KR0176415B1 - Farnesyl transferase inhibitor and derivative, process for the preparation thereof and composition containing same - Google Patents

Abstract

The present invention relates to a farnesyl transferase inhibitor, a derivative thereof, a method for preparing the same, and a composition containing the same, wherein the cinnamaldehyde derivative KRIBB-BP007 and derivatives synthesized from cinnamon according to the present invention and PRIBB0BP007-1 are synthesized therefrom. Silver may be usefully used as a cancer prevention and treatment agent by inhibiting the expression of las oncolytic enzymes.

Description

Panesyl transferase inhibitors and derivatives thereof, methods for their preparation and compositions containing them

1 is an ultraviolet absorbance spectrum of KRIBB-BP007, a farnesyl transferase inhibitor according to the present invention, FIG. 2 is a hydrogen NMR spectrum of KRIBB-BP007, and FIG. 3 is a carbon NMR spectrum of KRIBB-BP007, 4 is a hydrogen NMR spectrum of KRIBB-BP007-1, a derivative synthesized from KRIBB-BP007.

The present invention relates to a panesyl transferase inhibitor, a derivative thereof, a method for preparing the same, and a composition containing the same, and more particularly, to inhibit the action of panesyl transferase, which has been separated and purified from cinnamomum cassia Blume. The present invention relates to a cinnamic aldehyde derivative KRIBB-BP007 and a method for extracting the same, a derivative KRIBB-BP007-1 and a method synthesized therefrom, and an anticancer agent composition containing the same.

The incidence of cancer continues to increase with the development of civilization, but the cause of cancer has not been identified enough to suggest a fundamental treatment for cancer patients. Currently, the treatment of cancer patients mainly relies on surgical surgery, radiation therapy, and chemotherapy by the administration of 40 kinds of anticancer substances, but most of these treatments are limited to early cancer patients or certain cancers. Thus, cancer deaths are on the rise, and new levels of anti-cancer drugs are needed.

Recently, the Ras gene, first discovered as a cancer-expressing gene for Harvey (HaSV) and Kirsten (Ki) rat sarcoma viruses, is found in more than 30% of all human cancer cells, including 90% of pancreatic cancer and 50% of colorectal cancer. It is becoming. The Ras protein found in these cancer cells is found to be in a modified form unlike the Ras cells of normal cells. If the gas can be prevented from being modified, it is expected to be able to treat more than 30% of human cancers.

The Ras protein has a function similar to that of G-protein, which is known to be bound to the plasma membrane of the cell and to be combined with GTP (guanine triphosphate) in order to be activated. However, the Ras protein itself has a special lipophilic group that can interact with the plasma membrane. Since the Ras protein must have a lipophilic group to bind to the plasma membrane, it must undergo a certain modification process. In other words, the C-terminus of the Ras protein is CA1A2X (where C means cystine, A means aliphatic amino acids and X means all kinds of amino acids), and the action of farnesyl protein transferase. The panesyl group of the farnesyl pyrophosphate is bound to the C-terminal cystine, followed by hydrolysis of the three C-terminal amino acids and the C-terminal carboxyl group ester. Converted to a methyl ester compound.

The modified Ras protein binds strongly to the plasma membrane and continuously transmits a signal to the nucleus in the cell. As a result, the cells grow abnormally and ultimately develop into cancer cells. Therefore, if there is a substance that inhibits the panesylation of the Ras protein, it can be used as a new level of anticancer agent because it can prevent the normal Ras protein is modified to cause cancer cells.

Accordingly, the present inventors have been conducting research to develop inhibitors of panesyl protein transferase from edible resources, which have been secured for a long time, and use cinnamon to form new cinnamic derivatives different from the known cinnamic aldehyde compounds. It was found that the compound can inhibit the expression of cancer genes by inhibiting the action of panesyl transferase and thus preventing panesylation of Ras protein, thus completing the present invention.

It is therefore an object of the present invention to provide a novel cinnamic derivative compound that inhibits the action of farnesyl transferase and a method for extracting it from cinnamon.

Another object of the present invention is to provide a derivative synthesized from the compound and a method for synthesizing it.

Another object of the present invention is to provide a pharmaceutical composition containing the above compounds, which is useful as an anticancer agent by inhibiting the expression of a cancer gene.

In order to achieve the above object, the present invention provides a cinnamic aldehyde derivative having the following structural formula (I).

In the present invention, cinnamon is extracted with a mixture of chloroform and acetone, the extract is concentrated and dissolved in a basic aqueous solution, the aqueous solution is acidified and extracted with methylene chloride, and the methylene chloride extract is concentrated to silica gel chromatography and a high-speed liquid. It provides a method for extracting the compound of formula (I) comprising purification by chromatography.

In order to achieve the above another object, the present invention provides a compound having the following structural formula (II).

The present invention also provides a process for preparing the compound of formula (II) comprising reacting the compound of formula (I) with dibutylaluminum hydride in the presence of a tetrahydrofuran solvent.

In order to achieve the above another object of the present invention, the present invention provides a pharmaceutical composition for treating and preventing cancer, containing the compound of formula (I) or (II) as an active ingredient.

Hereinafter, the present invention will be described in detail.

The new cinnamic aldehyde derivative compound of the above formula (I) having a farnesyl transferase inhibitory activity, purified from cinnamon according to the present invention, was named KRIBB-BP007, which was a derivative chemically synthesized by reducing the compound. The compound of (II) was named KRIBB-BP007-1.

The compound KRIBB-BP007 can be obtained by extracting from edible cinnamon. The separation and purification process is as follows. Cinnamon was ground and extracted with a mixture of chloroform and acetone. The extract was concentrated under reduced pressure and extracted with 2% sodium hydroxide solution. The base layer is acidified and reextracted with methylene chloride, and the organic layers containing the active substance are collected and concentrated under reduced pressure. The obtained concentrate was purified using silica gel chromatography and HPLC to obtain compound KRIBB-BP007 having a farnesyl transferase inhibitory activity.

The derivative KRIBB-BP007-1 can be obtained by dissolving KRIBB-BP007 separated from cinnamon as described above in tetrahydrofuran (THF) and reacting with dibutylaluminum hydride to reduce it.

Since the cinnamic aldehyde derivatives of the present invention can inhibit the expression of the Ras cancer gene by inhibiting farnesyl transferase, it can be used as a new level of cancer prevention and treatment.

That is, KRIBB-BP007-1 and derivatives KRIBB-BP007-1 synthesized and purified from cinnamon as described above are purified, capsuleed, powdered, granulated, suspensioned, emulsion or parenteral dosage unit by conventional methods. Dosage forms or formulations may be formulated into multiple dose formulations and used as agents for the treatment and prevention of cancer.

The pharmaceutical composition containing the derivatives of the present invention as an active ingredient can be parenterally administered or orally as desired, 1 to 2 mg of KRIBB-BP007 per 1 kg of body weight per day, and 2 to 3 of KRIBB-BP007-1. It may be administered in an amount of mg. Dosage levels for a particular patient may vary depending on the particular compound to be used, weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion, drug mixing and the severity of the disease.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are only for illustrating the present invention, the present invention is not limited to these.

Example 1

[Isolation and Purification of KRIBB-BP007 from Cinnamon]

2 g of a mixture of chloroform and acetone (1: 1) was added to 200 g of finely ground crab (purchased from Geumsan, Chungcheongnam-do, Korea), and the mixture was left at room temperature for 24 hours, followed by stirring and filtering. The filtrate was collected, concentrated under reduced pressure, and then separated into an aqueous layer and an organic solvent layer by adding 1 L of ethyl acetate and 1 L of 2% sodium hydroxide solution. The separated aqueous layer was extracted three times with 1 L of ethyl acetate to remove the portion dissolved in the organic solvent, and then made acidic (pH 5) with 10% acetic acid solution. The acidified aqueous layer was extracted three times with 1 L of methylene chloride, and the organic solvent layer and the aqueous layer were separated using a separatory funnel. Subsequently, the inhibition rate of enzyme activity of the organic solvent layer and the aqueous layer was measured, and the organic solvent layers identified as containing the active substance were collected and concentrated to dryness under a reduced pressure to obtain a yellowish brown solid substance. The obtained solid was dissolved in methylene chloride and adsorbed onto silica gel (Merck, Art No. 9385), and then subjected to two silica gel column chromatography by changing the ratio of hexane and ethyl acetate from 1: 9 to 6: 4. The active fractions were separated and finally purified by HPLC (column: Ultracarb 10 ODS (250 x 21.2 mm) from Phenomenex): eluent: 80% methanol and 20% water), 100 g of cinnamon Pure KRIBB-BP007 was obtained with a yield of 11 mg sugar.

Example 2

[Synthesis of KRIBB-BP007-1]

20 mg of KRIBB-BP007 separated and purified from cinnamon was placed in a 50 ml round bottom flask, and 20 ml of tetrahydrofuran was added thereto to dissolve. The flask was placed in a dry ice-acetone bath with nitrogen filled to -70 ° C, and then 1 ml of dibutylaluminum hydride (DIBAL, 1M solution in hexane) was slowly added to room temperature. 50 mg of ammonium chloride was added thereto, then transferred to a separating funnel containing 100 ml of ethyl acetate, washed twice with 100 ml of water, and the organic solvent layer was dried using anhydrous sulfate and concentrated under reduced pressure. The concentrated solution was dissolved in methylene chloride and adsorbed onto silica gel (Merck, Art No. 9385), followed by two silica gel column chromatography using hexane and ethyl acetate in a ratio of 2: 8 to 6: 4. KRIBB-BP007-1 was obtained with a yield of 72%.

Example 3

[Structure Analysis of KRIBB-BP007 and KRIBB-BP007-1]

KRIBB-BP007 and KRIBB-BP007-1 obtained as described above were analyzed for UV absorbance using Shimadzu's UV-265 spectrophotometer, and high resolution MS was measured by VG70-SEQ mass spectrometry to determine molecular weight and molecular formula. It was. In addition, ¹ H, ¹³ C, Cozy, and HMQC spectra were obtained using a hex resonator (Bruker 300 MHz, 500 MHz NMR), and the structures were determined by comprehensive analysis of these spectra.

1 is an ultraviolet absorbance spectrum of KRIBB-BP007, a farnesyl transferase inhibitor according to the present invention, FIG. 2 is a hydrogen NMR spectrum of KRIBB-BP007, and FIG. 3 is a carbon NMR spectrum of KRIBB-BP007, 4 is a hydrogen NMR spectrum of KRIBB-BP007-1, a derivative synthesized from KRIBB-BP007.

The physicochemical characteristics of KRIBB-BP007 and KRIBB-BP007-1, which were analyzed as above, are as follows.

Example 4: Panesyl transferase inhibitory activity search

As an enzyme source of farnesyl protein transferase, 100 to 150 g of male Sprague Dawley rat brains were isolated, washed with physiological saline, homogenized, the homogenate was centrifuged and Q Sepharose high-speed column (Q Sepharose Fast Flow column) was used for partial purification.

The activity of the enzyme is H-Panesil Pyropostate ( H-farnesyl pyrophosphate (FPP) as a substrate using a scintillation proximity assay (Scintillation proximity assay, SPA) was measured by a modified method such as Brow (Blow). 10 μl sample solution, 10 μl assay buffer (50 mM Tris-HCl, pH7.5, 25 mM MgCl, 2 mM KCl, 5 mM DTT, 5 mM NaHPO, 0.01% Triton X-100), 20 μl of diluted 3HEPP (N -[1- [N- (NL-α-aspartyl-L-seryl-L-α-aspartyl] -L-propyl] -L-arginine; human 1 gE pentapeptide), 20 μl Biotin-Lamine B Peptide And 40 μl of farnesyl protein transferase and reacted at room temperature for 30 minutes. Then, 150 μl of SPA beads and boad / stop reaction solution were added and the degree of pansylation of biotin-lamin B peptide was measured using a liquid scintillation counter. Activity inhibition of farnesyl transferase was calculated by the following equation, measured in units of CPM (count per minute):

In the above formula, the blank sample is the case where there is no enzyme and inhibitor samples, and the control sample is measured after the reaction under the optimal conditions in the absence of the inhibitor sample.

According to the above method, the concentrations (IC ₅₀ ) of KRIBB-BP007 and KRIBB-BP007-1 of the present invention that inhibit 50% of the activity of farnesyl transferase are shown in Table 2 below.

As described above, the cinnamaldehyde derivative KRIBB-BP007 and the derivative KRIBB-BP007-1 synthesized and purified from cinnamon according to the present invention can inhibit the expression of the Ras cancer gene by inhibiting panesyl transferase. Therefore, it can be usefully used as a cancer prevention and treatment agent.

Claims (3)

Compound KRIBB-BP007 having the formula (I): Cinnamon is extracted with a mixture of chloroform and acetone, the extract is concentrated and dissolved in a basic aqueous solution, the aqueous solution is acidified and extracted with methylene chloroide, and the methylene chloride extract is concentrated by silica gel chromatography and high-performance liquid chromatography. A process for preparing a compound of formula (I) comprising the step of purifying. A pharmaceutical composition for treating or preventing cancer, comprising a compound of formula (I) or a compound of formula (II) as an active ingredient.