CN110218219A

CN110218219A - A kind of diketopiperazine compound, extracting method and its application

Info

Publication number: CN110218219A
Application number: CN201810173776.1A
Authority: CN
Inventors: 邹忠梅; 王孟华; 丁刚
Original assignee: Institute of Medicinal Plant Development of CAMS and PUMC
Current assignee: Institute of Medicinal Plant Development of CAMS and PUMC
Priority date: 2018-03-02
Filing date: 2018-03-02
Publication date: 2019-09-10

Abstract

The present invention provides a kind of diketopiperazine compound containing sulphur bridge, finds that the compound has light sensitivity for the first time, easily decomposing variation is other compounds, has extremely strong anti-tumor activity；The present invention also provides a kind of extracting methods of light sensitivity diketopiperazine compound, the extraction of special extracting method has been respectively adopted, column chromatographic elution and the preparation method being protected from light under processing, the sterling of compound is prepared suitable for rapid, high volume, purity is higher than 95%, can save 6 months under the conditions of low-temperature dark.

Description

A kind of diketopiperazine compound, extracting method and its application

Technical field

The invention belongs to field of plant extraction and pharmaceutical field, in particular to a kind of diketopiperazine compound and its extraction side Method.

Background technique

ETPs (Epipolythiodioxopiperazines), i.e., more sulphur bridge diketopiperazine Alkaloids, are by fungi The unique secondary metabolite with diketopiperazine parent nucleus and sulphur bridge structure of raw one kind.1936, in fungus Trichoderma It was found that first ETP class compound gliotoxin, has reported nearly 200 constituents so far.Studies have shown that ETP class It closes object common manifestation and goes out significant antitumor, antimycotic and immunosuppressive activity, and sulphur bridge is the important work of ETP class compound Property group.Because its extensively due to significant bioactivity and complicated and special structure, ETP constituents have been a concern, to it Always temperature does not subtract the fully synthetic trial of the further investigation of pharmacological activity mechanism, the explaination of Biosynthetic pathway, and chemistry. 2009, Movassaghi etc. reported ETP class compound (+) -11, and 11 '-dideoxyverticillin A's is fully synthetic (Science, 2009,324,238-241), then, chaetocin C, 12,12 '-dideoxychetracin A etc. ETP class Compound is also by success de novo formation.And for classical compound chetomin, there is not success since nineteen forty-four finds for the first time Fully synthetic report.

In vivo, ETP class compound is condensed by different amino acid precursors, not according to amino acid precursor With and each segment connection type, all ETP class compounds can be divided into 14 classes (Nat.Prod.Rep.2014,31, 1376-1404), the ETP of dimer type has 4 classes, including chetomin class, chaetocin class, verticillin class and Leptosin class, wherein the two of chetomin class compound monomer is connected to dimeric forms by C-N key, forms unique tool There is the dimer ETP class compound of asymmetry, and other three classes compounds are keyed by C-C into symmetrical dimer.Through Literature survey is crossed inventors have found that the dimer ETP class compound of symmetry all has relatively rich sulfide linkage compositional model, packet S2-S2 (referring in dimeric structure top half and lower half portion sulphur bridge that sulphur atom number is respectively 2 and 2, similarly hereinafter) is included, S2-S3, S3-S2, S3-S3, S4-S2, S4-S3, S4-S4 etc., and chetomin class only reports S2-S2 at present (J.Bacteriol.1944,48,527-530) and S2-S3 (J.Nat.Prod.2004,67,98-102) the two dimers Ingredient, immanent cause have not been reported.

It is retrieved and is found according to the prior art, there was only the two chemical combination of S2-S2 and S2-S3 in chetomin class compound at present Object, other compounds can not carry out chemical synthesis, rely only on biological extraction, and current data is shown, without higher number Amount sulphur bridge compound by biology extraction separate, wherein the technical issues of or technical difficulty it is very big, this greatlys restrict this Class compound is applied to the preparation field and its clinical application of anti-tumor drug.

Summary of the invention

In conclusion due to the structure-activity of chetomin class compound, industry and relatively rich sulphur bridge is clinically needed Class (Sn, n > 2) diketopiperazine compound, and found in the technical research of inventor's early period, when the quantity of sulphur bridge increases, This compound will become unstable, be easy to resolve into other compounds under light illumination, therefore such compound is with photosensitive Property.Explain 70 for many years chetomin class compound isolate and purify make slow progress, the immanent cause of fully synthetic failure.Hair Bright people passes through long felt, has obtained a kind of diketopiperazine compound using unique extracting method in some microorganisms, and two Ketone diethylenediamine compound has significant photosensitivity, i.e., photosensitive class diketopiperazine compound has general structure I structure, molecule Formula is C₃₁H₃₀N₆O₆S_xS_y, the value range of the x is the integer of 3-5, and the value range of y is the integer of 2-5

Above compound discloses special nature of the more sulphur bridge compounds of chetomin class to light sensitive, specific table for the first time Now the ingredient to be present in medicinal extract will not change, once obtaining monomer component, quickly become other homologues under light illumination. The present invention provides the whole process protection strategies of monomeric compound preparation, can quickly prepare the more sulphur of chetomin class of high-purity Endo compound.

The compound indicated in the form of Sx-Sy in the present invention indicates top half and the lower half portion of diketopiperazine parent nucleus Sulphur bridge in respectively contain x and y sulphur atom.Only when x and y are 2, compound can be stabilized, as long as x or y Greater than 2, then compound becomes unstable state, easily changes.It therefore, just can not passing through as more sulphur bridge Sn, n > 2 Synthesis mode is learned to be synthesized, at present also without document or patent to present in it animals and plants or extracting method report, because This can not synthesize and extract at present more sulphur bridge class compounds of chetomin.

And in the present invention, in above compound, further preferably, in the compound, wherein x is that 3 or 4, y is selected from The integer of 2-4.

In above compound, further preferably, in the compound, when wherein x is 3, y is selected from 2 and 3；Or when x is 4 When, y is the integer of 2-4.

In above compound, further preferably, in the compound, when wherein x is 3, y is selected from 2；Or when x is 4, Y is the integer of 2-4.

Further, a kind of extracting method of photosensitive class diketopiperazine compound is inventor provided, wherein the method It comprises the steps of:

1) it chooses Chaetomium fungi and carries out solid medium culture, obtain tunning；

2) the step 1) tunning is added water-free organic extraction solvent, extracting mode be extracted under room temperature or Heating and refluxing extraction, recycling design obtain total extract, the water-free organic extraction solvent be ethyl acetate, acetone, Methanol, ethyl alcohol, methylene chloride or volume ratio 1:0.3-3 methylene chloride/methanol mixed solvent one kind；

3) water is added in the step 2) total extract to be diluted, is then extracted with organic solvent, obtained corresponding Active component, the organic solvent are that one or more of mixing of ethyl acetate, hexamethylene, n-hexane or methylene chloride are molten Agent；

4) step 3) the methylene chloride active component is subjected to silica gel absorption, the silicagel column after the absorption carries out organic Mixed solvent gradient elution obtains respective components in conjunction with tlc analysis, and organic mixed solvent is petroleum ether-acetic acid second Ester, cyclohexane-ethyl acetate, cyclohexane-acetone, n-hexane-ethyl acetate, n-hexane-acetone or petroleum ether-acetone are mixed One kind of bonding solvent；

5) step 4) respective components are subjected to chromatography preparation and solvent is evaporated, it is molten that the respective components carry out chromatography methanol Solution is centrifuged, and then filtering, sample introduction is isolated and purified with preparative liquid chromatography, chromatography preparation process and solvent are evaporated It is protected from light, obtains the photosensitive class diketopiperazine compound.

All solvent inventions have carried out associated extraction experiment per capita in the above method, obtain the photosensitive class two Ketone diethylenediamine compound.

Inventor further provides a kind of extracting method, the method specifically:

1) it chooses Chaetomium fungi and carries out solid medium culture, be formulated as rice medium, formula rate is rice 60- 70g, distilled water 80-100mL；Or classical potato dextrose agar culture, it is formulated as potato 200g, Portugal Grape sugar 20g, agar 15-20g, distilled water 1000mL obtain tunning；

2) the step 1) tunning is added to the methylene chloride/methanol mixed solvent of volume ratio 1:0.3-3, under room temperature Soak extraction perhaps ultrasonic extraction or heating and refluxing extraction under room temperature, are extracted three times, recycling design is always extracted altogether Object；

3) water is added according to weight ratio 1:5-15 in the step 2) total extract to be diluted, then with isometric two Chloromethanes is extracted, and active component is obtained；

4) the step 3) active component is subjected to silica gel absorption, the silicagel column after the absorption carries out petroleum ether-acetone Gradient elution obtains respective components in conjunction with tlc analysis；

5) step 4) respective components are subjected to chromatography preparation and solvent is evaporated, it is molten that the respective components carry out chromatography methanol Solution is centrifuged, filtering, sample introduction, and liquid phase chromatogram condition is 65%-75% acetonitrile/25%-35% water, and chromatographic column is reverse phase silica gel half Column, flow velocity 2mL/min are prepared, chromatography preparation process and solvent are evaporated and are protected from light, and obtain the photosensitive class diketone Diethylenediamine compound.

In the studies above, the diketopiperazine compound polarity very little containing sulphur bridge, therefore water-containing organic solvent is to extraction two Ketone piperazines constituents extraction rate is low, thus uses water-free ethyl acetate, acetone, methanol, ethyl alcohol, methylene chloride or body Methylene chloride/methanol mixed solvent of the product than 1:0.3-3 extracts, and this extracting mode is after multi-solvents screen Obtained result.

Methylene chloride/methanol mixed solvent is added in 1:0.3-3 by volume, i.e. methylene chloride/methanol mixed solvent is hair The 1:0.3-3 of ferment bulk product.Other ratios in the present invention are also herewith managed.

In above-mentioned compound extracting method, wherein the preferred Chaetomium cochliodes of Chaetomium fungi, Chaetomium seminudum and Chaetomium globosum, preferably Chaetomium cochliodes.

Further, in above-mentioned compound extracting method, wherein the petroleum ether-ethyl acetate, hexamethylene-acetic acid The gradient elution volume ratio of ethyl ester and n-hexane-ethyl acetate is (1:0,8:1,3:1,1:1 and 0:1), hexamethylene-the third Ketone, n-hexane-acetone and petroleum ether-acetone gradient elution volume ratio are (1:0,10:1,5:1,2:1 and 0:1), often A gradient elution volume merges identical component according to tlc analysis for 3-5 silicagel column volume, obtains respective objects component.

Previous experiments discovery, because of such compound polarity very little, and polarity is close, and column chromatography distribution is concentrated very much, and It is easy and fatty acid ingredient mixes, so commonly used in the methylene chloride-methanol of the big extract of separating polar difference System is not suitable for separating such compound.Found by lab scale, petroleum ether, hexamethylene with or n-hexane combination ethyl acetate or Acetone forms mixed solvent system, can be sufficiently separated open fatty acid and such compound, available after removing fatty acid The good chetomin class compound of discrimination can be directly used for liquid phase preparation.

Wherein petroleum ether-acetone elution volume is (1:0,10:1,5:1,2:1,0:1), and each gradient elution volume is 3-5 A silicagel column volume merges identical component according to tlc analysis, obtains respective objects component, according to separating resulting map ratio Compared with for optimal eluent and gradient.

After the compounds purifying obtains sterling, being exposed to natural lighting lower 5 seconds can change, illumination 30 seconds can Serious variation occurs, entire liquid phase preparation and solvent are evaporated process needs and are strictly protected from light.

Further, in above-mentioned compound extracting method, wherein described to be protected from light processing include being blocked using physical method It isolates and purifies equipment to be protected from light, is integrally protected from light and uses free radical scavenger TEMPO (2,2,6,6- tetramethyl piperidines-using darkroom Nitrogen oxides) inhibit illumination cause variation.

Preparation of compounds need it is very strict be protected from light, physical method is protected from light for liquid phase preparatory phase, and darkroom is whole Body, which is protected from light, to be used to prepare liquid and is evaporated the stage, and free radical scavenger TEMPO can be used for the preservation of pure compounds, and when use passes through Liquid phase process removes TEMPO.

Liquid phase analysis is shown, by taking chetomin A as an example, same sample is equally divided into two parts of A and B, and sample A is not added Variation takes place in natural lighting 5 seconds in TMEPO, and degradation rate about 10%, degradation rate reaches 67% after 30 seconds；Sample B is added TEMPO, without visible degradation after illumination 5 seconds, degradation rate is only 6% after illumination 60 seconds.Measure is protected from light using low temperature (- 20 DEG C), is added After entering TMEPO, sample can long-term preservation at least six moon, purity be higher than 90%.

The present invention still further provides the above-mentioned diketopiperazine compound of one kind in preparation treatment anti-tumor drug Using.

The present invention provides a kind of pharmaceutical composition of diketopiperazine compound, described pharmaceutical composition includes above-mentioned diketone Diethylenediamine compound is active constituent, in addition the preparation that pharmaceutically acceptable auxiliary material is prepared, the preparation is tablet, ball Agent, powder, capsule, emulsion, solution, suspension, injection, drip solution or freeze-dried powder.

The tumour for the treatment of preferably includes: liver cancer, cervical carcinoma or breast cancer.

The invention has the beneficial effects that, it was found that more sulphur bridge chetomin analogs to the special nature of photo-labile, And a whole set of safeguard measure is creatively devised, for isolating and purifying for unstable compound, and then solve from nineteen forty-four Since chetomin has found, more than 70 years problems of Natural Medicine Chemistry and Synthetic Organic Chemistry researcher are perplexed.

The present invention is isolated and purified to more sulphur bridge compounds and other photo-labile compounds with critical guidance Meaning；And biosynthesis research fully synthetic to the chemistry of the constituents has revelatory effect；Due to chetomin class Closing object has anti-tumor activity very outstanding, and the present invention has important impetus to the research and development of antitumor lead compound.

Specifically, preparation method provided by the present invention also has guidance application extensively.

Extracting method of the present invention can be used for isolating and purifying the unstable reactive compound in natural products, and unstability is day The common phenomenon of right product, the reactive compound much from medicinal plant, microorganism etc. is often to temperature, illumination, oxygen etc. Extraneous factor has unstability, such as the main active Ligustilide from Chinese traditional medicine angelica, the antimalarial from artemisia annua Active constituent qinghaosu, the insecticidal constituent pyrethrins etc. from Pyrethrum plant have a degree of unstability, clearly These active constituents could be efficiently used after Chu's compound instable internal cause and external cause.

The present invention can be used for instructing fully synthetic chetomin of chemistry and the like and other more sulphur bridge compounds.

The present invention can be used for instructing the biosynthesis of chetomin and the like to study.

The present invention can be used for instructing chetomin and other contain more sulphur bridge structural compounds grinding for anti-tumor drug Hair.Unique sulphur bridge structure is the key that it with strength anti-tumor activity group in ETP class compound, document report such Ingredient reaches nanomole rank to the inhibiting effect of a variety of human tumor cells, solve more sulphur bridge structures instability problem be into One step deeply develops the important prerequisite of the antitumor lead compound of ETP class.

The present invention can be used for instructing chetomin and other contain more sulphur bridge structural compounds grinding for anti-tumor drug Hair.Unique sulphur bridge structure is the key that it with strength anti-tumor activity group in ETP class compound, solves more sulphur bridge knots The instability problem of structure is the important prerequisite for further deeply developing the antitumor lead compound of ETP class.

The present invention has obtained the noval chemical compound chetomin A (C containing more sulphur bridges from Chaetomium fungi₃₁H₃₀N₆O₆S₆)、 chetomin B(C₃₁H₃₀N₆O₆S₇)、chetomin C(C₃₁H₃₀N₆O₆S₈)、chetomin D(C₃₁H₃₀N₆O₆S₅) and chetomin E (C₃₁H₃₀N₆O₆S₆), by high resolution mass spectrum, a peacekeeping two dimensional NMR, which detects, determines its correct structure, and it was found that on Stating compound has anti-tumor activity much higher than positive drug, suitable for the research of anti-tumor activity lead compound and anti-swollen The preparation of tumor medicine.

More sulphur bridge diketopiperazine Alkaloids provided by the invention are a kind of unique fungal secondary metabolites, have spy Different dimeric structure and sulphur bridge structure, and there is completely new Anticancer Effect and Mechanism, it is expected to become a new class of antitumor elder generation Lead compound.In addition, compound of the present invention also provides reference for chemical synthesis related compound, can use In instructing design and research and development of the field of medicinal chemistry to antitumoral compounds.

The experimental procedure and experimental data not being described in detail in the present invention can according to the knowledge of those of ordinary skill in the art and Experience is adjusted, or is obtained with reference to relevant technical manual, and this field routine operation is belonged to.

Specific embodiment

The following examples are only used for further illustrating invention but are not limited to the present invention.It is all real based on invention above content institute Existing technology belongs to the scope of the invention.

Embodiment 1

The fermentation of Chaetomium fungi Chaetomium cochliodes

1) strain source

Fungi is Chaetomium fungi Chaetomium cochliodes

2) it ferments

Prepare the solid medium of fungi fermentation first, specific formula is that rice 60g is added in 500mL triangular flask, is steamed Distilled water 80mL, 121 DEG C spare after high pressure sterilization 30 minutes；PDA culture medium will be grown in, and (PDA is potato dextrose agar training Support base fungus block youngster expand is inoculated and is fermented on rice medium for the amplification of strain) on be in logarithmic phase Fungi be cut into small pieces, be inoculated on rice medium in superclean bench, 150 bottles of common fermentation.It is stored at room temperature fermentation 30 It.

Embodiment 2

The fermentation of Chaetomium fungi Chaetomium globosum

1) strain source

Fungi is Chaetomium fungi Chaetomium globosum

2) it ferments

Preparing the solid medium of fungi fermentation first, specific formula is potato dextrose agar culture, Formula is potato 200g, glucose 20g, agar 15-20g, distilled water 1000mL, the standby in 30 minutes of 121 DEG C of high pressure sterilizations With；Fungi in logarithmic phase is cut into small pieces, is inoculated into potato dextrose agar in superclean bench On, 100 bottles of common fermentation.It is stored at room temperature fermentation 30 days.

Embodiment 3

The preparation of the thick fraction of diketopiperazine in Chaetomium fungi Chaetomium cochliodes

After the completion of strain fermentation in embodiment 1, solid fermentation product is obtained, dose volume is than the dichloromethane for 1:0.5 Alkane/methanol mixed solvent impregnates solid fermentation object with the mixed solvent according to tunning and mixed solvent volume ratio 1:3, It extracts three times, recycling design obtains total extract 65g (medicinal extract), and total medicinal extract adds water 650mL to disperse the (weight ratio of total medicinal extract and water It for 1:10), is extracted 3 times with isometric methylene chloride, collects methylene chloride position, recycling design obtains methylene chloride extraction Object.The medicinal extract of ethyl acetate extract is uniformly mixed on silica gel (60-100 mesh), with the common column chromatography silica gel of 200-300 purpose (sample: silica gel weight ratio is 1:20) is separated, and (conventional column chromatography silica gel includes a variety of mesh numbers, there is 60-100 mesh, 100- The silica gel granularity of 200 mesh, 200-300 mesh and 300-400 mesh, different meshes is different, and mesh number is bigger, and granularity is thinner, separation effect Fruit is better, but dead absorption also can be more, and separation flow velocity is slower.Usually mix sample silica gel low mesh number, the higher mesh of separation silica gel Number).With petroleum ether-acetone (1:0,10:1,5:1,2:1,0:1) gradient elution, in conjunction with thin layer, (thin layer prefabricated board, solvent are Petroleum ether-acetone hybrid system, volume ratio 5:1-1:1) it analyzes, 3-5 column volume of each gradient elution there are after merging To 8 subfractions (Fr-A, Fr-B, Fr-C, Fr-D, Fr-E, Fr-F, Fr-G, Fr-H, according to gradient sequence, the subfraction Sequentially to flow out), wherein component Fr-C, Fr-D, Fr-E is the target components to further prepare purifying.

Embodiment 4

The preparation of the thick fraction of diketopiperazine in Chaetomium fungi Chaetomium globosum

After the completion of strain fermentation in embodiment 2, solid fermentation product is obtained, according to tunning and mixed solvent volume Than 1:3, solid fermentation object is impregnated with ethyl acetate, three times, recycling design obtains total extract 45g (leaching to ultrasonic extraction under room temperature Cream), total medicinal extract adds water 675mL dispersion (weight ratio of total medicinal extract and water is 1:15), it is extracted 3 times with isometric methylene chloride, Methylene chloride position is collected, recycling design obtains dichloromethane extract.The medicinal extract at methylene chloride position is uniformly mixed in silica gel On (60-100 mesh), separated with the common column chromatography silica gel of 200-300 purpose (sample: silica gel weight ratio is 1:20).With ring Hexane-ethyl acetate (1:0,8:1,3:1,1:1 and 0:1) gradient elution, in conjunction with thin layer, (thin layer prefabricated board, solvent are hexamethylene Alkane-ethyl acetate hybrid system, volume ratio 3:1-0:1) it analyzes, 3-5 column volume of each gradient elution there are after merging To 8 subfractions (Fr-A, Fr-B, Fr-C, Fr-D, Fr-E, Fr-F, Fr-G, Fr-H, according to gradient sequence, the subfraction Sequentially to flow out), wherein component Fr-C, Fr-D, Fr-E is the target components to further prepare purifying.

Embodiment 5

The preparation of compound liquid phase preparation

Several teat glass (8mm × 18mm or other specifications) are integrally wound into package, nozzle with opaque masking foil Upwards, it is put into common biological ice box, ice covering test tube is filled it up with into ice chest, and (the top where test tube mouth can not cover, and protect Demonstrate,prove nozzle and be higher than ice face), then entire ice chest is wrapped up with masking foil, is placed in sample preparation liquid in light protected environment.It takes common One piece of the syringe needle of syringe, is connected to the liquid outlet of semi-preparative liquid chromatogram, is fixed with sealed membrane, adhesive tape or other materials, Ensure no leakage when liquid phase operation.

Embodiment 6

Compound chetomin A liquid phase preparation flow

Compound liquid phase preparation flow is carried out according to embodiment 5, and obtained component Fr-D is in embodiment 3 or 4 with chromatography methanol Dissolution, centrifugation are crossed after 0.22 μm of filter membrane for use, and liquid phase chromatogram condition is -35% water of 65% acetonitrile, collects t_R26.9min's Chromatographic peak, the syringe needle that liquid phase liquid outlet will be connected to when collection pierce through masking foil so that preparation solution flow into masking foil package, It is placed in the test tube in ice chest.After the completion of collection, it is evaporated preparation solution in dark indoor Rotary Evaporators, obtains monomeric compound Chetomin A (S4-S2) (purity is higher than 95%).It is evaporated after sample and will be evaporated the round-bottomed flask tinfoil paper of sample in darkroom Paper integrally wraps up, and is put into -20 DEG C of refrigerators and is kept in dark place, and takes out partially dried sample for nuclear-magnetism and mass spectrometric measurement.

The structure of compound chetomin A is identified by high resolution mass spectrum and one-dimensional and two dimensional NMR means.

Chetomin A: white powder, HR-ESI-MS (m/z 797.0447 [M+Na]⁺, calcd.797.0449)；

Nuclear magnetic resonance method (1D and 2D NMR) further confirms that the compound structure:¹H-NMR (500MHz, CDCl₃)δ 3.16 (3H, s, CH₃- 2), 4.37 (1H, m, CH₂OH-3), 4.27 (1H, m, CH₂OH-3), 3.49 (1H, m, CH₂OH-3), 6.10 (1H, s, H-5), 5.48 (1H, s, H-6), 6.81 (1H, d, J=7.9Hz, H-7), 7.26 (1H, m, H-8), 6.86 (1H, t, J=7.5Hz, H-9), 7.10 (1H, d, J=7.7Hz, H-10), 4.34 (1H, d, J=16.1Hz, H-11a), 3.18 (1H, d, J=16.1Hz, H-11b), 3.05 (3H, s, CH₃- 2'), 4.48 (1H, dd, J=10.7,4.5Hz, CH₂OH-3'), 4.07 (1H, dd, J=10.7,3.9Hz, CH₂OH-3'), 4.34 (1H, m, CH₂OH-3'), 3.17 (3H, s, CH₃- 5'), 4.14 (1H, d, J=16.1Hz, H-7'), 3.43 (1H, d, J=16.1Hz, H-7'), 7.28 (1H, s, H-9'), 7.35 (1H, dd, J =6.4,2,9Hz, H-11'), 7.13 (1H, m, H-12'), 7.13 (1H, m, H-13'), 7.56 (1H, d, J=6.2,2.9Hz, H-14')。

¹³C-NMR (125MHz, CDCl₃) δ 166.2 (C-1), 27.6 (CH₃- 2), 75.7 (C-3), 60.3 (CH₂OH-3), 162.8 (C-4), 80.2 (C-5), 148.0 (C-6a), 110.7 (C-7), 131.1 (C-8), 120.7 (C-9), 124.2 (C- 10), 127.0 (C-10a), 73.6 (C-10b), 43.5 (C-11), 73.3 (C-11a), 167.5 (C-1'), 29.2 (CH₃- 2'), 77.9 (C-3'), 62.4 (CH₂OH-3'), 167.9 (C-4'), 29.5 (CH₃- 5'), 77.3 (C-6'), 30.2 (C-7'), 108.9 (C-8'), 122.2 (C-9'), 134.8 (C-10a'), 111.4 (C-11'), 120.3 (C-12'), 123.1 (C- 13'), 118.9 (C-14'), 129.2 (C-14a').

Liquid-phase chromatographic analysis condition: sample room temperature: 20 DEG C, column temperature: 35 DEG C, chromatographic column: Acquity UPLC BEH C18 (2.1 × 100mm, 1.7 μm, Waters Corp., Milford, USA), mobile phase: acetonitrile/water, chromatographic process: 40% Acetonitrile/60% water, flow velocity: 0.3mL/min.

Show that Chetomin A can change for 5 seconds under natural lighting through liquid-phase chromatographic analysis, degradation 10%, Have occurred and that serious variation, degradation reach 67% after 30 seconds.

Embodiment 7

The preparation of compound chetomin B and C in embodiment 3 or 4

Compound liquid phase preparation flow is carried out according to embodiment 5, and embodiment 3 and 4 obtained component C of embodiment are with chromatography methanol Dissolution, liquid phase chromatogram condition are -25% water of 75% acetonitrile, collect t_RThe chromatographic peak of 18.8min obtains single after being evaporated in darkroom Body compound chetomin B (S4-S3) (purity is higher than 95%)；Collect t_RThe chromatographic peak of 20.6min, is evaporated in darkroom Obtain monomeric compound chetomin C (S4-S4) afterwards (purity is higher than 95%).

By high resolution mass spectrum and one-dimensional and two dimensional NMR means identify compound chetomin B and The structure of chetomin C.

Chetomin B: white powder, HR-ESI-MS (m/z 829.0168 [M+Na]⁺, calcd.829.0169)；

Nuclear magnetic resonance method further confirms that the compound structure:¹H-NMR (500MHz, CDCl₃) δ 3.28 (3H, s, CH₃- 2), 4.30 (1H, dd, J=13.0,3.9Hz, CH₂OH-3), 3.98 (1H, dd, J=13.6,5.0Hz, CH₂OH-3), 3.98 (1H, m, CH₂OH-3), 6.05 (1H, s, H-5), 5.57 (1H, s, H-6), 6.86 (1H, d, J=7.9Hz, H-7), 7.25 (1H, m, H-8), 6.78 (1H, t, J=7.6Hz, H-9), 7.00 (1H, d, J=7.5Hz, H-10), 4.06 (1H, m, H- 11a), 3.39 (1H, d, J=14.3Hz, H-11b), 3.15 (3H, s, CH₃- 2'), 4.60 (1H, dd, J=10.4,3.8Hz, CH₂OH-3'), 4.15 (1H, m, CH₂OH-3'), 4.04 (1H, m, CH₂OH-3'), 3.18 (3H, s, CH₃- 5'), 4.18 (1H, M, H-7'), 3.47 (1H, d, J=15.9Hz, H-7'), 7.28 (1H, s, H-9'), 7.45 (1H, dd, J=6.3,2,6Hz, H- 11'), 7.13 (1H, m, H-12'), 7.14 (1H, m, H-13'), 7.54 (1H, d, J=6.3,2.6 Hz, H-14').

¹³C-NMR (125MHz, CDCl₃) δ 169.5 (C-1), 28.0 (CH₃- 2), 75.2 (C-3), 62.0 (CH₂OH-3), 165.0 (C-4), 79.5 (C-5), 149.4 (C-6a), 110.9 (C-7), 131.6 (C-8), 120.6 (C-9), 124.5 (C- 10), 125.9 (C-10a), 71.7 (C-10b), 50.1 (C-11), 77.9 (C-11a), 167.6 (C-1'), 29.5 (CH₃- 2'), 78.0 (C-3'), 62.5 (CH₂OH-3'), 167.9 (C-4'), 29.4 (CH₃- 5'), 76.6 (C-6'), 30.4 (C-7'), 109.1 (C-8'), 121.9 (C-9'), 135.1 (C-10a'), 111.5 (C-11'), 120.3 (C-12'), 123.2 (C- 13'), 118.8 (C-14'), 129.1 (C-14a').

Chetomin C: white powder, HR-ESI-MS (m/z 860.9902 [M+Na]⁺, calcd.860.9890)；

Nuclear magnetic resonance method further confirms that the compound structure:

¹H-NMR (500MHz, CDCl₃) δ 3.11 (3H, s, CH₃- 2), 4.40 (1H, m, CH₂OH-3), 3.93 (1H, m, CH₂OH-3), 4.55 (1H, m, CH₂OH-3), 6.22 (1H, s, H-5), 5.25 (1H, s, H-6), 6.68 (1H, d, J=7.8Hz, H-7), 7.22 (1H, m, H-8), 6.82 (1H, t, J=7.6Hz, H-9), 7.20 (1H, m, H-10), 3.93 (1H, m, H- 11a), 3.33 (1H, d, J=14.5Hz, H-11b), 3.00 (3H, s, CH₃- 2'), 4.37 (1H, m, CH₂OH-3'), 4.03 (1H, d, J=14.1Hz, CH₂OH-3'), 4.26 (1H, m, CH₂OH-3'), 3.12 (3H, s, CH₃- 5'), 4.10 (1H, d, J= 15.6Hz, H-7'), 3.41 (1H, d, J=15.6Hz, H-7'), 7.12 (1H, s, H-9'), 7.08 (1H, m, H-11'), 7.06 (1H, m, H-12'), 7.02 (1H, t, J=7.6Hz, H-13'), 7.59 (1H, d, J=7.8Hz, H-14').

¹³C-NMR (125MHz, CDCl₃) δ 167.6 (C-1), 29.0 (CH₃- 2), 79.2 (C-3), 61.9 (CH₂OH-3), 166.9 (C-4), 82.4 (C-5), 148.6 (C-6a), 110.0 (C-7), 131.4 (C-8), 120.4 (C-9), 124.9 (C- 10), 127.4 (C-10a), 72.5 (C-10b), 48.3 (C-11), 74.5 (C-11a), 168.0 (C-1'), 29.4 (CH₃- 2'), 78.0 (C-3'), 62.4 (CH₂OH-3'), 168.8 (C-4'), 29.8 (CH₃- 5'), 77.3 (C-6'), 30.2 (C-7'), 108.8 (C-8'), 123.1 (C-9'), 134.9 (C-10a'), 111.6 (C-11'), 120.5 (C-12'), 123.2 (C- 13'), 119.2 (C-14'), 129.3 (C-14a').

Liquid-phase chromatographic analysis condition: sample room temperature: 20 DEG C, column temperature: 35 DEG C, chromatographic column: Acquity UPLC BEH C18 (2.1 × 100mm, 1.7 μm, Waters Corp., Milford, USA), mobile phase: acetonitrile/water, chromatographic process: 40% Acetonitrile/60% water, flow velocity: 0.3mL/min

Show that Chetomin B can change for 5 seconds under natural lighting through liquid-phase chromatographic analysis, degradation 15%, Have occurred and that serious variation, degradation reach 55% after 30 seconds.Chetomin C can change for 5 seconds under natural lighting, Have occurred and that serious variation, degradation reach 58% after degradation 15%, 30 seconds.

Embodiment 8

The preparation of compound chetomin D in embodiment 3 or 4

Compound liquid phase preparation flow is carried out according to embodiment 5, and embodiment 3 or 4 obtained component Fr-E are molten with chromatography methanol Solution, liquid phase chromatogram condition is -35% water of 65% acetonitrile, as shown in following liquid chromatogram, collects t_R16.7min chromatography Peak obtains monomeric compound chetomin D (S3-S2) after being evaporated in darkroom (purity is higher than 95%).

The structure of compound chetomin D is identified by high resolution mass spectrum and one-dimensional and two dimensional NMR means.

Chetomin D: white powder, HR-ESI-MS (m/z 765.0728 [M+Na]⁺, calcd.765.0728)；

Nuclear magnetic resonance method further confirms that the compound structure:¹H-NMR (500MHz, CD₃OD) 3.23 (3H, s, CH of δ₃- 2), 4.43 (1H, d, J=12.6Hz, CH₂OH-3), 4.32 (1H, d, J=12.6Hz, CH₂OH-3), 6.17 (1H, s, H-5), 6.76 (1H, dd, J=8.0,3.9Hz, H-7), 7.25 (1H, m, H-8), 6.90 (1H, m, H-9), 7.33 (1H, m, H-10), 4.27 (1H, d, J=15.3Hz, H-11a), 3.19 (1H, d, J=15.3Hz, H-11b), 3.09 (3H, s, CH₃- 2'), 4.53 (1H, d, J=12.1Hz, CH₂OH-3'), 3.94 (1H, d, J=12.0Hz, CH₂OH-3'), 3.32 (3H, s, CH₃- 5'), 3.95 (1H, d, J=16.0Hz, H-7'), 3.45 (1H, d, J=16.0Hz, H-7'), 6.88 (1H, s, H-9'), 7.38 (1H, M, H-11'), 7.19 (1H, m, H-12'), 7.17 (1H, m, H-13'), 7.69 (1H, d, J=7.1Hz, H-14').

¹³C-NMR (125MHz, CD₃OD) δ 167.7 (C-1), 28.3 (CH₃- 2), 78.4 (C-3), 60.4 (CH₂OH-3), 163.5 (C-4), 81.6 (C-5), 150.7 (C-6a), 111.9 (C-7), 132.3 (C-8), 120.7 (C-9), 126.3 (C- 10), 128.1 (C-10a), 75.3 (C-10b), 43.3 (C-11), 77.9 (C-11a), 168.5 (C-1'), 29.7 (CH₃- 2'), 78.3 (C-3'), 62.8 (CH₂OH-3'), 169.0 (C-4'), 29.0 (CH₃- 5'), 77.8 (C-6'), 31.2 (C-7'), 109.7 (C-8'), 126.3 (C-9'), 135.5 (C-10a'), 112.4 (C-11'), 123.6 (C-12'), 121.4 (C- 13'), 120.4 (C-14'), 131.4 (C-14a').

Show that Chetomin D can change for 5 seconds under natural lighting through liquid-phase chromatographic analysis, degradation 10%, Have occurred and that serious variation, degradation reach 69% after 30 seconds.

Embodiment 9

The preparation of compound chetomin E

Compound liquid phase preparation flow is carried out according to embodiment 5, and obtained component Fr-E is in embodiment 3 or 4 with chromatography methanol Dissolution, liquid phase chromatogram condition are -35% water of 65% acetonitrile, collect t_RThe chromatographic peak of 20.0min obtains single after being evaporated in darkroom Body compound chetomin E (S3-S3) (purity is higher than 95%).

Chetomin E: white powder, HR-ESI-MS (m/z 797.0452 [M+Na]⁺,calcd.797.0449)；

It is C that compound molecule formula, which can be obtained, according to the accurate molecular weight that high resolution mass spectrum provides₃₁H₃₀N₆O₆S₆, according to The cleavage of mass spectrum rule of chetomin class compound, can speculate in conjunction with the fragment ion m/z 396,380 occurred in mass spectrogram The top half of chetomin E contains three sulfide linkages, and three sulfide linkages are contained in lower half portion.

Nuclear magnetic resonance method further confirms that the compound structure:¹H-NMR (500MHz, CD₃OD) 3.33 (3H, s, CH of δ₃- 2), 4.36 (1H, d, J=12.3Hz, CH₂OH-3), 3.94 (1H, m, CH₂OH-3), 6.12 (1H, d, J=4.8Hz, H-5), 6.82 (1H, m, H-7), 7.25 (1H, t, J=8.2Hz, H-8), 6.80 (1H, m, H-9), 7.19 (1H, m, H-10), 4.02 (1H, d, J=14.3Hz, H-11a), 3.28 (1H, d, J=14.3Hz, H-11b), 3.13 (3H, s, CH₃- 2'), 4.56 (1H, D, J=12.1Hz, CH₂OH-3'), 3.88 (1H, m, CH₂OH-3'), 3.10 (3H, s, CH₃- 5'), 3.97 (1H, m, H-7'), 3.45 (1H, d, J=15.4Hz, H-7'), 6.91 (1H, s, H-9'), 7.48 (1H, d, J=8.0 Hz, H-11'), 7.18 (1H, M, H-12'), 7.14 (1H, m, H-13'), 7.66 (1H, d, J=7.0Hz, H-14').

¹³C-NMR (125MHz, CD₃OD) δ 170.5 (C-1), 28.3 (CH₃- 2), 81.2 (C-3), 61.7 (CH₂OH-3), 165.3 (C-4), 80.1 (C-5), 152.4 (C-6a), 112.0 (C-7), 132.7 (C-8), 120.5 (C-9), 126.5 (C- 10), 127.2 (C-10a), 73.5 (C-10b), 49.5 (C-11), 80.0 (C-11a), 168.6 (C-1'), 29.5 (CH₃- 2'), 80.7 (C-3'), 62.7 (CH₂OH-3'), 167.8 (C-4'), 30.1 (CH₃- 5'), 77.9 (C-6'), 31.2 (C-7'), 109.9 (C-8'), 126.1 (C-9'), 135.8 (C-10a'), 112.6 (C-11'), 123.7 (C-12'), 121.3 (C- 13'), 120.2 (C-14'), 131.2 (C-14a').

Show that Chetomin E can change for 5 seconds under natural lighting through liquid-phase chromatographic analysis, degradation 9%, 30 Have occurred and that serious variation, degradation reach 62% after second.

Embodiment 10

Antitumor activity of compound tests (mtt assay)

Mtt assay principle: i.e. tetrazolium salts colorimetric test is a kind of method for detecting cell survival.Tetrazolium salts (trade name thiazole Orchid, abbreviation MTT) it is a kind of dyestuff that can receive hydrogen, in living cells, the succinate dehydrogenase on mitochondria can make external source MTT It is reduced to bluish violet crystallization first a ceremonial jade-ladle, used in libation (Formazan) of indissoluble and is deposited in cell, but dead cell is without this function.Dimethyl is sub- Sulfone (DMSO) can dissolve the bluish violet in cell and crystallize and have absorption maximum in 570nm, pass through the light absorption value at measurement 570nm Can indirect reaction living cells quantity, thus available mtt assay evaluation drug is to the inhibiting effect of Living cells proliferation.

Tumor cell line: human hepatoma cell strain (HepG2), Breast cancer lines (MCF-7) and human cervical carcinoma cell lines (HeLa)

Experimental procedure: it is thin to be inoculated in 96 holes according to the density in 5000-10000/hole for the tumour cell of logarithmic growth phase On born of the same parents' culture plate, at 37 DEG C, 5%CO₂Under the conditions of cultivate 24 hours after be added sample to be tested, each sample sets 5 concentration ladders Degree 10^-4, 10^-5, 10^-6, 10^-7With 10^-8Mol/L, each concentration set three in parallel, and every hole adds 2 μ L of sample solution or blank solution. The MTT solution that 10 μ L concentration are 5mg/mL is added in every hole after culture 48 hours, continues culture 4 hours, discards supernatant liquid, every hole The DMSO of 100 μ L is added, after crystal is completely dissolved, measures absorbance OD value of each hole at 570nm with microplate reader, presses Cytostatic to tumor cell rate is calculated according to following formula, and is pressed down with Logit method with the half that pharmacology statistical software calculates sample Concentration IC processed₅₀Value.

Cell proliferation inhibition rate=(OD_{Negative control cell mean}-OD_{Sample cell mean})÷OD_{Negative control cell mean}× 100%

Test result is shown in Table

Inhibited proliferation of 1 compound of table to three plants of human tumor cells

Experimental result: as shown in Table 1, compound chetomin A~E in general formula I of the present invention is to three plants of human tumours Cell has extremely strong inhibitory activity, and compared with positive drug cis-platinum, inhibiting effect improves about 1000 times, has reached nanomole Rank.In addition, the compound inhibiting effect containing more sulphur bridges is better than disulfide-stabilized conjunction object chetomin, illustrate that sulphur bridge is The crucial pharmacophoric group of such compound, and the more long acting sulphur bridge the stronger.

The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously Limitations on the scope of the patent of the present invention therefore cannot be interpreted as, it is noted that come for those skilled in the art It says, without departing from the inventive concept of the premise, several deformations and transformation can also be made, these belong to guarantor of the invention Range is protected, therefore, the scope of protection of the patent of the present invention is determined by the appended claims.

Claims

1. a kind of diketopiperazine compound containing sulphur bridge, which is characterized in that the diketopiperazine compound has significant photosensitive Perception has general structure I structure, molecular formula C₃₁H₃₀N₆O₆S_xS_y, molecular formula C₃₁H₃₀N₆O₆S_xS_y, the value model of the x The integer for 3-5 is enclosed, the value range of y is the integer of 2-5

2. compound according to claim 1, which is characterized in that wherein x is 3 or 4, and y is selected from the integer of 2-4.

3. compound according to claim 1, which is characterized in that when wherein x is 3, y is selected from 2 and 3；Or when x is 4, y For the integer of 2-4.

4. compound according to claim 1, which is characterized in that when wherein x is 3, y is selected from 2；Or when x is 4, y 2- 4 integer.

5. a kind of extracting method of diketopiperazine compound, which is characterized in that the method comprises the steps of:

2) water-free organic extraction solvent is added in the step 1) tunning, extracting mode is to extract or heat under room temperature Refluxing extraction, recycling design obtain total extract, the water-free organic extraction solvent be ethyl acetate, acetone, methanol, One kind of the methylene chloride/methanol mixed solvent of ethyl alcohol, methylene chloride or volume ratio 1:0.3-3；

3) water is added in the step 2) total extract to be diluted, is then extracted with organic solvent, obtained corresponding effective Position, the organic solvent are one or more of mixed solvents of ethyl acetate, hexamethylene, n-hexane or methylene chloride；

4) step 3) the methylene chloride active component is subjected to silica gel absorption, the silicagel column after the absorption carries out organic mixing Solvent gradient elution obtains respective components in conjunction with tlc analysis, and organic mixed solvent is petroleum ether-ethyl acetate, hexamethylene Alkane-ethyl acetate, cyclohexane-acetone, n-hexane-ethyl acetate, n-hexane-acetone or petroleum ether-acetone mixed solvent It is a kind of；

5) step 4) respective components being subjected to chromatography preparation and solvent is evaporated, the respective components carry out the dissolution of chromatography methanol, from Then the heart, filtering, sample introduction are isolated and purified with preparative liquid chromatography, chromatography preparation process and solvent are evaporated and are kept away Light processing obtains the photosensitive class diketopiperazine compound.

6. extracting method according to claim 5, which is characterized in that the Chaetomium fungi can be selected from Chaetomium Cochliodes, Chaetomium seminudum and Chaetomium globosum, preferably Chaetomium cochliodes。

7. extracting method according to claim 5, which is characterized in that the method comprises the steps of:

1) it chooses Chaetomium fungi and carries out solid medium culture, be formulated as rice medium, formula rate is rice 60-70g, Distilled water 80-100mL；Or classical potato dextrose agar culture, it is formulated as potato 200g, glucose 20g, agar 15-20g, distilled water 1000mL obtain tunning；

2) the step 1) tunning is added to the methylene chloride/methanol mixed solvent of volume ratio 1:0.3-3, is impregnated under room temperature Ultrasonic extraction or heating and refluxing extraction under perhaps room temperature are extracted, is extracted three times altogether, recycling design obtains total extract；

3) water is added according to weight ratio 1:5-15 in the step 2) total extract to be diluted, then with isometric dichloromethane Alkane is extracted, and active component is obtained；

5) step 4) respective components being subjected to chromatography preparation and solvent is evaporated, the respective components carry out the dissolution of chromatography methanol, from The heart, filtering, sample introduction, liquid phase chromatogram condition are 65%-75% acetonitrile/25%-35% water, and chromatographic column is prepared by reverse phase silica gel half Column, flow velocity 2mL/min, chromatography preparation process and solvent are evaporated and are protected from light, and obtain the photosensitive class diketopiperazine Compound.

8. compound extracting method according to claim 7, which is characterized in that the gradient volume of the petroleum ether-acetone Ratio is 1:0-0:1, and each gradient elution volume is that 3-5 silicagel column volume merges identical component, obtain according to tlc analysis To respective objects component.

9. compound extracting method according to claim 5, which is characterized in that described to be protected from light processing including using physics side Method, which is blocked, isolates and purifies equipment, is integrally protected from light using darkroom and inhibits the pair of illumination initiation anti-using TEMPO free radical scavenger It answers.

10. a kind of application of any one of claim 1-4 diketopiperazine compound in preparation treatment anti-tumor drug.