KR100288584B1 - Taxane derivatives and its manufacturing method - Google Patents

Abstract

The present invention relates to 13-diacetyl-texkinin I and its preparation which can be used in the preparation of taxane derivatives having anticancer effects.

Description

Taxane derivatives and preparation method thereof

The present invention relates to novel taxene compounds that can be used in the preparation of taxol and other anti-cancer taxane derivatives and methods of producing the same.

Taxol is known to be effective in the treatment of leukemia and cancer and has antitumor effects. Baxtin III, 10-deacetylbaccatin III and 10-diacetyl have similar structures to Taxol. Taxol (10-deacetyltaxol) and cephlomannine are taxan derivatives, among which they can be used as important precursors for use in fac- texel semisynthesis, some with their own and anti-tumor effects.

Methods for obtaining taxol include separation of taxol from the bark and leaves of yew trees, production by tissue culture of plant cells, semi-synthesis method of producing taxol using relatively large amounts of baccatin present in plants. have.

The present invention relates to compounds that can be used as synthetic raw materials or useful intermediates of taxane-based anticancer substances.

When Taxol is produced by plant cell tissue culture, various Taxol derivatives are produced in addition to Taxol. The present inventors purified 13-deacetyl-taxchinin I (13-deacetyl-taxchinin I) from these derivatives to determine the chemical structure and confirmed that it is a new material.

13-Diacetyl-texkinin I is a colorless needle-like crystal with melting point of 216 ° C, Fabmass resultant molecular weight of 692, and molecular formula represented by the following structural formula as C ₃₈ H ₄₄ O ₁₂ .

Wherein Bz is a benzoyl group and Ac is an acetyl group.

The present invention also relates to a method for preparing a compound which can be used as a synthetic raw material or useful intermediate of a taxan-based anticancer substance.

For example, 13-diacetyl-taxkinin I can be isolated from plant cell culture by modifying the method described in Korean Patent Application No. 96-2621 or 96-2622.

Dichloromethane was added to the concentrated methanol extract obtained by adding methanol to the Taxus plant cells, and the mixture was left under reduced pressure. The methanol layer was removed to obtain a dichloromethane extract. Dichloromethane is added in an amount of about 10-50% by volume to the concentrated methanol extract. Both methanol extraction and dichloromethane extraction can be repeated as many times as necessary.

The dichloromethane extract is treated with an adsorbent such as activated carbon or clay to remove impurities such as tar present in the obtained dichloromomecan extract, and then the filtrate obtained is concentrated under reduced pressure.

The obtained dichloromethane concentrate was added to about 500-1500% of hexane in volume ratio to obtain a precipitate, and then the filtrate obtained by filtration was concentrated to obtain a dried product.

The obtained dried material was dissolved in CH ₂ Cl ₂ , loaded on a silica gel column, and eluted sequentially with Ch ₂ Cl ₂ , Ch ₂ Cl ₂ + methanol to obtain a fraction containing 13-diacetyl-taxkinin I.

This fraction is dissolved in 50% methanol and loaded again on an octadecysilica (ODS) column and eluted with methanol in turn to obtain 13-diacetyl-taxkinin I. Pure 13-diacetyl-taxkinin was repeated twice with preparative TLC (Prep-TLC) eluting the 13-diacetyl-taxkinin I fraction with a hexane / ethyl acetate = 2: 1 developing solvent using a silica gel column. Obtain I.

Although the present invention will be described in detail with reference to examples, the scope of the present invention is not limited by these examples.

Example 1.

SYG-1 (Yuseong-gu, Daejeon, Korea) incubated for 14 days in 1500L of B5 medium (Gamborg et al., Exp. Cell Res., 50: 151: 158 (1968)) with 10uM of AgNO3 added Incubated with 500 L of the accession number KCTC 0232BP dated March 14, 1966, in the KCTC affiliated to the Biotechnology Research Institute located in the Biotechnology Research Institute, and added 3% (w / v) sugar with the start of the culture Was incubated at 24 ° C. On day 7 and day 21 after incubation, 1% and 2% maltose were added. After 50 days of incubation, the culture was terminated and centrifuged to separate cells.

Example 2

8 L of methanol was added to 8 kg of plant cells obtained in Example 1, stirred at room temperature for 30 minutes, and filtered to obtain a methanol extract. After this extraction operation was repeated three more times, the obtained methanol extracts were combined and concentrated under reduced pressure at about 30 mmHg and about 35 ° C to obtain a concentrate of about 8 L. About 2 L of dichloromethane was added to 8 L of the obtained concentrate, and stirred at room temperature for about 15 minutes to prevent, and the methanol layer was removed. This extraction operation was repeated two more times. The dichloromethane extract was concentrated completely under reduced pressure at about 30 mmHg and about 35 ° C., and 10 g of the obtained dried material was dissolved in 56 ml of dichloromethane, and then dissolved in clay (active clay, F-1: Japan). g was added, stirred at about 40 ° C. for 20 minutes, and filtered to obtain a filtrate and a filtrate. The filtrate was washed with 285 ml of dichloromethane, and the resulting wash was combined with the previous filtrate and concentrated under reduced pressure to about 100 ml at about 20 mmHg and about 35 ° C. The concentrated solution was slowly added to about 1,000 ml of nucleic acid to obtain a precipitate, followed by leisure to obtain a filtrate. Concentrate the filtrate with a rotary evaporator, take about 7 g of dry matter, dissolve in CH ₂ Cl ₂ , load it on a silica gel column, and add 300 ml of CH ₂ Cl ₂ (fraction 1), and 300 ml of 1% methanol in CH ₂ Cl ₂ (fraction 2). Elution was in turn. Fraction 3 was obtained by elution again with 500 ml of 2% methanol in CH ₂ Cl ₂ , followed by washing with 500 ml of 1% methanol in CH ₂ Cl ₂ again to obtain fraction 4. TLC (5% methanol in developing solvent CH ₂ Cl ₂ ) was repeated three times each of fractions 1-4, and the Rf value on the silica gel plate of 13-diacetyl-texkinin I was about 0.18, and Rf of Taxol. The values were separated from each other as about 0.20. In addition, it was confirmed that all 13-diacetyl-texkinin I transitioned to fraction 3. In addition, 13-diacetyl-texkinin I showed a deep blue violet while taxol showed a light brown color and was easily distinguished when 10% H 2 SO 4 was sprayed onto the developed TLC plate and heated on a hot plate.

TLC of 13-diacetyl-texkinin I and taxol with octadecysilica (ODS) gel plates (developing solvent: 70% methanol), with Rf values of 0.22 and 0.20, respectively, and repeated TLC plates three or four times If so, these two materials were distinguished from each other. Dissolve fraction 3 in 050% methanol and load it on an ODS gel column, eluting sequentially with 200 ml of 50% methanol (fraction 31), 60% methanol (fraction 32), 70% methanol (fraction 33) and 100% methanol (fraction 34). It was. Fractions 31-34 were analyzed by HPLC, respectively, and fractions 32 (320 mg) and fractions 33 (360 mg) contained 11% and 41% of 13-diacetyl-texkinin I, respectively. Fraction 33 was subjected to silica gel column chromatography twice using hexane / ethyl acetate = 2: 1 as the developing solvent to give about 100 mg of 13-diacetyl-texkinin I in the form of white acicular crystals.

The HPLC profile of purified 13-diacetyl-texkinin I is shown in FIG. 1. Purified 13-diacetyl-texkinin I was colorless needle-like crystals with a melting point of 216 占 폚. Fabmass results (FIG. 2) were 692 (C ₃₈ H ₄₄ O ₁₂ ) molecular weight.

1H-NMR and 13C-NMR data measured by dissolving 13-diacetyl-texkinin I in CDCl ₃ are shown in Table 1 and Table 2, respectively.

Reported 11 (15 → 1) -Aveo-Taxane (11 (15 → 1) -abeo-taxane) -based compound (Chi. Chem. Lett. 4, 695 (1993); J. Nat. Prod. 56, 1520 (1993); PC 39, 861 (1995); Tetrahedron 37, 10175 (1995);). Analysis of spectral data revealed that 13-diacetyl-texkinin I was a novel substance that has not been reported.

Example 3

To support the chemical structure analysis of 13-diacetyl-texkinin I, 13-diacetyl-texkinin I was partially taken and acetylated to prepare acetylated 13-diacetyl-texkinin I. That is, 25 mg of 13-diacetyl-texkinin I was dissolved in pyridine, and acetic anhydride (Ac ₂ O) was added thereto, and the mixture was left at room temperature for 12 hours. The reaction nuclei were suspended in excess water and extracted with ethyl acetate. The ethyl acetate layer was concentrated and silica gel column chromatography (developing solvent: hexane / EtOAc = 4: 1) gave 20 mg of acetylated 13-diacetyl-texkinin I. Acetylated 13-diacetyl-texkinin I was dissolved in CDCl ₃ to measure 1H-NMR and 13C-NMR, and spectral data of related 11 (15 → 1) -abeo-taxane-based compounds (Tetrahedron 37, 10175 (1995) ), It was confirmed that the results exactly match the spectral data of Texkinin I acetate.

1 is an HPLC profile of purified 13-diacetyl-texkinin I.

2 is a Fabmass spectrum of 13-diacetyl-texkinin I.

13-Diacetyl-Texkinin I can be used as a synthetic raw material or useful intermediate of taxan-based anticancer substances.

Claims (2)

13-Diacetyl-texkinin represented by the following structural formula Ⅰ. Wherein Bz is a benzoyl group and Ac is an acetyl group. Iii) extracting a sample of the plant of Taxus with methanol to process the methanol extract; Ii) extracting the methanol extract extracted in step 1 with dichloromethane to obtain a dichloromethane extract; V) treating the dichloromethane extract obtained in step 2 with a synthetic adsorbent and filtering to obtain a filtrate; Iii) adding the filtrate obtained in step 3 to hexane and filtering to obtain a filtrate; V) silica gel chromatography of the filtrate obtained in step 4, wherein 13-diacetyl-texkinin I is prepared.