CN1169802C - Corter pseudolaricis acetic acid derivative and its medicinal composite and their antineoplastic and antifungal application - Google Patents

Abstract

The present invention relates to a cortex pseudolaricis acetic acid derivative and a composition thereof, which have antitumor cell activity, antiviral activity and antifungal activity. The compound can be salt which is permitted medically, wherein the dotted lines independently represent omissible bonds, Y respectively and independently represents O or NH, R1 independently represents a methyl group or COXR', wherein X independently represents O or NH, R' independently represents H, an alkyl group, an alkenyl group, an aryl group (the aryl group which is not substituted, or one or two aryl groups which are independently selected from chlorine, bromine, fluorine, a methoxy group and a nitryl aralkyl group), a heterocyclic group, a heterocyclic alkyl group and goniotriol; R3 respectively and independently represents H, an alkyl group, an alkenyl group, an aryl group (the aryl group which is not substituted, or one or two aryl groups which are independently selected from chlorine, bromine, fluorine, a methoxy group and a nitryl aralkyl group), a heterocyclic group, a heterocyclic alkyl group and goniotriol; R2 represents H, an alkyl group, an alkyl acyl group, an aryl alkanoyl group, an aryl acyl group, a heterocyclic acyl group, an alkenyl acyl group and an aryl (the aryl group which is not substituted or one or two aryl groups which are selected from chlorine, bromine, fluorine, a methoxy group and a nitryl aralkyl group) acyl group.

Description

Corter pseudolaricis acetic acid compound and preparation method thereof

Technical field

The invention belongs to medicine, relate to some and have and natural and synthetic corter pseudolaricis acetic acid derivative and drug regimen thereof, its purposes in antitumor, antiviral, anti-mycotic activity pharmacy.

Background technology

Pseudolaricis is Golden Larch Bark, golden larch, Cortex Glyptostrobi Pensilis, wattle skin again, originates to be pinaceae plant golden larch Pseudolarixamabilis (Nelson) Regd.[P.kaempferi Gord] root skin and near root bark.Pseudolaricis suffering, temperature.Poisonous, Anti-Puritic and insecticidal.See the supplementary Amplifications of the Compendium of Materia Medica of Qing Dynasty's Zhao Xuemin the earliest, be meant the skin of the rose of Sharon that produce in area in addition, Sichuan, be used for the treatment of ringworm.Pseudolaricis develops into antifungal drug since the fifties, is mainly used in treatment trick tinea, neurodermatitis, eczema, leprosy dysentery first-class (Chinese medicinal materials, 1994.02.15; 17 (2): 15 ~ 19).Native rose of Sharon acid wherein, have the common pathomycete effect of China that suppresses (Chinese journal of dermatology, 1960,8:18).Afterwards, through silica gel column chromatography further separate pseudolaric acid (1), corter pseudolaricis acetic acid (2) and native rose of Sharon propionic acid (3) etc.Wherein corter pseudolaricis acetic acid (pseudolaric acid B) is a main component, molecular formula C ₂₃H ₃₀O ₈, structural formula is 2. (chemical journal, 1982,40,447). and pseudolaric acid glycosides and corter pseudolaricis acetic acid glycosides are still arranged in addition.Pharmacological research shows that native rose of Sharon total acid has the effect that suppresses the common pathomycete of China.

R ₁ R ₂ R ₃

1. CH ₃ COCH ₃ H

2. COOCH ₃ COCH ₃ H

3. COOH COCH ₃ H

4. COOH H H

5. CH ₃ H H

6. CH ₃ COCH ₃ CH ₃

7. COOCH ₃ H CH ₃

8. CH ₃ H CH ₃

At present use the more antifungal antibiotic that has clinically, read mycin etc. as grisovin, nystatin, amphotericin B, gram; The antimycotic azole drug of salicylic acid such as Whitfield's ointment, salicylic amide and chemosynthesis is as clotrimazole, miconazole, econazole, KETOKONAZOL, Yi Kang azoles, fluconazole etc.Wherein especially rapidly with the azole drug development, oxime health azoles, fenticonazole, health azoles, Travogyn, bifonazole etc. successively occur, synthesized lipophilic novel triazole antifungal agent in addition, but how said medicine exists toxicity or the bigger shortcoming of pungency to some extent, limited its clinical application, therefore, people have turned to natural product with sight.

Summary of the invention

The objective of the invention is to overcome deficiency of the prior art, a kind of corter pseudolaricis acetic acid compound antimycotic, antitumor action that has is provided;

Another object of the present invention provides a kind of preparation method of corter pseudolaricis acetic acid compound.

Technical scheme of the present invention is summarized as follows:

A kind of corter pseudolaricis acetic acid compound, represent with following structural formula:

A kind of preparation method of corter pseudolaricis acetic acid compound, it comprises the steps:

(1) with pseudolaricis meal 95% ethanol percolation, makes medicinal extract;

(2), obtain extract after boiling off solvent with ether or benzene extraction;

(3) post silica gel column chromatography is collected 7: 3 sherwood oil-ether flow points, gets corter pseudolaricis acetic acid, and at sherwood oil-ether

Middle recrystallization gets corter pseudolaricis acetic acid;

(4) corter pseudolaricis acetic acid with 0.35g is dissolved in the anhydrous methylene chloride of 100ml, add successively 2.4g furfuryl alcohol,

1.9g 1.3-bicyclohexane base carbon two imido and the 4-N-dimethyl aminopyridine of catalytic amount, room temperature

Stirred 24 hours, through diatomite filtration, steaming desolventizes, and silica gel column chromatography obtains a kind of structure that obtains

Formula is

Compound, that is: corter pseudolaricis acetic acid furans-2 methyl esters.

Can use the alkali salify that pharmaceutically is suitable for.The manufacture method of the salt that pharmaceutically allows of The compounds of this invention with use will be in the Synthetic Organic Chemistry field normally used method carry out the composition that forms after the appropriate combination.

The present invention also comprises the pharmaceutical composition of an application formula (I), or the pharmaceutical composition of multiple drug excipient.Specifically, as can basic solution to containing the solution salify of free The compounds of this invention.

Medicament form when The compounds of this invention is used as anti-tumor agent or antifungal preparation can be selected various forms, for example tablet, capsule, powder, granular preparation, liquid preparation etc., medicament form when The compounds of this invention is used as oral anti-tumor agent or antifungal preparation, can select various forms, for example tablet, capsule, powder, granular preparation, liquid preparation etc.

The compounds of this invention as non-oral formulation etc., can be selected various forms such as injection, the creme of external application, suppository, liquid preparation etc.The figuration preparation generally contains 0.1～100 weight %, preferably contains the effective constituent of 1～100 weight %.

The actual ideal dosage of The compounds of this invention can be determined according to the kind of the composition of the kind of the compound that uses, cooperation, suitable frequency and the position and the patient of treatable disease.

Embodiment

The compounds of this invention can extract in the following manner, separates, and is synthetic.

Embodiment 1: corter pseudolaricis acetic acid furans-2 methyl esters synthetic

Pseudolaricis 10kg is ground into meal, uses 95% ethanol percolation, and gained medicinal extract is with ether or benzene extraction, boil off behind the solvent the 1.6kg extract.Through the post silica gel column chromatography, collect 7: 3 sherwood oil-ether flow points, get Pseudolarix acid B 7.2g through column chromatography repeatedly, recrystallization gets column crystal m.p.127.3-128.5 ℃ (document 127-128 ℃) and mixture A (main component is a pseudolaricis formic acid) in sherwood oil-ether.IR (KBr) cm ^-1: 3550 (COOH), 2950,1700 (C=O), 1220. corter pseudolaricis acetic acid 0.35g (0.9mmol) are dissolved in the anhydrous CH of 100ml ₂Cl ₂In, add furfuryl alcohol 2.4g (24mmol) successively, DCC (1.3-bicyclohexane base carbon two imido) 1.9g (30mmol) and catalytic amount DMAP (4-N-dimethyl aminopyridine), stirring at room 24h.Reactant is through diatomite filtration, and steaming desolventizes, silica gel column chromatography (ether: sherwood oil 1: 1), colorless solid corter pseudolaricis acetic acid furans-2-methyl esters 0.29g, recrystallization in yield 68.85%. ether-sherwood oil, colourless cylindrulite, mp.117～118 ℃.

¹HNMR (300MHz, CDCl ₃) δ ppm:1.62 (3H, s, C ₁₁Me); 1.95 (3H, d, J=1.2Hz, C ₁₆Me); 2.12 (3H, s, OCOCH ₃); 1.62-2.12,2.61-3.28 (11H, m, CH); 3.71 (3H, s, C ₁₉OCH ₃); 5.13 (2H, s, C '-H ₂O); 5.86 (1H, d, J=15.9Hz, C ₁₃-H), 6.35 (1H, d, J=2.1, C ' ₄-H), 6.35 (1H, d, J ₁=2.1, C ' ₆-H) 6.51 (1H, dd, J=3.6 15.9Hz, C ₁₄-H), 7.14-7.19 (2H, m, C ₈-H; C ₁₅-H 3 ', 5 '-H), 7.42 (1H, d, d J=0.6,0.6Hz, 5 '-H) .C ₂₈H ₃₂O ₉[α] ¹⁶ _D-35.1 (EtOH, c 0.11), ultimate analysis: M:512.55.Calculated value %:C 65.61 H 6.29; Measured value %:C, the H solvability: dissolve in organic solvents such as methyl alcohol, dimethyl sulfoxide (DMSO), water-soluble.

The difference of acidity, neutrality, alkaline matter: alkaline matter.

Thin-layer chromatography Rf value: 0.67, launch solvent: ether: sherwood oil (2: 1).

A kind of corter pseudolaricis acetic acid compound of the present invention is promptly: corter pseudolaricis acetic acid furans-2-methyl esters is using as anti-tumor agent or anti-virus formulation or antifungal preparation.

Experimental example 1

Material RPMI1640 substratum is a GIBCO company product, and Hoechst33342 and propidium iodide (PI) are Sigma company product, and foetal calf serum is that Inst. of Hematology, Chinese Academy of Medical Sciences produces.Agarose and ethidium bromide (EB) are Sigma company product, and cell line k562 clone is presented by consonance medical university basis, and routine is incubated in the RPMI1640 substratum of 10% heat-inactivated fetal bovine serum 37 ℃, 5%CO ₂Incubation 24h goes down to posterity, take the logarithm vegetative period cell be used for the experiment.

The apoptotic experimental group cell of inducing is respectively through 1 * 10 ^-4Mol/L, 1 * 10 ^-5Mol/L, 1 * 10 ^-6Mol/L, 1 * 10 ^-7The mol/L corter pseudolaricis acetic acid is handled, and other establishes control group and does not add corter pseudolaricis acetic acid continuation cultivation 30h, and collecting cell is made apoptotic cell and detected.

Detect the apoptosis ratio with the two methods of dying of Hoechst33342/PI fluorescence.

The DNA electrophoretic analysis collects 2 * 10 ⁶Individual cell, DNA is extracted in the PBS washing according to a conventional method, is dissolved among the 30ulTE; 1% sepharose (containing the pyridine of 0.5ug/ml bromination second), voltage 50v behind the electrophoresis 4h, takes pictures under the ultraviolet lamp.

Being inverted light microscope observation K562 cell is suspension growth, and cell is rounded, and corter pseudolaricis acetic acid is 1 * 10 ^-4M～1 * 10 ^-6The concentration range innerlich anwenden 19h of M, the visible cell volume-diminished, cell becomes ellipse, and is shaft-like, dumbbell shape, the cell endoparticle increases, and cell density descends, and has film bag apoptotic body to form.

Fluorescence microscope is through the two methods of dying of fluorescence, and visible cellular control unit nuclear uniformity is dyed blue look fluorescence by hoechst 33342.Behind the cultured continuously 40h, as seen there are 40% above nucleus chromatic agglutination, limit to move, a plurality of chromatic agglutination corpusculums occur, dyed blue look fluorescence by hoechst33342.

The mtt assay measurement result the results are shown in shown in (table 1).The different concns corter pseudolaricis acetic acid is handled cell 48h, obviously suppresses the growth of K562 cell, and inhibiting rate is the concentration dependence, its IC ₅₀Value is 5 * 10 ^-5M.

Inhibiting rate behind the table 1 corter pseudolaricis acetic acid effect K562 cell 48h

Group drug level (mol/L) A ₅₇₀Inhibiting rate (%)

Blank group 0.359 ± 0.019

Control group 1.430 ± 0.062

Experimental group 5 * 10 ^-40.580+0.044* 79.37

1×10 ^-4 0.743±0.081* 64.15

5×10 ^-5 0.794±0.105* 59.38

1×10 ^-5 1.019±0.089* 38.38

5×10 ^-6 1.170±0.052* 24.28

1×10 ^-6 1.179±0.069* 23.44

5×10 ^-7 1.286±0.094 13.45

1×10 ^-7 1.330±0.098 9.34

* compare P＜0.01. with control group

It is nucleic acid molecule by endonuclease at " the DNA ladder " that be cut into about 180bp between the nucleosome and the dna fragmentation of multiple is formed that the DNA agarose gel electrophoresis is analyzed one of typical biochemical change of apoptotic cell.1 * 10 ^-5M and 1 * 10 ^-6Two kinds of concentration corter pseudolaricis acetic acids of M are handled the K562 cell, occur when 40h " DNA ladder " clearly.

Experimental example 2

The reference literature method becomes 1 * 10 with the logarithmic phase cell dilution ⁴Cell/ml is inoculated in 96 well culture plates immediately, and the 0.1ml/ hole adds the substratum that contains different concns then in experimental port, parallel 3 holes of every concentration, and control group adds equal-volume and holds agent, puts 37 ℃ of CO ₂Incubator was cultivated 96 hours, it is centrifugal that (1000rpm 20min), abandons supernatant liquor, every hole adds the serum free medium of the freshly prepared 0.2mg/ml MTT of 0.2ml, continue to cultivate 4 hours for 37 ℃, centrifugal again, incline serum after, 0.2ml DEIMSO dissolving MTT first hairpin precipitation, with miniature ultrasonic oscillator concussion 5min mixing, on MR700 type microplate reader, measure the optical density(OD) at 570nm place, calculate and suppress growth of tumour cell rate IC ₅₀Corter pseudolaricis acetic acid furans-2 methyl esters is 3.12 * 10 to the inhibiting rate of A2780 ^-5Mol/L, K562 cell inhibiting rate is 7.15 * 10 ^-5Mol/L.

Experimental example 3

The anti-mycotic activity of corter pseudolaricis acetic acid furans-2 methyl esters

Trichophyton (BMU-16467), and alpha fungus (BMU-16466, ATCC-28185), Candida albicans (BMU-16443, ATCC-11006)

1. experimental technique

Test with reference to tube dilution method.

2 results

Bacterial strain MIC (mg/ml) MFC (mg/ml)

Trichophyton (BMU-16467) 18.8 22.6

Alpha fungus (BMU-16466, ATCC-28185) 45.3 49.5

Candida albicans (BMU-16443, ATCC-11006) 73.6 95.2

Claims (2)

1. corter pseudolaricis acetic acid compound is characterized in that it represents with following structural formula:

2. the preparation method of a corter pseudolaricis acetic acid compound is characterized in that it comprises the steps:

(1) with pseudolaricis meal 95% ethanol percolation, makes medicinal extract;

(2), obtain extract after boiling off solvent with ether or benzene extraction;

(3) post silica gel column chromatography is collected 7: 3 sherwood oil-ether flow points, get corter pseudolaricis acetic acid, and recrystallization gets corter pseudolaricis acetic acid in sherwood oil-ether;

(4) corter pseudolaricis acetic acid with 0.35g is dissolved in the anhydrous methylene chloride of 100ml, add the furfuryl alcohol of 2.4g, 1.3-bicyclohexane base carbon two imido of 1.9g and the 4-N-dimethyl aminopyridine of catalytic amount successively, stirring at room 24 hours, through diatomite filtration, steaming desolventizes, silica gel column chromatography obtains structural formula and is

Compound.