5,6-dihydro-11H-benzo[5,6]arhepta[1,2-b]pyridin-11-yl]-1 -(4-thiomorpholinoacetyl)piperidine

Step A

At -5°C, 4-(8-chloro-3-bromo-5,6-dihydro-11H-benzo[5,6]arhepta[1,2-b]pyridine-11-ylidene Base)-1-piperidine-1-carboxylic acid ethyl ester (25.86g, 55.9mmol) was mixed with 250ml of concentrated sulfuric acid, then 4.8g (56.4mmol) of sodium nitrate was added and stirred for 2 hours. The mixture was poured onto 600 g of ice and basified with concentrated aqueous ammonium hydroxide. The compound was filtered, washed with 300ml of water and extracted with 500ml of dichloromethane. The extract was washed with 200ml of water, dried over magnesium sulfate, then filtered and concentrated in vacuo to a residue. Chromatography (silica gel, 10% ethyl acetate/dichloromethane) of the residue gave 24.4 g (86% yield) of product. mp = 165-167°C, mass spectrum: MH ⁺ = 506,508 (CI).

Elemental Analysis: Calculated: C, 52.13; H, 4.17; N, 8.29

Found: C, 52.18; H, 4.51; N, 8.16.

Step B:

The product of step A (20 g, 40.5 mmol) was mixed with 200 ml of concentrated sulfuric acid at 20 °C, and the mixture was then cooled to 0 °C. To this mixture was added 7.12 g (24.89 mmol) of 1,3-dibromo-5,5-dimethyl-hydantoin, and stirred at 20° C. for 3 hours. After cooling to 0°C, dibromohydantoin (1.0 g, 3.5 mmol) was added and stirred at 20°C for 2 hours. The mixture was poured onto 400 g of ice, basified at 0°C with concentrated aqueous ammonium hydroxide and the resulting solid collected by filtration. The solid was washed with 300 mL of water, slurried in 200 mL of acetone and filtered to give 19.79 g (85.6% yield) of product. mp=236-237°C, mass spectrum: MH ⁺ =586 (CI).

Elemental Analysis: Calculated: C, 45.11; H, 3.44; N, 7.17

Found: C, 44.95; H, 3.57; N, 7.16.

Step C:

A suspension of 25 g (447 mmol) of iron filings, 10 g (90 mmol) of calcium chloride and 20 g (34.19 mmol) of the product of step B in 700 ml of 90:10 ethanol/water was mixed at 50°C. The mixture was heated at reflux overnight and filtered through Celite ^(R) , washing the filter cake with 2 x 200 mL of hot ethanol. The filtrate and washings were combined and concentrated in vacuo to a residue. The residue was extracted with 600 ml of dichloromethane, washed with 300 ml of water and dried over magnesium sulfate. Filtration and concentration in vacuo to a residue followed by chromatography (silica gel, 30% ethyl acetate/dichloromethane) afforded 11.4 g (60% yield). mp=211-212°C, mass spectrum: MH ⁺ =556 (CI).

Elemental Analysis: Calculated: C, 47.55; H, 3.99; N, 7.56

Found: C, 47.45; H, 4.31; N, 7.49.

Step D:

The product from Step C (20 g, 35.9 mmol) was slowly added (in portions) to 8 g (116 mmol) of sodium nitrite in 120 ml of concentrated aqueous hydrochloric acid at -10°C. The resulting compound was stirred at 0°C for 2 hours and then slowly added (dropwise) to 150 ml (1.44 mol) of 50% hypophosphorous acid at 0°C over 1 hour. Stir at 0°C for 3 hours, then pour into 600 g of ice and basify with concentrated aqueous ammonium hydroxide. Extracted with 2 x 300 mL of dichloromethane, dried over magnesium sulfate and concentrated to a residue. Chromatography (silica gel, 25% ethyl acetate/hexanes) of the residue afforded 13.67 g (70% yield). mp=163-165°C, mass spectrum: MH ⁺ =541 (CI).

Elemental Analysis: Calculated: C, 48.97; H, 4.05; N, 5.22

Found: C, 48.86; H, 3.91; N, 5.18.

Step E:

The product from step D (6.8 g, 12.59 mmol) and 100 ml of concentrated aqueous hydrochloric acid were combined and stirred overnight at 85°C. The mixture was cooled, poured into 300 ml of ice and basified with concentrated aqueous ammonium hydroxide. Extract with 2 x 300 mL of dichloromethane, then dry the extract over magnesium sulfate. Filtration and concentration in vacuo to a residue followed by chromatography (silica gel, 10% methanol/ethyl acetate + 2% aqueous ammonium hydroxide) afforded 5.4 g (92% yield) of the title compound. mp = 172-174°C, mass spectrum: MH ⁺ = 469 (FAB).

Elemental Analysis: Calculated: C, 48.69; H, 3.65; N, 5.97

Found: C, 48.83; H, 3.80; N, 5.97.

Step F:

At 20° C., the compound (13 g, 33.3 mmol) of Step E of Example 1 was mixed with 300 ml of toluene, and then 32.5 ml (32.5 mmol) of 1M DIBAL toluene solution was added. The mixture was heated at reflux for 1 hour, cooled to 20°C, 32.5 ml of 1M DIBAL solution were added and heated at reflux for 1 hour. The mixture was cooled to 20°C and poured into a mixture of 400 g of ice, 500 ml of ethyl acetate and 300 ml of 10% aqueous sodium hydroxide. The aqueous layer was extracted with dichloromethane (3 x 200ml), the organic layer was dried over magnesium sulfate, then concentrated in vacuo to a residue. Chromatography (silica gel, 12% methanol/ethyl acetate + 4% aqueous ammonium hydroxide) gave 10.4 g of the title compound as a racemate. Mass spectrum: MH ⁺ = 469/471 (FAB).

Step G:

4-[8-Chloro-3,7-dibromo-5,6-dihydro-11H-benzo[5,6]arhepta[1,2-b]pyridin-11-yl]-1 -(Hydroxyacetyl)piperidine

At room temperature, add 0.17ml of N-methylmorpholine (NMM), 0.075g of 1-hydroxybenzene to 0.235g (0.5mmol) of the substance of step F of Example 1 in 3ml of anhydrous dimethylformamide (DMF) solution Triazole (HOBT), 0.145g 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (DEC) and 0.06g 80% technical grade glycolic acid. The resulting yellow solution was stirred for 24 hours, then evaporated under high vacuum. The residue was partitioned between dichloromethane and saturated brine, and the aqueous layer was extracted with dichloromethane (3 x 25ml). The combined organic layers were dried over magnesium sulfate, filtered and concentrated. The residue was purified by flash chromatography on 30 g of silica gel, eluting with 60% ethyl acetate/hexane and 3% methanol/dichloromethane (0.5 L each) to afford the title compound (0.255 g, 96.5% yield).

MS: 529 (M+H).

Step H

4-[8-Chloro-3,7-dibromo-5,6-dihydro-11H-benzo[5,6]arhepta[1,2-b]pyridin-11-yl]-1 -(Chloroacetyl)piperidine

A mixture of the compound from Step G of Example 1 (0.11 g, 0.2 mmol) and 1.5 ml of thionyl chloride was stirred at room temperature for 18 hours, then concentrated under reduced pressure. The residue was dissolved in dichloromethane and toluene was added to give a cloudy solution which was concentrated again and dried under high vacuum to give the title compound (0.11 g) as a yellow puff.

MS: 547/549 (M/M+2).

step 1

To about 0.11g 4-[8-chloro-3,7-dibromo-5,6-dihydro-11H-benzo[5,6]arhepta[1,2-b]pyridine-11- 0.12 ml of thiomorpholine was added to a solution of -1-(chloroacetyl)piperidine in 5 ml of dichloromethane, and the mixture was stirred at room temperature for 24 hours and diluted with 20 ml of distilled water. The layers were separated and the aqueous layer was back extracted with 10 mL of dichloromethane. The combined organic layers were washed once with saturated brine, dried over magnesium sulfate, filtered and evaporated to a brown residue. After 20 g of silica gel flash chromatography, eluting with 200 ml of 3% methanol/dichloromethane, 0.12 g of the target compound was obtained, and the two-step yield was 93.8%. MS: 614.5 (M+H).

FPT _IC50 = 0.0089 μM.

Example 2

4-[8-Chloro-3,7-dibromo-6,11-dihydro-5H-benzo[5,6]arhepta[1,2-b]pyridin-11-yl]-1 -(4-thiomorpholinoacetyl)piperidine S-oxide

To 0.09g (0.15mmol) of 4-[8-chloro-3,7-dibromo-5,6-dihydro-11H-benzo[5,6]arhepta[1 123 μl of trifluoroacetic acid (TFA) and 121 μl of 30% hydrogen peroxide were added to a solution of 2-b]pyridin-11-yl]-1-(4-thiomorpholinyl)piperidine in 10 ml of distilled tetrahydrofuran (THF). The resulting solution was stirred at room temperature for 20 hours, then concentrated. The residue was extracted with 2 x 10 ml portions of dichloromethane and 10 ml distilled water. The combined organic layers were washed once with saturated brine, dried over magnesium sulfate, filtered and evaporated to a colorless residue. Preparative TLC developed with 10% methanol (ammonia)/dichloromethane afforded 0.03 g of the title compound as a white solid. Yield: 32.6%.

MS: 630(M).

FPT _IC50 = 0.0068 μM.

Example 3

(+,-)-1-(3-bromo-8,10-dichloro-5-ethyl-6,11-dihydro-5H-benzo[5,6]arhepta[1,2 -b]pyridin-11-yl)-4-(4-pyridylacetyl)piperidine N4-oxide

Step A

n-Butyllithium (2.5M in hexane, 7.3ml, 18.25mmol) was added to diisopropylamine (2.8ml, 20.13mmol) in tetrahydrofuran (THF, distilled, 20ml) solution. Stir at 0°C for 30 minutes, then cool to -78°C. Add N-(1,1-dimethylethyl)-3-methyl-5-bromo-2-pyridinecarboxamide (2.0g, 7.38mmol) in THF (10ml) at -78°C, in- The resulting purple solution was stirred for an additional half hour at 78°C. A solution of 3,5-dichlorobenzyl chloride (2.8 g, 14.32 mmol) in tetrahydrofuran (10 ml) was added dropwise at -78°C. The dry ice-acetone bath was replaced with an ice/water bath and the reaction mixture was stirred at 0°C for 11/2 hours. Thin layer chromatography (3% V/V ethyl acetate:hexanes) determined the reaction was complete. The reaction mixture was quenched with water (100ml) and extracted with ethyl acetate (2x200ml). The organic layer was separated, washed with water (100ml), dried over magnesium sulfate, filtered and the solvent was evaporated to give an oil which was chromatographed on silica gel (3% v/v ethyl acetate: hexanes) to give a colorless oil which was obtained at Suction filtration under high vacuum (0.2 mm) solidified to obtain a white solid (2.7 g, yield 96.7%).

Step B

To a solution of diisopropylamine (0.82ml, 5.86mmol) in tetrahydrofuran (10ml, distilled from sodium) was added n-butyllithium (2.5M in hexane, 2.3ml, 5.75mmol) at -78°C. Stir at -78°C for 20 minutes. The product from Example 3, Step A (1.0 g, 2.64 mmol) in tetrahydrofuran (5 mL) was added and the resulting purple solution was stirred for 1/2 hour at -78°C. Neat ethyl bromide (0.7ml, 9.37mmol) was added, the dry ice bath was replaced by an ice/water bath, and the reaction mixture was stirred at 0°C for 1 hour. Reaction completion was determined by silica gel thin layer chromatography (3% V/V ethyl acetate: hexane). Water (50ml) and ethyl acetate (100ml) were added to the reaction mixture. The organic layer was separated, washed with water (5 ml), dried over sodium sulfate, filtered and the solvent was evaporated to give a light yellow oil which was chromatographed on silica gel (3% V/V ethyl acetate: hexanes) under high vacuum ( 0.2 mm) for 2 hours to obtain a colorless oil (0.95 g, yield 88.7%). MS: CIMH 459.

Step C

Phosphorus oxychloride (5ml, 53.6mmol) was added to a solution of the product from Step B of Example 3 (0.9g, 1.96mmol) in toluene (10ml) and stirred at reflux for 5 hours. The reaction mixture was cooled to room temperature, and the solvent was evaporated under reduced pressure. Toluene (20ml) was added and the solvent was evaporated again. Water (30ml), ethyl acetate (100ml) and 10% sodium hydroxide (10ml) were added sequentially to the residual oil. The organic layer was separated, washed with brine (50ml), dried over magnesium sulfate, filtered and the solvent was evaporated to give an oil which was used in the next reaction (0.8g, 100% yield) MS: CIMH 383/385.

Step D

The product from Step C of Example 3 (0.8 g, 2.08 mmol) was mixed with aluminum trichloride (2.7 g, 20.2 mmol) at 20°C, then stirred at 175°C on a preheated oil bath for 10 minutes. (1:1 V/V ethyl acetate:hexane) was determined to be complete by silica gel thin layer chromatography. The reaction was cooled to 0°C and water (10ml) was added followed by 2 equivalents of hydrochloric acid (15ml). The mixture was basified by adding 20% sodium hydroxide under ice cooling, then extracted with dichloromethane (2 x 100ml). The organic layer was separated, dried over magnesium sulfate, filtered and the solvent was evaporated to give a white solid (0.6 g, 75% yield). MS: CI MH (383/385).

Step E

A solution of the product from Example 3, Step D (0.3g, 7.79 x 10-4m) in 6N hydrochloric acid (10ml) and methanol (5ml) was stirred at reflux temperature for 24 hours. The reaction was cooled to 20°C, added to ice (100 g) and basified to pH 14 with 25% NaOH at 0°C. The precipitated white solid was collected, washed with water (20ml), dissolved in dichloromethane (100ml), dried over magnesium sulfate, filtered and the solvent was evaporated. The residual solid was chromatographed on silica gel (1:1 V/V ethyl acetate:hexane) to give a white solid (200 mg, 66% yield). MS: CI MH (384/386).

Step F

Sodium borohydride (100 mg, 2.66 mmol) was added to a solution of the product from Step E of Example 3 (130 mg, 0.338 mmol) in ethanol (5 ml) at 0°C, and the reaction mixture was stirred for 5 minutes. The reaction was determined to be complete by thin layer chromatography (silica gel, 30% V/V ethyl acetate: hexane). Water (20ml) and dichloromethane (30ml) were added, the organic layer was separated, dried over magnesium sulfate, filtered and the solvent was evaporated to give an oil which was chromatographed on silica gel (20% V/V ethylacetate: hexane), as a 70:30 mixture of the two diastereomers (130 mg, 100% yield). MS: CI (MH 388).

Step G

Thionyl chloride (0.15ml, 2.05mmol) was added to a solution of the product from step F of Example 3 (60mg, 0.155mmol) in toluene (3ml) at 0°C. The reaction mixture was stirred at 0°C for 1 hour and at 20°C for an additional hour. The reactant was cooled to 0°C, and water (10ml), ethyl acetate (20ml) and 10% sodium hydroxide (5ml) were added sequentially. The organic layer was separated, washed with brine (5ml), dried over magnesium sulfate, filtered and the solvent evaporated to give an oil (65mg).

Step H

Piperazine (100 mg, 1.16 mmol) was added to a solution of the product from step G of Example 3 (60 mg, 0.155 mmol) in acetonitrile (5 ml) at 0°C. Triethylamine (0.2ml, 1.43mmol) was added and the reaction mixture was stirred at 20°C for 2 hours. Water (20ml) and dichloromethane (50ml) were added. The organic layer was separated, washed with brine (20ml), dried over magnesium sulfate, filtered and the solvent evaporated to give the crude product as a white solid (60mg). MS: CI MH (456). NMR showed an 80/20 mixture of the two diastereomers.

Step I

At 0°C, to a solution of the product (50 mg, 0.109 mmol) in Step H of Example 3 in dimethylformamide (5 ml) was added 1-hydroxybenzotriazole (40 mg, 0.296 mmol), 1-(3-di Methylaminopropyl)-3-ethylcarbodiimide hydrochloride (DEC1, 50mg, 0.26mmol) and 4-pyridyl N-oxide acetic acid (40mg, 0.26mmol), add N-methylmorpholine (0.5ml, 4.03mmol), the reaction mixture was stirred at 20°C for 24 hours. The solvent was evaporated under reduced pressure and the residual oil was extracted with dichloromethane (40ml), washed with water (20ml) and brine (20ml), dried over magnesium sulfate, filtered and the solvent evaporated to give an oil which was chromatographed on silica gel (10% V /V methanol:dichloromethane) gave the product as a white solid, dried under high vacuum (0.2 mm) for 3 hours (50 mg, yield 78).

NMR: 80:20 mixture of diastereomers.

MS FABS MH 590.8/588.9

Exact mass spectrum: MH ⁺ 587.0616 calculated for C ₂₇ H ₂₆ N ₄ O ₂ BrCl ₂ ; found 587.0612

Step 3J

Separation of diastereomers

A mixture of two diastereomers (200 mg, 80:20 mix) was separated on a Chiralpack AD column from Chiral Technologies Inc., Exton, Pennsylvania, using ethanol as the eluting solvent. Chiralpack AD is amylose tris(3,5-dimethylphenylcarbamate) coated on a 10 μm silica matrix.

1) The major diastereomer (120 mg) was obtained as a racemate, white solid

FABS MH 588.9/590.8

_FABS MS MH ⁺ _Calcd . for _{C27H26N4O2BrCl2 587.0616} ; found MH ⁺ _587.0612

FPT _IC50 = 0.006 μM.

2) The minor diastereomer (20 mg) was obtained as a racemate as a white solid

MS FABS 588.9/590.8

Example 4

(+)-4-(3-Bromo-8,10-dichloro-6,11-dihydro-5H-benzo[5,6]arepta[1,2-b]pyridine-11- Base)-1-[2-(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)-1-oxoethyl]piperidine

Step A

The product from step B of Example 5 (9.90 g, 18.9 mmol) was dissolved in 150 ml of dichloromethane and 200 ml of acetonitrile and heated to 60°C. Add 2.77 g (20.8 mmol) N-chlorosuccinimide, heat to reflux for 3 hours, monitor the reaction by TLC (30% ethyl acetate/water). Then 2.35 g (10.4 mmol) of N-chlorosuccinimide was added, and the mixture was refluxed for 45 minutes. The reaction mixture was cooled to room temperature, extracted with 1N sodium hydroxide and dichloromethane. The dichloromethane layer was dried over magnesium sulfate, filtered and flash chromatographed (1200 mL normal phase silica gel, eluting with 30% ethyl acetate/water) to give 6.24 g of the desired product. M.p.193-195.4°C.

Step B:

At -10°C, 2.07 g (30.1 mmol) of sodium nitrite was added to 160 ml of concentrated hydrochloric acid, and stirred for 10 minutes. The product from Example 4, Step A (5.18 g, 10.1 mmol) was added and the reaction mixture was heated from -10°C to 0°C for 2 hours. The reactant was cooled to -10°C, 100ml of hypophosphorous acid was added, and left overnight. To extract the reaction mixture, it was poured into crushed ice and basified with 50% sodium hydroxide/dichloromethane. The organic layer was dried over magnesium sulfate, filtered and concentrated to dryness. Purification by flash chromatography (600 mL normal phase silica gel, eluting with 20% ethyl acetate/hexanes) afforded 3.98 g of product. Mass Spectrum: MH ⁺ 497.2.

Step C:

3.9 g of the product from step B of Example 4 were dissolved in 100 ml of concentrated hydrochloric acid and refluxed overnight. The mixture was cooled, basified with 50% w/w sodium hydroxide and the resulting mixture extracted with dichloromethane. The dichloromethane layer was dried over magnesium sulfate, the solvent was evaporated and dried under vacuum to give 3.09 g of the desired product. Mass spectrum: MH ⁺ = 424.9.

Step D:

Using a method similar to Steps F and G of Example 5, 1.73 g of the desired product was obtained, mp 169.6-170.1°C; [α] _D ²⁵ +48.2° (c=1, MeOH).

Step E

The product (0.5g, 1.2mmol) of step D of Example 5 was dissolved in 10ml of DMF, and 0.3g (1.5mmol) of phthaloyl (pthaloyl) glycine, 0.29g (1.5mmol) of DEC, 0.20 g (1.5 mmol) HOBT and 0.15 g (1.5 mmol) N-methylmorpholine. The reaction mixture was stirred overnight, warming to room temperature. Remove volatiles. Partition between water and dichloromethane. The aqueous phase was extracted with dichloromethane. The dichloromethane fractions were combined, dried over magnesium sulfate and concentrated. Purification by flash chromatography, eluting with 50% ethyl acetate-hexanes, afforded the title compound. Mass spectrum MH+=614 mp=144-145°C.

FPT _IC50 = 0.0186 μM.

Example 5

(+)-4-(3,10-dibromo-8-chloro-6,11-dihydro-5H-benzo[5,6]arepta[1,2-b]pyridine-11( R)-yl)-1-[(1-oxopropyl-4-piperidinyl)acetyl]piperidine

Step A:

At -5°C, 4-(8-chloro-3-bromo-5,6-dihydro-11H-benzo[5,6]arhepta[1,2-b]pyridine-11-ylidene Base)-1-piperidine-1-carboxylic acid ethyl ester (15g, 38.5mmol) was mixed with 150ml of concentrated sulfuric acid, then 3.89g (38.5mmol) of potassium nitrate was added and stirred for 4 hours. The mixture was poured into 3 L of ice and basified with 50% concentrated aqueous sodium hydroxide. Extract with dichloromethane, dry over magnesium sulfate, then filter and concentrate in vacuo to a residue. Recrystallization of the residue from acetone gave 6.69 g of product. ¹ H NMR (CDCl ₃ , 200MHz): 8.5(s,1H); 7.75(s,1H); 7.6(s,1H); 7.35(s,1H); 4.15(q,2H); ); 3.5-3.1(m,4H); 3.0-2.8(m,2H); 2.6-2.2(m,4H); 1.25(t,3H).

Step B

The product of step A (6.69 g, 13.1 mmol) was combined with 100 ml of 85% ethanol/water, then 0.66 g (5.9 mmol) of calcium chloride and 6.56 g (117.9 mmol) of iron were added, and the mixture was then heated at reflux overnight . The hot reaction mixture was filtered through celite ^(R) , and the filter cake was washed with hot ethanol. The filtrate was concentrated in vacuo to give 7.72 g of product. Mass spectrum MH ⁺ = 478.0.

Step C:

7.70 g of the product of Step B were mixed with 35 mL of acetic acid, then 45 mL of bromine in acetic acid was added and the mixture was stirred overnight at room temperature. 300 ml of 1N aqueous sodium hydroxide solution was added, followed by 75 ml of 50% aqueous sodium hydroxide solution, and extraction was performed with ethyl acetate. The extract was dried over magnesium sulfate and concentrated in vacuo to a residue. Chromatography (silica gel, 20%-30% ethyl acetate/hexanes) of the residue gave 3.47 g of product (and 1.28 g of partially purified product). Mass spectrum MH ⁺ =555.9.

¹ H NMR (CDCl ₃ , 300MHz): 8.5(s,1H); 7.5(s,1H); 7.15(s,1H),4.5(s,2H); 4.15(m,3H); 3.8(br s, 2H); 3.4-3.1(m, 4H); 9-2.75(m, 1H); 2.7-2.5(m, 2H); 2.4-2.2(m, 2H); 1.25(m, 3H).

Step D:

Sodium nitrite (1.86 g, 27 mmol) was added to 300 ml HCl (cooled to -10°C) and stirred for 5 minutes. To this mixture was added the compound of Example 5, Step C (5 g, 9 mmol) portionwise over 10 minutes. The reaction was warmed to 0-3°C and stirred for 2 hours. To this mixture was slowly added 75 ml of hypophosphorous acid, and the reaction mixture was kept in the refrigerator for about 16 hours. The mixture was poured onto ice and the pH was adjusted to 9-10 with 50% (v/v) sodium hydroxide. Extract with ethyl acetate. The ethyl acetate layer was dried and concentrated in vacuo to a residue. Chromatography (silica gel, 10%-20% ethyl acetate/hexanes) of the residue gave 3.7 g of product. Mass spectrum MH ⁺ = 541.0.

¹ H NMR (CDCl ₃ , 200MHz): 8.52(s, 1H); 7.5(d, 2H); 7.2(s, 1H), 4.15(q, 2H); 3.9-3.7(m, 2H); 3.5-3.1 (m,4H); 3.0-2.5(m,2H); 2.4-2.2(m,2H); 2.1-1.9(m,2H); 1.26(t,3H).

Step E:

The product of Step D (0.70 g, 1.4 mmol) was mixed with 8 ml of concentrated aqueous hydrochloric acid and the mixture was heated at reflux overnight. Add 30 ml of 1N aqueous sodium hydroxide solution, then add 5 ml of 50% aqueous sodium hydroxide solution, and extract with dichloromethane. The extract was dried over magnesium sulfate and concentrated in vacuo to give 0.59 g of the title compound. Mass spectrum M ⁺ = 468.7. mp=123.9-124.2°C.

Step F:

A toluene solution of the compound from Example 5, Step E (8.1 g) was prepared and 17.3 ml of 1M DIBAL in toluene was added. The mixture was heated under reflux and 21 ml of a 1M DIBAL/toluene solution was added slowly (dropwise) over 40 minutes. The reaction mixture was cooled to about 0°C, and 700 ml of 1M aqueous hydrochloric acid was added. Separate and discard the organic phase. The aqueous phase was washed with dichloromethane, the extract was discarded, and then the aqueous phase was basified by adding 50% aqueous sodium hydroxide solution. Extraction with dichloromethane, drying of the extract over magnesium sulfate and concentration in vacuo afforded 7.30 g of the title compound as a racemic mixture of enantiomers.

Step G - Separation of Enantiomers

The racemic target compound in step F of Example 5 was separated by preparative chiral chromatography (Chiralpack AD, 5cm×50cm column, eluting with 20% isopropanol/hexane+0.2% diethylamine) to obtain the above (+)-isomers and (-)-isomers of compounds.

Physicochemical data of (+)-isomer: mp=148.8℃; mass spectrum MH ⁺ =472; [α] _D ²⁵ =+65.6°(12.9mg/2ml methanol)

Physicochemical data of (-)-isomer: mp=112℃; mass spectrum MH ⁺ =472; [α] _D ²⁵ =-65.2°(3.65mg/2ml methanol)

Step H:

(+)-isomer

The (+)-isomer product from step G of Example 5 (3.21 g, 6.80 mmol) was mixed with 150 ml of anhydrous DMF. Make 1.33g (+)-isomer with 1.37g 1-N-tert-butoxycarbonylpiperidinyl-4-acetic acid, 1.69g (8.8mmol) DEC, 1.19g (8.8mmol) HOBT and 0.97ml (8.8 mmol) 4-methylmorpholine, and the mixture was stirred overnight at room temperature. Concentrate in vacuo to remove DMF, and add 50 ml of saturated aqueous sodium bicarbonate solution. Extracted with dichloromethane (2 x 250ml), washed the extract with 50ml brine and dried over magnesium sulfate. Concentration in vacuo to a residue and chromatography (silica gel, eluting with 2% methanol/dichloromethane + 10% ammonium hydroxide) gave 2.78 g of product. Mass spectrum MH ⁺ =694.0 (FAB); [α] _D ²⁵ =+34.1° (5.45mg/2ml methanol).

Step I:

Treat 2.78g of the compound from Example 5, Step H and 60ml of dichloromethane, then cool to 0°C and add 55ml of TFA. The mixture was stirred at 0°C for 3 hours, and 500 ml of 1N aqueous sodium hydroxide solution was added, followed by 30 ml of 50% aqueous sodium hydroxide solution. Extraction with dichloromethane, drying over magnesium sulfate, and concentration in vacuo gave 1.72 g of product. mp=104.1°C. Mass spectrum M ⁺ =597; [α] _D ²⁵ =+53.4° (11.42mg/2ml methanol).

Step J:

To the compound from step I of Example 5 (0.11 g, 0.19 mmol) and triethylamine (0.08 ml, 0.57 mmol) dissolved in anhydrous dichloromethane (6 ml) was added propionyl chloride (0.04 ml, 2 eq). After stirring at room temperature overnight, 1M hydrochloric acid was added and the mixture was shaken. The organic phase was separated, washed with 1N sodium hydroxide and dried over magnesium sulfate. Filtration and concentration in vacuo afforded the title compound (0.10 g, 83% yield, mp 106.4-109.3°C).

FPT _IC50 = 0.0068 μM.

Raw material preparation

Starting materials useful in preparing compounds of the invention are illustrated by the following preparative examples, which should not be construed to limit the scope of the disclosure. This type of raw material can be prepared by methods known in the art, as seen in Bioorganic & Medicinal Chemistry Letters (volume 3, No. 6, page 1073-1078, (1993), U.S. 4355036, PCT/US94/11390 (WO95/10514), PCT/US94/11391 (WO95/10515), PCT/US94/11392 (WO95/10516); Organic Functional Group Preparations by Stanley R. Sandler and Wolf Karo (2nd Edition , Academic Press, Inc., San Diego, California, Vol. 1-3, (1983)) and J March, Advanced Organic Chemistry, Reactions & Mechanisms, and Structure, 3rd ed., John Wiley & Sons, New York, p. 1346. (1985) et al. Other mechanistic pathways and analogous structures within the scope of the invention will be apparent to those skilled in the art.

determination

In vitro enzyme assay: FPTIC ₅₀ (inhibition of farnesyl protein transferase, in vitro enzyme assay) was determined according to the method disclosed in WO/10515 or WO95/10516. The data in the Examples demonstrate that the compounds of the invention are inhibitors of partially purified rat brain farnesyl protein transferase (FPT) Ras-CVLS farnesylation. The data also indicate that the compounds of the present invention are potent inhibitors ( _IC50 <<10 μM) of farnesylation of partially purified rat brain FPT Ras-CVLS.

When preparing pharmaceutical compositions from the compounds described by this invention, inert, pharmaceutically acceptable carriers can be either solid or liquid. Solid preparations include powders, tablets, dispersible granules, capsules, cachets and suppositories. Powders and tablets may contain from about 5 to about 70% active ingredient. Suitable solid carriers are known in the art, such as magnesium carbonate, magnesium stearate, talc, sucrose, lactose. Tablets, powders, cachets, and capsules are solid dosage forms suitable for oral administration.

When preparing a suppository, a low-melting wax such as fatty acid glyceride or a mixture of cocoa butter is first melted, and the active ingredient is uniformly dispersed therein with stirring. The molten homogeneous mixture is then poured into convenient sized molds and allowed to cool and thereby solidify.

Liquid formulations include solutions, suspensions and emulsions. Examples suitable for parenteral injection are water or water-propylene glycol solutions.

Liquid preparations may also include solutions for intranasal administration.

Aerosol preparations suitable for inhalation may include solutions and solids in powder form, which may be in combination with a pharmaceutically acceptable carrier such as an inert compressed gas.

Also included are solid form preparations which are to be converted, shortly before use, to liquid preparations for either oral or parenteral administration. Such liquid preparations include solutions, suspensions and emulsions.

The compounds of the invention may also be administered transdermally. Transdermal compositions may be in the form of ointments, lotions, aerosols and/or emulsions, including transdermal patches of the matrix type or depot type commonly used for this purpose in the art.

Preferably the compound is administered orally.

Preferably the pharmaceutical formulation is in unit dosage form. In such form, the preparation is divided into unit doses containing appropriate quantities of the active component, eg, an effective amount to achieve the desired purpose.

The amount of the active compound in the unit dosage form of the preparation can be varied or adjusted between about 0.1 mg-1000 mg, more preferably about 1 mg-300 mg, according to the specific usage.

The actual dosage employed will vary according to the requirements of the patient and the severity of the condition being treated. Those skilled in the art can readily determine the appropriate dosage for a particular situation. In general, treatment is initiated with doses slightly lower than the optimum dose of the compound. Thereafter, the dose is gradually increased to achieve the optimum effect in the particular case. For convenience, the total daily dosage may be divided and administered in portions throughout the day as required.

The amount and frequency of administration of the compounds of the present invention and pharmaceutically acceptable salts thereof are adjusted according to the physician's judgment after taking into account various factors such as the age, physical condition and size of the patient and the severity of the symptoms being treated. Generally, the recommended dosage regimen for oral administration is 10 mg-2000 mg per day, preferably 10-1000 mg per day, divided into 2-4 times throughout the day to block tumor growth. When administered within this dosage range, the compounds are nontoxic.

The following are examples of pharmaceutical dosage forms containing the compounds of the invention. The scope of the pharmaceutical composition aspects of the invention is not limited by the examples provided.

Examples of pharmaceutical dosage forms

Example A - Tablets serial number Element mg/tablet mg/tablet 1 active ingredient 100 500 2 Lactose USP 122 113 3 Corn starch, food grade, 10% paste in pure water 30 40 4 cornstarch, food grade 45 40 5 Magnesium stearate 3 7 total 300 700

Preparation

Mix ingredients Nos. 1 and 2 in a suitable mixer for 10-15 minutes. The mixture with No. 3 ingredient was granulated. Grind the wet granules through a coarse sieve (eg 1/4", 0.63 cm) if desired. Dry the wet granules. If desired, sieve the dry granules and allow to mix with the ingredients of No. 4 for 10-15 minutes. Add No. 5 The ingredients are mixed for 1-3 minutes.The mixture is compressed on a suitable tablet machine to the appropriate size and weight.

Example B - Capsules serial number Element mg/capsule mg/capsule 1 active ingredient 100 500 2 lactose 106 123 3 cornstarch, food grade 40 70 4 Magnesium stearate NF 7 7 total 253 700

Manufacturing method

The ingredients of numbers 1, 2 and 3 are mixed in a suitable mixer for 10-15 minutes. Add ingredient number 4 and mix for 1-3 minutes. The mixture is filled into two-section hard gelatin capsules on a suitable encapsulation machine.

Although the invention has been described in conjunction with the specific embodiments set forth above, it is apparent that many alternatives, modifications and variations thereof will be apparent to those skilled in the art. All such alternatives, modifications and variations are considered to be within the spirit and scope of the invention.

Claims (7)

1. be compound or its pharmacy acceptable salt of following formula structure:

2. suppress the medicinal compositions of abnormal growth of cells, it comprises the compound and the pharmaceutically acceptable carrier of the claim 1 of significant quantity.

3. suppress the method for abnormal growth of cells, this method comprises the compound of the claim 1 that gives significant quantity.

4. the method for claim 3, wherein repressed cell is for expressing the tumour cell of activated ras oncogene.

5. the method for claim 3, wherein repressed cell is pancreas tumour cell, lung carcinoma cell, myelomatosis tumour cell, thyroid follicle tumour cell, myelodysplasia tumour cell, epidermal carcinoma tumour cell, bladder cancer tumour cell and colon tumor cell.

6. the method for claim 3, wherein the inhibition of abnormal growth of cells produces by suppressing the ras farnesyl-protein transferase.

7. the method for claim 6, wherein said inhibition is the inhibition to tumour cell, wherein Ras albumen since other gene cancer sudden change of non-Ras gene be activated.