Conjugated enyne compound, preparation method and application thereof, pharmaceutical composition and application thereof

Abstract

The invention provides a conjugated enyne compound, a preparation method and application thereof, a pharmaceutical composition and application thereof, and relates to the technical field of biological medicines. The conjugated enyne compound provided by the invention has the structural characteristics that the positions 5 and 6 and the positions 9 and 10 are acetylenic bonds, the C-12 position is substituted with acetylcysteine residues, the conjugated enyne compound is a derivative of endiyne biosynthesis precursor 1, 3, 5, 7, 9, 11, 13-pentadecene, the biosynthesis route of endiyne relates to an actinomycete mercaptan (MSH) detoxification mechanism, and the conjugated enyne compound can be used for preparing an actinomycete thiol compound. It is revealed that an MSH-dependent enzymatic detoxification mechanism participates in biosynthesis of endiyne, plays an important role in antibiotic resistance and environmental adaptation of endiyne strains, and also brings brand new reference and thinking for analysis of biosynthesis pathways of endiyne. In addition, the conjugated enyne compound provided by the invention has good anti-tumor activity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a conjugated enediyne compound and a preparation method and application thereof, a pharmaceutical composition and application thereof. BACKGROUND

[0002] Enediyne antibiotics isolated from actinomycetes are considered to be the most active anti-tumor antibiotics so far. The biosynthetic mechanism of the enediyne core nine-membered ring and ten-membered ring skeleton has been a hot and difficult point of research. Through comparative analysis of the reported enediyne gene clusters, it is found that five conserved genes may be responsible for the synthesis of the core ring, including polyketide synthase (PKSE), thioester hydrolase (E10) and three conserved proteins (E3, E4, E5). Researchers have obtained fifteen-carbon conjugated heptene or various enone derivatives thereof by heterologous expression or in vitro catalysis of polyketide synthase PKSE and thioester hydrolase E10, which are considered to be the key precursors of enediyne ring formation, responsible for the formation of the enediyne carbon chain skeleton. However, in addition to polyketide synthase E and thioester hydrolase E10, the functions of other candidate genes (E3, E4, E5) responsible for the biosynthesis of the core ring are unknown, and there is a lack of related intermediates, which hinders the speculation and research of the biosynthesis of the enediyne core part. Therefore, it is of great significance to explore related enediyne precursor structures from actinomycete strains with enediyne biosynthetic gene clusters for the analysis of their biosynthetic pathways. SUMMARY

[0003] Therefore, the purpose of the present application is to provide a conjugated enediyne compound and a preparation method and application thereof, a pharmaceutical composition and application thereof. The conjugated enediyne compound provided by the present application provides technical support and a new synthesis route for the biosynthesis of enediyne as an intermediate.

[0004] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0005] The present application provides a conjugated enediyne compound, which has the structure shown in formula I or formula II:

[0006]

[0007] The present application also provides a preparation method of the conjugated enediyne compound according to the above technical solutions, which comprises the following steps:

[0008] The fermentation broth of Micromonospora eburnea is subjected to solid-liquid separation to obtain fermentation bacteria and fermentation supernatant, respectively;

[0009] extracting the fermentation supernatant with an extractant to obtain an organic extract phase, removing the solvent in the organic extract phase to obtain the M. eburnea fermentation product; the extractant comprises one or more of ester solvents and halogenated hydrocarbons; the extraction is performed under light-avoiding conditions;

[0010] subjecting the M. eburnea fermentation product to solid phase extraction column separation to obtain component Fr. 7; the solid phase extraction column separation conditions comprise: the stationary phase comprises normal phase silica gel; the elution mode is gradient elution, and the eluent used in the gradient elution comprises n-hexane, ethyl acetate and methanol, and the volume ratio of the n-hexane, ethyl acetate and methanol is 20:1:0-0:0:1;

[0011] subjecting the component Fr. 7 to semi-preparative HPLC separation to obtain the conjugated enediyne compound having the structure shown in Formula I or Formula II; the semi-preparative HPLC separation conditions comprise: the chromatographic column comprises a C18 chromatographic column; the mobile phase system comprises mobile phase A and mobile phase B, the mobile phase A is 0.05-0.2 v / v% formic acid aqueous solution, and the mobile phase B is acetonitrile; the elution mode is gradient elution; the gradient elution program is as follows: 0-5 min, the volume fraction of the mobile phase B is linearly increased from 8-12% to 18-22%; 5-35 min, the volume fraction of the mobile phase B is linearly increased from 18-22% to 65-75%; 35-45 min, the volume fraction of the mobile phase B is linearly increased from 65-75% to 85-92%; 45-46 min, the volume fraction of the mobile phase B is linearly increased from 85-92% to 95-100%; 46-60 min, the volume fraction of the mobile phase B is 95-100%; 60-61 min, the volume fraction of the mobile phase A is linearly decreased from 95-100% to 8-12%; 61-70 min, the volume fraction of the mobile phase B is 8-12%.

[0012] Preferably, the M. eburnea fermentation product further comprises a fermentation cell extract, and the fermentation cell extract is obtained by extracting the fermentation cell with an extraction agent; the extraction agent comprises one or more of lower alcohols, ketone solvents, ester solvents and nitrile solvents; the extraction is performed under light-avoiding conditions.

[0013] Preferably, the lower alcohols comprise at least one of methanol and ethanol;

[0014] The ketone solvents comprise at least one of acetone and butanone;

[0015] The ester solvents comprise at least one of ethyl acetate, ethyl formate, ethyl butyrate and propyl acetate;

[0016] The nitrile solvents comprise acetonitrile;

[0017] The halogenated hydrocarbon includes at least one of chloroform, dichloromethane and dichloroethane.

[0018] Preferably, in the gradient elution process of the solid phase extraction column separation, the volume ratio of n-hexane, ethyl acetate and methanol is 20:1:0, 5:1:0, 3:1:0, 1:1:0, 0:1:0, 0:20:1, 0:10:1, 0:5:1 and 0:0:1 in turn.

[0019] Preferably, the column temperature of the semi-preparative HPLC separation is 20-30 DEG C, the flow rate of the mobile phase is 2-5 mL / min, the detector is a diode array detector, and the detection wavelength is 200-600 nm.

[0020] The application also provides application of the conjugated enyne compound as an intermediate for enediyne biosynthesis.

[0021] The application also provides a derivative of the conjugated enyne compound, which includes a pharmaceutically acceptable salt, a hydrate, a solvate and a crystal form.

[0022] The application also provides a pharmaceutical composition, which includes an active component and a pharmaceutically acceptable adjuvant, and the active component includes one or more of the conjugated enyne compound and the derivative of the conjugated enyne compound.

[0023] The application also provides application of the conjugated enyne compound, the derivative of the conjugated enyne compound or the pharmaceutical composition in preparation of an antitumor drug or an antibiotic drug.

[0024] The conjugated enyne compound provided by the application is a natural alkynyl compound, and formula I and formula II are Δ 11,12The double bond E / Z configuration isomer, which is characterized in that the 5, 6 and 9, 10 positions are acetylene bonds, and the C-12 position is substituted with an acetylcysteine residue, is a derivative (downstream modification product) of enediyne biosynthetic precursor 1, 3, 5, 7, 9, 11, 13-pentadecapentaene, and the biosynthetic pathway of the enediyne involves a mycothiol (MSH) detoxification mechanism: the conjugated enediyne precursor is conjugated with the thiol group of MSH to produce a detoxification product, and after removing one molecule of myo-inositol and glucosamine, it is extruded to the outside of the cell to obtain a conjugated enediyne compound with the structures shown in formula I and formula II. The conjugated enediyne compound provided by the application first discloses that the MSH-dependent enzymatic detoxification mechanism is involved in the biosynthesis of enediyne, which plays an important role in the antibiotic resistance and environmental adaptation of enediyne strains, and also brings new ideas and thoughts for the analysis of the biosynthetic pathway of enediyne. Moreover, the conjugated enediyne compound provided by the application has good antitumor activity, and has good application prospect in the preparation of antitumor drugs and antibiotic drugs.

[0025] The preparation method of the conjugated enediyne compound provided by the application is simple in process, simple in operation, green and environmentally friendly, low in production cost, and suitable for industrialized production. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 UV spectrum of compound 1 (formula I);

[0027] Figure 2 HR-ESI-MS spectrum of compound 1;

[0028] Figure 3 H-NMR spectrum of compound 1; 1 H-NMR spectrum of compound 1;

[0029] Figure 4 C-NMR spectrum of compound 1; 13 C-NMR spectrum of compound 1;

[0030] Figure 5 DEPT spectrum of compound 1;

[0031] Figure 6 HSQC spectrum of compound 1;

[0032] Figure 7 HMBC spectrum of compound 1;

[0033] Figure 8 H- 1 H- 1 HCOSY spectrum of compound 1;

[0034] Figure 9 ROESY spectrum of compound 1;

[0035] Figure 10 UV spectrum of compound 2 (formula II);

[0036] Figure 11 HR-ESI-MS spectrum of compound 2;

[0037] Figure 12 H-NMR spectrum of compound 2; 1 H-NMR spectrum of compound 2;

[0038] Figure 13 C-NMR spectrum of compound 2; 13 C-NMR spectrum of compound 2;

[0039] Figure 14 DEPT spectrum of compound 2;

[0040] Figure 15 HSQC spectrum of compound 2;

[0041] Figure 16 HMBC spectrum of compound 2;

[0042] Figure 17 H- 1 H- 1 HCOSY spectrum of compound 2;

[0043] Figure 18 ROESY spectrum of compound 2;

[0044] Figure 19 Two-dimensional nuclear magnetic correlation signal of compound 1;

[0045] Figure 20 Two-dimensional nuclear magnetic correlation signal of compound 2;

[0046] Figure 21 3E * ,7E * ,11E * ,13E * ,3'R * -1 configuration in the PCM (methanol) model MPW1PW91 / 6-311+G(d,p) level of linear regression analysis of experimental values and theoretical calculation values;

[0047] Figure 22 3E * ,7E * ,11E * ,13E * ,3'S * -1 configuration in the PCM (methanol) model MPW1PW91 / 6-311+G(d,p) level of linear regression analysis of experimental values and theoretical calculation values;

[0048] Figure 23 Figure 3 is a graph showing the relative deviation of the calculated fit values from the experimental values for the 3E * ,7E * ,11E * ,13E * ,3′R * -1 configuration and 3E * ,7E * ,11E * ,13E * ,3′S * -1 configuration;

[0049] Figure 24 Figure 4 is an HPLC profile of a fermentation broth extract and a fermentation supernatant extract of Micromonospora eburnea;

[0050] Figure 25 Figure 5 is an MS analysis profile of a biosynthesis intermediate MSH-S-toxin complex of compound 1 and compound 2 in a fermentation broth extract of Micromonospora eburnea;

[0051] Figure 26 Figure 6 is an HPLC detection profile of a click chemistry reaction of a fermentation product of Micromonospora eburnea;

[0052] Figure 27 Figure 7 is an LC-MS analysis profile of a cycloaddition product of a click chemistry reaction. DETAILED DESCRIPTION

[0053] The present application provides a conjugated ene-yne compound, having a structure shown in formula I or formula II:

[0054]

[0055] The present application also provides a preparation method of the conjugated ene-yne compound described in the above technical solution, comprising the following steps:

[0056] The fermentation broth of Micromonospora eburnea is subjected to solid-liquid separation to obtain fermentation broth and fermentation supernatant, respectively;

[0057] The fermentation supernatant is extracted by an extractant to obtain an organic extraction phase, and the solvent in the organic extraction phase is removed to obtain a fermentation product of M.eburnea; the extractant includes one or more of ester solvents and halogenated hydrocarbons; the extraction is carried out in the dark;

[0058] The M. eburnea fermentation product is subjected to solid phase extraction column separation to obtain component Fr. 7; the solid phase extraction column separation conditions include: the stationary phase comprises normal phase silica gel; the elution mode is gradient elution, and the eluent used in the gradient elution comprises n-hexane, ethyl acetate and methanol, and the volume ratio of the n-hexane, ethyl acetate and methanol is 20:1:0-0:0:1;

[0059] The component Fr. 7 is subjected to semi-preparative HPLC separation to obtain a conjugated enediyne compound having a structure shown in Formula I or Formula II; the semi-preparative HPLC separation conditions include: the chromatographic column comprises a C18 chromatographic column; the mobile phase system comprises mobile phase A and mobile phase B, the mobile phase A is 0.05-0.2 v / v % formic acid aqueous solution, and the mobile phase B is acetonitrile; the elution mode is gradient elution; and the gradient elution program is as follows: 0-5 min, the volume fraction of the mobile phase B is linearly increased from 8-12 % to 18-22 %; 5-35 min, the volume fraction of the mobile phase B is linearly increased from 18-22 % to 65-75 %; 35-45 min, the volume fraction of the mobile phase B is linearly increased from 65-75 % to 85-92 %; 45-46 min, the volume fraction of the mobile phase B is linearly increased from 85-92 % to 95-100 %; 46-60 min, the volume fraction of the mobile phase B is 95-100 %; 60-61 min, the volume fraction of the mobile phase A is linearly decreased from 95-100 % to 8-12 %; and 61-70 min, the volume fraction of the mobile phase B is 8-12 %.

[0060] Unless otherwise specified, the materials and equipment used in the present application are commercially available in the art.

[0061] In the present application, the fermentation broth of the Micromonospora eburnea is subjected to solid-liquid separation to obtain fermentation bacteria and fermentation supernatant, respectively.

[0062] In the present application, the solid-liquid separation is not particularly limited, and any solid-liquid separation method known to those skilled in the art can be used, such as centrifugation, filtration or suction filtration. In the present application, the rotation speed of the centrifugation is preferably 3000-8000 r / min, and can be specifically 3000 r / min, 4000 r / min, 5000 r / min, 6000 r / min, 7000 r / min or 8000 r / min; and the centrifugation time is preferably 10-15 min, and can be specifically 10 min, 11 min, 12 min, 13 min, 14 min or 15 min.

[0063] In the present application, the preparation method of the fermentation broth of the Micromonospora eburnea preferably comprises the following steps:

[0064] Spores of Micromonospora eburnea are inoculated into a seed culture medium for propagation culture to obtain a seed liquid;

[0065] The seed liquid is inoculated into a fermentation culture medium for fermentation culture to obtain a fermentation product of Micromonospora eburnea.

[0066] In the present application, spores of Micromonospora eburnea are inoculated into a seed culture medium for propagation culture to obtain a seed liquid. In the present application, the seed culture medium preferably comprises a TSB culture medium or a GYM culture medium. In the present application, the volume of the seed culture medium used by a single spore is preferably 20-150 mL, which can be specifically 20 mL, 50 mL, 80 mL, 100 mL, 120 mL or 150 mL. In the present application, the temperature of the propagation culture is preferably 27-30℃, which can be specifically 27℃, 28℃, 29℃ or 30℃; the time of the propagation culture is preferably 12-28 h, which can be specifically 12 h, 15 h, 18 h, 20 h, 22 h, 25 h or 28 h; the propagation culture is preferably carried out under shaking of a shaker, and the rotating speed of the shaker is preferably 150-220 r / min, which can be specifically 150 r / min, 180 r / min, 200 r / min or 220 r / min.

[0067] After obtaining the seed liquid, the seed liquid is inoculated into a fermentation medium for fermentation culture to obtain the fermentation product of Micromonospora eburnea. In the present application, the composition of the fermentation medium preferably comprises: glucose 35-45 g / L, which can be specifically 35 g / L, 38 g / L, 40 g / L, 42 g / L or 45 g / L; acid hydrolysis casein 4-6 g / L, which can be specifically 4 g / L, 4.5 g / L, 5 g / L, 5.5 g / L or 6 g / L; NaCl 4-6 g / L, which can be specifically 4 g / L, 4.5 g / L, 5 g / L, 5.5 g / L or 6 g / L; MgSO4 2-3 g / L, which can be specifically 2 g / L, 2.2 g / L, 2.5 g / L, 2.8 g / L or 3 g / L; K2HPO4 0.8-1.2 g / L, which can be specifically 0.8 g / L, 0.9 g / L, 1 g / L, 1.1 g / L or 1.2 g / L; CaCO3 1.5-2.5 g / L, which can be specifically 1.5 g / L, 1.8 g / L, 2 g / L, 2.2 g / L or 2.5 g / L; CH3COONa 0.2-1 g / L, which can be specifically 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L or 1 g / L; NaI 0.1-1 g / L, which can be specifically 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L or 1 g / L; the solvent is water; the pH value of the fermentation medium is preferably 7-7.4, which can be specifically 7, 7.1, 7.2, 7.3 or 7.4. In the present application, the inoculation amount of the seed liquid is preferably 5-15 v / v%, which can be specifically 5 v / v%, 8 v / v%, 10 v / v%, 12 v / v% or 15 v / v%. In the present application, the temperature of the fermentation culture is preferably 27-30°C, which can be specifically 27°C, 28°C, 29°C or 30°C; the time of the fermentation culture is preferably 5-14 days, which can be specifically 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days; the fermentation culture is preferably carried out under shaking of a shaking table, and the rotating speed of the shaking table is preferably 150-220 r / min, which can be specifically 150 r / min, 180 r / min, 200 r / min or 220 r / min.

[0068] After obtaining the fermentation product of M. eburnea, the present application preferably further comprises detecting the fermentation product of M. eburnea by click chemistry. In the present application, the click chemistry detection preferably comprises mixing the fermentation product of M. eburnea, azide, polar organic solvent and ruthenium-based catalyst, and then detecting by HPLC after click chemistry reaction.

[0069] In the present application, the azide preferably includes one or more of ethyl 2-azidoacetate, azidobenzene, and 1-azido-4-methylbenzene. In the present application, the mass ratio of the M. eburnea fermentation product to the substance amount of ethyl 2-azidoacetate is preferably 1 mg: 0.03 to 0.07 µmol, and can be specifically 1 mg: 0.03 µmol, 1 mg: 0.04 µmol, 1 mg: 0.05 µmol, 1 mg: 0.06 µmol, or 1 mg: 0.07 µmol.

[0070] In the present application, the ruthenium-based catalyst preferably includes one or more of (1,5-cyclooctadiene)(pentamethylcyclopentadienyl)ruthenium(II) chloride (abbreviated as Cp*RuCl), pentamethylcyclopentadienylbis(triphenylphosphine)ruthenium(II) chloride, and chloro(pentamethylcyclopentadienyl)ruthenium(II) tetramer. In the present application, the mass ratio of the M. eburnea fermentation product to the substance amount of (1,5-cyclooctadiene)(pentamethylcyclopentadienyl)ruthenium(II) chloride is preferably 1 mg: 0.0001 to 0.0005 µmol, and can be specifically 1 mg: 0.0001 µmol, 1 mg: 0.0002 µmol, 1 mg: 0.00025 µmol, 1 mg: 0.0003 µmol, 1 mg: 0.0004 µmol, or 1 mg: 0.0005 µmol.

[0071] In the present application, the polar organic solvent preferably includes one or more of acetone, N,N-dimethylformamide, dimethyl sulfoxide, and acetonitrile. In the present application, the mass ratio of the M. eburnea fermentation product to the volume of the polar organic solvent is preferably 1 g: 20 to 100 mL, and can be specifically 1 g: 20 mL, 1 g: 50 mL, 1 g: 80 mL, or 1 g: 100 mL.

[0072] In the present application, the mixing preferably comprises: dissolving the M. eburnea fermentation product in a first portion of the polar organic solvent to obtain a M. eburnea fermentation product solution; dissolving the ethyl 2-azidoacetate in a second portion of the polar organic solvent to obtain an ethyl 2-azidoacetate solution; dissolving the (1,5-cyclooctadiene)(pentamethylcyclopentadienyl)ruthenium(II) chloride in a third portion of the polar organic solvent to obtain a (1,5-cyclooctadiene)(pentamethylcyclopentadienyl)ruthenium(II) chloride solution; mixing the M. eburnea fermentation product solution and the ethyl 2-azidoacetate solution, and then adding the (1,5-cyclooctadiene)(pentamethylcyclopentadienyl)ruthenium(II) chloride solution, and finally adding the remaining polar organic solvent. In the present application, the concentration of the M. eburnea fermentation product solution is preferably 10-50 g / L, and can be specifically 10 g / L, 20 g / L, 30 g / L, 40 g / L or 50 g / L. In the present application, the concentration of the ethyl 2-azidoacetate solution is preferably 2.5-12.5 mM (mmol / L), and can be specifically 5 mM. In the present application, the concentration of the (1,5-cyclooctadiene)(pentamethylcyclopentadienyl)ruthenium(II) chloride solution is preferably 0.0125-0.0625 mM, and can be specifically 0.0125 mM, 0.02 mM, 0.025 mM, 0.03 mM, 0.04 mM, 0.05 mM, 0.06 mM or 0.0625 mM.

[0073] In the present application, the temperature of the click chemistry reaction is preferably 20-30°C, and can be specifically 20°C, 22°C, 25°C, 28°C or 30°C; and the time of the click chemistry reaction is preferably 14-24 h, and can be specifically 14 h, 16 h, 18 h, 20 h, 22 h or 24 h.

[0074] After the completion of the click chemistry reaction, the present application preferably further comprises: concentrating and drying the click chemistry reaction liquid obtained from the click chemistry reaction, and then re-dissolving, and then performing HPLC detection on the obtained click chemistry reaction product solution. The present application does not have special limitations on the concentration and drying, and the solvent can be completely removed. In the present application, the conditions of the HPLC detection are preferably the same as those of the semi-preparative HPLC separation.

[0075] The present application carries out click chemistry detection in the presence of azide 2-azidoacetic acid ethyl ester and ruthenium-based catalyst (1,5-cyclooctadiene) (pentamethylcyclopentadienyl) ruthenium (II) chloride, and the ruthenium-catalyzed click chemistry means can catalyze the cycloaddition reaction of the alkyne group inside the compound with azide to generate 1,4,5-trisubstituted 1,2,3-triazole derivatives, so as to target and explore relevant conjugated ene-yne ingredients from candidate strains with enediyne metabolic potential. The present application verifies the existence of the conjugated ene-yne compounds in the M.eburnea fermentation product through click chemistry detection.

[0076] After obtaining the fermentation supernatant, the present application extracts the fermentation supernatant with an extractant to obtain an organic extraction phase, removes the solvent in the organic extraction phase, and obtains the M.eburnea fermentation product.

[0077] In the present application, the extractant includes one or more of ester solvents and halogenated hydrocarbons, and more preferably ester solvents; the ester solvents preferably include at least one of ethyl acetate, ethyl formate, ethyl butyrate and propyl acetate; and the halogenated hydrocarbons preferably include at least one of chloroform, dichloromethane and dichloroethane. In the present application, the volume ratio of the fermentation supernatant to the extractant for single extraction is preferably 1:0.8-1.2, which can be specifically 1:0.8, 1:0.9, 1:1, 1:1.1 or 1:1.2. In the present application, the number of extractions is preferably 2-4 times, and more preferably 3 times; and the extraction is carried out in the dark.

[0078] The method for removing the solvent in the present application is not particularly limited, and the method for removing the solvent known to those skilled in the art can be used, such as concentration and drying in sequence.

[0079] In the present application, the M.eburnea fermentation product preferably further includes a fermentation bacterial extract, which is preferably obtained by extracting the fermentation bacteria with an extraction agent. Specifically, the fermentation bacteria and the extraction agent are mixed for extraction, solid-liquid separation is carried out to obtain an extraction phase, and the solvent in the extraction phase is removed to obtain the fermentation bacterial extract.

[0080] In the present application, the extraction agent preferably comprises one or more of lower alcohol, ketone solvent, ester solvent, nitrile solvent and halogenated hydrocarbon, more preferably lower alcohol; the lower alcohol preferably comprises at least one of methanol and ethanol; the ketone solvent preferably comprises at least one of acetone and butanone; the ester solvent preferably comprises at least one of ethyl acetate, ethyl formate, ethyl butyrate and propyl acetate; the nitrile solvent preferably comprises acetonitrile; the halogenated hydrocarbon preferably comprises at least one of chloroform, dichloromethane and dichloroethane. In the present application, the ratio of the mass of the fermentation bacteria to the volume of the extraction agent for single extraction is preferably 1 g:0.1-1 L, which can be specifically 1 g:0.1 L, 1 g:0.2 L, 1 g:0.3 L, 1 g:0.4 L, 1 g:0.5 L, 1 g:0.6 L, 1 g:0.7 L, 1 g:0.8 L, 1 g:0.9 L or 1 g:1 L.

[0081] In the present application, the mixing preferably comprises vortex mixing, and the present application does not have special limitations on the conditions of the vortex mixing, which can uniformly mix the raw materials.

[0082] In the present application, the extraction preferably comprises ultrasonic extraction; the power of the ultrasonic extraction is preferably 20-60 W, which can be specifically 20 W, 30 W, 40 W, 50 W or 60 W; the temperature of the extraction is preferably 25-40℃, which can be specifically 25℃, 30℃, 35℃ or 40℃; the number of times of the extraction is preferably 2-4, more preferably 3; the time of single extraction is preferably 30-90 min, which can be specifically 30 min, 40 min, 50 min, 60 min, 70 min, 80 min or 90 min; the extraction is preferably carried out under light shielding conditions.

[0083] The present application does not have special limitations on the solid-liquid separation, which can adopt a solid-liquid separation mode well known to those skilled in the art, such as centrifugation, filtration or suction filtration. In the present application, the rotation speed of the centrifugation is preferably 3000-8000 r / min, which can be specifically 3000 r / min, 4000 r / min, 5000 r / min, 6000 r / min, 7000 r / min or 8000 r / min; the time of the centrifugation is preferably 5-15 min, which can be specifically 5 min, 10 min, 11 min, 12 min, 13 min, 14 min or 15 min.

[0084] The present application does not have special limitations on the method for removing the solvent, which can adopt a method for removing the solvent well known to those skilled in the art, such as sequentially carrying out concentration and drying.

[0085] The present application can also combine the organic extraction phase and the extraction phase first, and then remove the solvent in the obtained mixture to obtain the M. eburnea fermentation product. The method for removing the solvent in the present application is not particularly limited, and any method for removing the solvent known to those skilled in the art can be used, such as concentration and drying in sequence.

[0086] After obtaining the M. eburnea fermentation product, the present application separates the M. eburnea fermentation product by solid phase extraction column (SPE column) to obtain component Fr. 7. In the present application, the conditions for the solid phase extraction column separation include: the stationary phase includes normal phase silica gel; the elution mode is gradient elution, and the eluent used in the gradient elution includes n-hexane, ethyl acetate and methanol, and the volume ratio of the n-hexane, ethyl acetate and methanol is 20:1:0-0:0:1, and in specific embodiments, the volume ratio can be 20:1:0, 5:1:0, 3:1:0, 1:1:0, 0:1:0, 0:20:1, 0:10:1, 0:5:1 and 0:0:1 in sequence. In the present application, the volume of each ratio of eluent in the gradient elution process is preferably 250 mL, each fraction is collected, concentrated and then subjected to HPLC analysis, and the seventh elution fraction (i.e. component Fr. 7) is selected for subsequent semi-preparative HPLC separation. In the present application, the conditions for the HPLC analysis are preferably the same as those for the semi-preparative HPLC separation.

[0087] After obtaining the component Fr. 7, the present application performs semi-preparative HPLC separation on the component Fr. 7 to obtain the conjugated enediyne compound having the structure shown in Formula I or Formula II.In the present application, the conditions of the semi-preparative HPLC separation include: the chromatographic column comprises a C18 chromatographic column, which can be specifically a COSMOSIL C18 Column (Φ10*250 mm, 5 μm); the column temperature is preferably 20-30 °C, which can be specifically 20 °C, 22 °C, 25 °C, 28 °C or 30 °C; the mobile phase system comprises mobile phase A and mobile phase B, the mobile phase A is 0.05-0.2 v / v% formic acid aqueous solution, the volume fraction of formic acid in the 0.05-0.2 v / v% formic acid aqueous solution can be specifically 0.05%, 0.1%, 0.15% or 0.2%; the mobile phase B is acetonitrile; the flow rate of the mobile phase is preferably 2-5 mL / min, which can be specifically 2 mL / min, 3 mL / min, 4 mL / min or 5 mL / min; the detector is preferably a diode array detector, and the detection wavelength is 200-600 nm; the elution mode is gradient elution; the program of the gradient elution is as follows: 0-5 min, the volume fraction of the mobile phase B is linearly increased from 8-12% (which can be specifically 8%, 9%, 10%, 11% or 12%) to 18-22% (which can be specifically 18%, 19%, 20%, 21% or 22%); 5-35 min, the volume fraction of the mobile phase B is linearly increased from 18-22% (which can be specifically 18%, 19%, 20%, 21% or 22%) to 65-75% (which can be specifically 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74% or 75%); 35-45 min, the volume fraction of the mobile phase B is linearly increased from 65-75% (which can be specifically 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74% or 75%) to 85-92% (which can be specifically 85%, 86%, 87%, 88%, 89%, 90%, 91% or 92%); 45-46 min, the volume fraction of the mobile phase B is linearly increased from 85-92% (which can be specifically 85%, 86%, 87%, 88%, 89%, 90%, 91% or 92%) to 95-100% (which can be specifically 95%, 96%, 97%, 98%, 99% or 100%); 46-60 min, the volume fraction of the mobile phase B is 95-100% (which can be specifically 95%, 96%, 97%, 98%, 99% or 100%); 60-61 min, the volume fraction of the mobile phase A is linearly decreased from 95-100% (which can be specifically 95%, 96%, 97%, 98%, 99% or 100%) to 8-12% (which can be specifically 8%, 9%, 10%, 11% or 12%); 61-70 min, the volume fraction of the mobile phase B is 8-12%, which can be specifically 8%, 9%, 10%, 11% or 12%.

[0088] The application further provides application of the conjugated ene-yne compound as an intermediate in biosynthesis of enediyne.

[0089] The application further provides a derivative of the conjugated ene-yne compound, which includes a pharmaceutically acceptable salt, a hydrate, a solvate and a crystal form. In the application, the pharmaceutically acceptable salt preferably includes one or more of hydrochloride, sulfonate, tartrate, maleate and citrate. The application does not have special limitation on the pharmaceutically acceptable salt, and a salt formation method well known to those skilled in the art can be used. The application does not have special limitation on the hydrate, solvate and crystal form, and a hydrate, solvate and crystal form well known to those skilled in the art can be used.

[0090] The application further provides a pharmaceutical composition including an active component and a pharmaceutically acceptable adjuvant; the active component includes one or more of the conjugated ene-yne compound and the derivative of the conjugated ene-yne compound. The application does not have special limitation on the pharmaceutically acceptable adjuvant, and a pharmaceutically acceptable adjuvant well known to those skilled in the art can be used. In the application, the dosage form of the pharmaceutical composition preferably includes injection, tablet, capsule, pill, suspension or emulsion. In the application, the administration route of the pharmaceutical composition preferably includes oral administration, spray, transdermal injection, intravenous injection or intramuscular injection. In the application, the mass content of the active component in the pharmaceutical composition is preferably 1-99%, which can be specifically 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%.

[0091] The application further provides application of the conjugated ene-yne compound, the derivative of the conjugated ene-yne compound or the pharmaceutical composition in preparation of an antitumor drug or an antibiotic drug.

[0092] In order to further illustrate the application, the conjugated ene-yne compound, the preparation method and application thereof, the pharmaceutical composition and the application thereof provided by the application are described in detail below with reference to the examples, but they should not be understood as limiting the protection scope of the application.

[0093] In the following examples, Micromonospora eburnea (DSM 44814) is purchased from the DSMZ company.

[0094] Example 1

[0095] (1) Extraction, separation and purification of compound 1 (formula I) and compound 2 (formula II)

[0096] Micromonospora eburnea single spore was inoculated in a 250 mL flask containing 100 mL TSB medium, and cultured at 30°C, 200 r / min for 36 h to obtain seed liquid. The fermentation medium was prepared by adding acid hydrolysis casein 5 g, NaCl 5 g, MgSO4 2.5 g, K2HPO4 1 g, CaCO3 2 g, CH3COON a 400 mg in 899 mL water, and the pH was adjusted to 7.2. The mixture was sterilized in a pot at 121°C. Then, 100 mL of 400 g / L glucose aqueous solution filtered through a 0.22 μm membrane was added, and the mixture was stirred. Then, 1 mL of 100 g / L NaI aqueous solution filtered through a 0.22 μm membrane was added, and the mixture was stirred. Finally, 1 L of fermentation medium was obtained. The whole experiment was carried out in the dark.

[0097] The seed liquid was inoculated in a 250 mL flask containing 100 mL fermentation medium (total 25 L) at an inoculation amount of 10 v / v%, and cultured at 30°C, 200 r / min for 7 days to obtain fermentation broth. The fermentation broth was centrifuged at 4000 r / min for 10 min to collect the fermentation broth and the fermentation supernatant. Methanol was added to the fermentation broth, and the mixture was vortexed and ultrasonically extracted at 30°C for 30 min. The mixture was centrifuged at 4000 r / min for 10 min to collect the methanol extract. The extraction was repeated three times. The fermentation supernatant was extracted with an equal volume of ethyl acetate to obtain the ethyl acetate extract. The extraction was repeated three times. The methanol extract and the ethyl acetate extract were combined, concentrated under reduced pressure, and dried to constant weight to obtain the M. eburnea fermentation product. The whole experiment was carried out in the dark.

[0098] The M. eburnea fermentation product was preliminarily separated by normal phase silica gel SPE column, and the specific steps were as follows: a sample mixing: the M. eburnea fermentation product was dissolved in 100 mL methanol, and then 100-200 mesh normal phase silica gel was added to the methanol. The mixture was stirred and dried by rotary evaporation. The sample volume was measured. b dry column packing: 3 times the sample volume of 200-300 mesh normal phase silica gel was packed into the SPE column, and the column was compacted. c sample loading: the sample was added to the upper layer of the silica gel column, and the sample layer was compacted. Then, cotton was placed on the sample layer. d gradient elution: the normal hexane-ethyl acetate-methanol three-phase system (volume ratio: 20:1:0, 5:1:0, 3:1:0, 1:1:0, 0:1:0, 0:20:1, 0:10:1, 0:5:1, 0:0:1) was used as the eluent for gradient elution. The volume of each ratio of eluent was 250 mL. Each fraction was collected, concentrated, and analyzed by HPLC. The 7th elution fraction (component Fr. 7) was selected for subsequent semi-preparative HPLC separation.

[0099] Semi-preparative HPLC separation of fraction Fr. 7 afforded compound 1 (2.4 mg, t R = 35.5 min) and compound 2 (2.1 mg, t R = 34.2 min) as orange-yellow amorphous powders.

[0100] HPLC analysis and semi-preparative HPLC separation conditions: Agilent 1260 semi-preparative liquid chromatograph, COSMOSIL C18 Column (Φ10 x 250 mm, 5 μm) as the chromatographic column, 3 mL / min as the flow rate of mobile phase, DAD detector, 25 °C as the column temperature, 0.1 v / v% formic acid aqueous solution as the mobile phase A, acetonitrile as the mobile phase B, and the gradient elution program as shown in Table 1.

[0101] Table 1 Gradient elution program

[0102] Time / min Mobile phase A volume fraction / % Mobile phase B volume fraction / % 0 90 10 5 80 20 35 30 70 45 10 90 46 0 100 60 0 100 61 90 10 70 90 10

[0103] (2) Structure identification of compound 1 and compound 2

[0104] According to the UV spectrum of compound 1 ( Figure 1 ), HR-ESI-MS ( Figure 2 ), and nuclear magnetic resonance spectrum with CD3OD as the solvent: 1 H-NMR ( Figure 3 ), 13 C-NMR ( Figure 4 ), DEPT spectrum ( Figure 5 ), HSQC spectrum ( Figure 6 ), HMBC spectrum ( Figure 7 ), 1 H- 1 HCOSY spectrum ( Figure 8 ), and ROESY spectrum ( Figure 9 ), the structure of compound 1 was determined. According to the UV spectrum of compound 2 ( Figure 10 ), HR-ESI-MS ( Figure 11 ), and nuclear magnetic resonance spectrum with CD3OD as the solvent: 1 H-NMR ( Figure 12 ), 13 C-NMR ( Figure 13 ), DEPT spectrum ( Figure 14 ), HSQC spectrum ( Figure 15 ), HMBC spectrum ( Figure 16 ), 1 H- 1 HCOSY spectrum ( Figure 17 ), and ROESY spectrum ( Figure 18The analysis results of compound 2 were determined by HRESIMS.

[0105] Compound 1 was an orange yellow amorphous powder, HRESIMS showed [M+H]+m / z 356.1309 (calcd. for C 20 H 22 NO3S,356.1315), combined with 1 H and 13 C-NMR information (Table 2), the molecular formula of compound 1 was determined as C 20 H 21 NO3S, with 11 degrees of unsaturation. The UV spectrum had a maximum absorption at λ max 362nm.

[0106] According to the analysis of nuclear magnetic spectrum, the molecular formula of compound 1 was determined as C 1 H NMR (600MHz, MeOD-d4) spectrum showed one terminal olefinic hydrogen proton signal [δ H 5.36 (1H, dd, J = 16.8, 1.8 Hz), 5.22 (1H, dd, J = 9.5, 1.8 Hz), H2-1]; one pair of conjugated olefinic hydrogens [δ H 6.19 (1H, d, J = 16.7 Hz, H-7), 6.10 (1H, d, J = 16.7 Hz, H-8)] in trans; and the remaining six olefinic hydrogen signals [δ H 6.69 (1H, d, J = 10.4 Hz, H-2), 6.61 (1H, m, H-3), 6.44 (1H, m, H-2), 6.31 (1H, m, H-14), 5.84 (1H, d, J = 14.5 Hz, H-4), 5.72 (1H, s, H-11)]. Two methyl signals were shown in the high field region [δ H 1.86 (3H, d, J = 5.6 Hz, H3-15), 1.97 (3H, s H3-7′)], and three hydrogen signals at δ H 4.47 (1H, brs), 3.37 (1H, m), 3.08 (1H, m) were speculated to be one secondary amine hydrogen proton and one thia-methylene proton signal, combined with the molecular formula information. 13 C NMR (150MHz, MeOD-d4) spectrum combined with DEPT and two-dimensional spectrum showed 20 carbon signals, which were determined by HSQC to be related to hydrogen, including one amide carbonyl carbon (δ C 172.9, C-6′), one carboxyl carbon (δ C 172.0, C-4′), one substituted olefinic carbon (δ C 148.7), nine unsubstituted olefinic carbons (δC 143.9, 137.6, 133.0, 129.1, 121.8, 120.6, 120.4, 112.7, 105.2), 2 methyl groups (δ C 22.7, 18.6), 1 nitrogen-carbon signal (δ) C 55.3) and one sulfur-carbon signal (δ C 35.8), 4 substituted alkyne carbon signals (δ C The values ​​95.8, 95.1, 94.0, and 92.2 indicate the presence of two alkyne bonds in the structure.

[0107] exist 1 H- 1 The H COSY spectrum shows that The presence of relevant signals, combined with the terminal olefinic hydrogen signal of H-1, indicates that compound 1 has a conjugated diene structure at its end, which is confirmed by the relevant peaks in the HMBC spectrum; in addition, H-4 and C-5 (δ C 92.2) The presence of HMBC correlation indicates that the conjugated diene is linked to the alkyne bond. The conjugated alkene hydrogen H-7 (δ H 6.19) and H-8 (δ) H 6.10) shows no correlation with other hydrogen signals, suggesting that both ends are connected to alkyne bonds. The H-7 and C-6 (δ) peaks observed in the HMBC spectrum... C 92.2) / C-9(δ C 94.0), and H-8 and C-10 (δ C The existence of long-range correlations in 95.8 confirms this structure. The relevant peaks indicate that a terminal methyl group is attached to the olefin chain to form a propylene structure; H-11 (δ H The singlet information of 5.72 (s) suggests that its two ends are connected to alkyne bonds or substituted alkene carbons. The positions of the remaining two alkene carbons are determined by HMBC information. H-11 (δ H 5.72) and C-10 (δ C 95.8) / C-12(δ C 148.7) / C-13(δ C 129.1), and H-13 (δ H 6.69) and C-11 (δ C 105.2) / C-12(δ C 148.7) shows long-range correlation, indicating that C-11 is connected to the alkyne bond and C-12 is connected to the terminal propylene bond.

[0108] exist 1 H- 1H COSY correlations, combined with HMBC spectrum H-7' (δ H 1.97) to amide carbonyl carbon C-6' (δ C 172.9), H-2' (δ H 3.37) to carboxyl carbon C-4' (δ C 172.0) respectively, suggested the presence of an N-acetylcysteine moiety in compound 1. HMBC correlation of H-2' (δ H 3.08) to C-12 (δ C 148.7) indicated the acetylcysteine moiety was at C-12. The planar structure of compound 1 was thus determined and the carbon and hydrogen signals were fully assigned. The 1D NMR data assignment is shown in Table 2.

[0109] In the NOESY spectrum, H-2 (δ H 6.44) / H-4 (δ H 5.84) and H-1 (δ H 5.36) / H-3 (δ H 6.61) were correlated, indicating a trans conjugated diene structure; the coupling constant of 16.7 Hz between conjugated olefinic hydrogens H-7 / H-8 suggested the double bond Δ 7,8 was E type; H-11 (δ H 5.72) correlated with H-2' (δ H 3.37 / 3.08) / H-3' (δ H 4.47) of the N-acetylcysteine moiety, while H-11 did not correlate with H-13 (δ H 6.69), indicating the double bond Δ 11,12 was E type; H-13 (δ H 6.69) correlated with H-15 (δ H 1.86), indicating the double bond Δ 11,12 was E type.

[0110] Table 2. NMR data of compound 1 and compound 2 (MeOD-d4; hydrogen spectrum, 600 MHz; carbon spectrum, 150 MHz)

[0111]

[0112]

[0113]

[0114] Figure 19 are two-dimensional nuclear magnetic correlation signals of compound 1, Figure 20For the 2D NMR correlation signals of compound 2, it was known that compound 2 had the same high resolution mass spectrum information as compound 1, and the molecular formula was C 20 H 21 NO3S([M+H]+m / z356.1312), and the NMR data of both were similar, suggesting that it was a diastereoisomer of compound 1. The main difference between the two was C-11(112.7, Δδ C +7.5) and C-13(132.4, Δδ C +3.3), suggesting that the Δ 11,12 double bond configuration of compound 2 was opposite to that of compound 1, which was Z type, and the presence of obvious NOE correlation between H-11 and H-13 in the NOESY spectrum confirmed this structure.

[0115] (3) Determination of the absolute configuration of compound 1

[0116] For the R / S absolute configuration of C-3' of compound 1, initial conformation analysis was performed by Monte Carlo search algorithm under MMFF94 molecular mechanics force field using Spartan 14.0 software, and the relatively favorable conformation was obtained under an energy window of Δ<6kcal / mol. Further optimization was performed on the force field minimum energy conformation obtained by density functional theory (DFT) at B3LYP / 6-31G(d) energy level under vacuum conditions using Gaussian 09 software. At the same time, harmonic vibration frequency test was performed to determine that the final optimized conformation had no virtual frequency. Gauge-independent atomic orbital (GIAO) NMR theoretical calculation was performed on two candidate structures (3E * ,7E * ,11E * ,13E * ,3′R * -1; 3E * ,7E * ,11E * ,13E * ,3′S * -1) at the MPW1PW91 / 6-311+G(d,p) level, and the results are shown in Figures 21 to 23 , wherein, Figure 21 is the linear regression analysis diagram of the experimental value and the theoretical calculation value of 3E * ,7E * ,11E * ,13E * ,3′R * -1 conformation at the PCM (methanol) model MPW1PW91 / 6-311+G(d,p) level.Figure 22 3E * 7E * 11E * 13E * 3'S * Linear regression analysis of experimental and theoretical calculation values of compound 1 at PCM (methanol) model MPW1PW91 / 6-311+G(d,p) level, Figure 23 The relative deviation of the calculated and experimental values of the two configurations. Based on the DP4+ probability analysis results of the NMR data, the candidate structure 3E * 7E * 11E * 13E * 3' R * has the highest fitting degree of the calculated and experimental values, thus determining the stereochemical configuration of compound 1 as 3E, 7E, 11E, 13E, 3' R.

[0117] (4) Determination of the absolute configuration of compound 2

[0118] Due to the serious overlap of the H-7 / H-8 signals of compound 2, it is impossible to determine its Δ 7,8 double bond E / Z configuration by coupling constant, therefore, the NMR theoretical calculation of the 7-position E / Z configuration and the C-3' position R / S absolute configuration of compound 2 was carried out by Gaussian 09. First, the initial conformation analysis was carried out by Monte Carlo search algorithm under MMFF94 molecular mechanics force field using Spartan 14.0 software, and the relatively favorable conformation was obtained under the energy window of Δ<6kcal / mol. Further optimization of the force field minimum energy conformation was carried out by Gaussian 09 software using the density functional theory (DFT) of B3LYP / 6-31G(d) energy level under vacuum condition. At the same time, harmonic vibration frequency test was carried out to determine that the final optimized conformation had no imaginary frequency. At the MPW1PW91 / 6-311+G(d,p) level, four candidate structures (3E * 7E * 11Z * 13E * 3' R * -2; 3E * 7E * 11Z * 13E * 3' S * -2; 3E * 7Z * 11Z * 13E *,3′R * -2;3E * ,7Z * ,11Z * ,13E * ,3′S * -2) were performed. The DP4+ probability analysis based on the NMR data showed that the calculated value of candidate structure 3E * ,7E * ,11Z * ,13E * ,3′R * was the highest, thus determining the stereochemical configuration of compound 2 as 3E,7E,11Z,13E,3′R.

[0119] Example 2

[0120] Analysis of secondary metabolites of Micromonospora eburnea

[0121] MSH is a key low molecular weight sulfhydryl compound in actinomycetes, which is similar in function to glutathione (GSH) in eukaryotes, and is involved in oxidative stress defense and toxin detoxification, playing a key role in antibiotic resistance and environmental adaptation of actinomycetes.

[0122] The methanol extract phase prepared in Example 1 was concentrated and dried to constant weight to obtain a fermentation mycelium extract. The ethyl acetate extract phase prepared in Example 1 was concentrated and dried to constant weight to obtain a fermentation supernatant extract. The fermentation mycelium extract was subjected to LC-MS analysis, and the fermentation supernatant extract was subjected to HPLC analysis. The HPLC analysis conditions were the same as the semi-preparative HPLC separation conditions in Example 1. LC-MS analysis: liquid phase conditions: Agilent 1260 semi-preparative liquid chromatograph, chromatographic column COSMOSIL C18 Column (Φ10×250mm, 5μm), mobile phase flow rate 3mL / min, DAD detector, detection wavelength 200-800nm, column temperature 25℃, mobile phase A 0.1v / v% methanol water solution, mobile phase B acetonitrile, gradient elution program as shown in Table 3; mass spectrometry conditions: positive ion mode, ion source ESI, mass range 50-2000, ion spray voltage 3500V, atomization gas temperature 350℃, de-clustering voltage 60V, collision energy 30eV, collision energy range 5eV. The results are shown in Figures 24 to 25 .

[0123] Table 3 Gradient elution program

[0124]

[0125]

[0126] From HPLC chromatogram ( Figure 24 It can be seen that compounds 1 and 2 are mainly present in the fermentation supernatant of *M. eburnea*. LC-MS analysis showed that a product peak (MSH-S-toxin complex) with a molecular weight of 678.2, matching the complex formed by the conjugation of trace amounts of mycothiol (MSH) thiol groups with compound 2, could be detected in *M. eburnea* cells. The LC-MS results for this peak (…) Figure 25 The mass spectrometry results show a quasi-molecular ion peak at 679.4 Å, a fragment ion peak at 340.2 Å (after the removal of one molecule of inositol and one molecule of glucosamine), and a fragment ion at 192.1 Å (after the cleavage of sulfur bonds and the removal of MSH). These peaks indicate that the complexes are formed by the conjugation of MSH with compounds 1 / 2. Therefore, it can be confirmed that the generation of compounds 1 and 2 involves an MSH-dependent enzymatic detoxification mechanism. The conjugated enyne precursor conjugates with the thiol groups of MSH to produce detoxification products. After hydrolysis and the removal of one molecule of inositol and glucosamine, these products are excreted extracellularly, yielding the detoxification byproducts—compounds 1 and 2.

[0127] Example 3

[0128] Detection of natural alkynyl components in fermentation products using ruthenium-catalyzed click chemistry.

[0129] The specific operating steps are as follows: Dissolve 20 mg of *M. eburnea* fermentation product in 1 mL of acetone to obtain a *M. eburnea* fermentation product solution. Add 200 μL of acetone solution containing 5 mmol / L ethyl 2-azidoethyl, mix well, and then add 200 μL of acetone solution containing 0.025 mmol Cp*RuCl. Make up the reaction volume to 2 mL with acetone. Stir the reaction at room temperature for 16 h, concentrate by rotary evaporation, and dry to constant weight. Redissolve in 50 μL LDMSO. Perform HPLC and LC-MS analysis on the obtained click chemistry reaction product. The conditions for HPLC and LC-MS analysis are the same as those in Example 2. The HPLC analysis results are shown below. Figure 26 The LC-MS analysis results are shown in [link to LC-MS analysis]. Figure 27 .

[0130] from Figure 26 As can be seen from the data, the fermentation product peak with a retention time of approximately 34 min disappeared after the click chemical reaction, and a new peak with a retention time of approximately 30–31 min was generated; LC-MS analysis ( Figure 27 The newly generated peak (retention time 20–21 min) [M+H] is displayed. += 485.2, which matches the molecular mass of the product of the cycloaddition reaction of the disappeared fermentation product peak ([M+H] + = 356.2) with ethyl 2-azidoacetate, thus indicating that the fermentation product peak at about 34 min is an alkyne-containing compound.

[0131] The present application employs Cp*RuCl to catalyze the cycloaddition reaction of azide and alkyne in natural products to generate 1,2,3-triazole, so that the natural conjugated enyne components in the fermentation product of Micromonospora can be targetedly explored.

[0132] Test Example 1

[0133] Antitumor activity of compounds 1 and 2

[0134] The CCK-8 method was used to determine the proliferation inhibition rate of compounds 1 and 2 on human cervical cancer cells Hela and human hepatoma cells HepG2: logarithmic growth period human cervical cancer cells Hela and human hepatoma cells HepG2 were taken, and after being digested with 0.25% trypsin, single cell suspensions were prepared with DMEM culture solution; 1.0×10 4 cells per well were inoculated in a 96-well culture plate, and the volume of each well was 90 μL. The culture plate was moved into a CO2 incubator, and incubated at 37℃, 5% CO2 and saturated humidity for 4 h, then 10 μL of different final concentrations of doxorubicin and compound were added, with gradients of 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.12 μg / mL, 1.56 μg / mL, 0.78 μg / mL, 0.39 μg / mL, and each group had 2 repeated wells. After 48 h of continuous culture in the cell incubator, 10 μL of CCK-8 reagent was added, and incubated at 37℃ for 2 h, and the absorbance at 490 nm was measured, and tumor cells, drugs and blank medium were set as controls.

[0135] The inhibition rate of the drug on the corresponding tumor cells was calculated according to the measured absorbance value: tumor inhibition rate = (OD 对照组 - OD 实验组 ) / (OD 对照组 - OD 空白组 )×100%. The results are shown in Table 4.

[0136] Table 4 Inhibition of tumor cells by compounds 1 and 2

[0137]

[0138] The experimental results prove that compounds 1 and 2 have good activity in inhibiting the proliferation of human cervical cancer cells Hela and human hepatoma cells HepG2.

[0139] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. A conjugated enediyne compound, characterized by, having the structure of Formula I or Formula II:

2. The method for preparing the conjugated enyne compound according to claim 1, characterized in that, comprising the steps of: solid-liquid separation of fermentation broth of Micromonospora eburnea to obtain fermentation mycelium and fermentation supernatant respectively; extraction of the fermentation supernatant with an extractant to obtain an organic extract phase, removal of solvent in the organic extract phase to obtain M. eburnea fermentation product; the extractant comprises one or more of ester solvent and halogenated hydrocarbon; the extraction is carried out in the dark; solid phase extraction column separation of the M. eburnea fermentation product to obtain component Fr. 7; the solid phase extraction column separation conditions comprise: the stationary phase comprises normal phase silica gel; the elution mode is gradient elution, the eluent used in the gradient elution comprises n-hexane, ethyl acetate and methanol, and the volume ratio of the n-hexane, ethyl acetate and methanol is 20:1:0-0:0:1; semi-preparative HPLC separation of the component Fr. 7 to obtain the conjugated enediyne compound having the structure of Formula I or Formula II; the semi-preparative HPLC separation conditions comprise: the chromatographic column comprises a C18 chromatographic column; the mobile phase system comprises mobile phase A and mobile phase B, the mobile phase A is 0.05-0.2 v / v% formic acid aqueous solution, and the mobile phase B is acetonitrile; the elution mode is gradient elution; the gradient elution program is as follows: 0-5 min, the volume fraction of the mobile phase B is linearly increased from 8-12% to 18-22%; 5-35 min, the volume fraction of the mobile phase B is linearly increased from 18-22% to 65-75%; 35-45 min, the volume fraction of the mobile phase B is linearly increased from 65-75% to 85-92%; 45-46 min, the volume fraction of the mobile phase B is linearly increased from 85-92% to 95-100%; 46-60 min, the volume fraction of the mobile phase B is 95-100%; 60-61 min, the volume fraction of the mobile phase A is linearly decreased from 95-100% to 8-12%; 61-70 min, the volume fraction of the mobile phase B is 8-12%.

3. The preparation method according to claim 2, characterized in that, The M. eburnea fermentation product further comprises fermentation mycelium extract, the fermentation mycelium extract is obtained by extraction of the fermentation mycelium with an extractant; the extractant comprises one or more of lower alcohol, ketone solvent, ester solvent, nitrile solvent and halogenated hydrocarbon; the extraction is carried out in the dark.

4. The production method according to claim 2 or 3, characterized by, The lower alcohol comprises at least one of methanol and ethanol; The ketone solvent comprises at least one of acetone and butanone; The ester solvent comprises at least one of ethyl acetate, ethyl formate, ethyl butyrate and propyl acetate; The nitrile solvent comprises acetonitrile; The halogenated hydrocarbon comprises at least one of chloroform, dichloromethane and dichloroethane.

5. The preparation method according to claim 2, characterized in that, In the gradient elution process of the solid phase extraction column separation, the volume ratio of n-hexane, ethyl acetate and methanol is 20:1:0, 5:1:0, 3:1:0, 1:1:0, 0:1:0, 0:20:1, 0:10:1, 0:5:1 and 0:0:1 in turn.

6. The preparation method according to claim 2, characterized in that, The column temperature of the semi-preparative HPLC separation is 20-30 DEG C; the flow rate of the mobile phase is 2-5 mL / min, the detector is a diode array detector, and the detection wavelength is 200-600 nm.

7. Use of the conjugated ene-yne compound of claim 1 as an intermediate of enediyne biosynthesis.

8. The derivative of the conjugated enediyne compound according to claim 1, characterized by The derivative includes a pharmaceutically acceptable salt, a hydrate, a solvate and a crystal form.

9. A pharmaceutical composition, characterized by, The active ingredient includes one or more of the conjugated ene-yne compound of claim 1 and the derivative of the conjugated ene-yne compound of claim 8.

10. Use of the conjugated ene-yne compound of claim 1, the derivative of the conjugated ene-yne compound of claim 8 or the pharmaceutical composition of claim 9 in the preparation of an antitumor drug or an antibiotic drug.

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