CN115043790A - Symmetric heterocyclic compound, preparation method and application thereof - Google Patents
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Abstract
The application relates to a symmetric heterocyclic compound in the technical field of microorganisms, and the structure of the symmetric heterocyclic compound is shown as the following formula:
the compound is obtained by fermenting and extracting the phaeosphaeria rubra, the phaeosphaeria rubra is named as phaeosphaeria rubra Tubeufia PF02-2, and the preservation unit is as follows: china center for type culture Collection with the preservation number of CCTCC NO: m2019957. The compound has the application of preparing tumor drug resistance reversal agent or tumor drug sensitizer.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a symmetrical heterocyclic compound, a preparation method and application thereof, and especially relates to a novel symmetrical heterocyclic dirubicin A consisting of 16 atoms, a preparation method and application thereof in reversing drug-resistant tumor cell activity.
Background
Cancer has become one of the serious health and life threatening diseases for human beings. The treatment of tumors mainly comprises chemotherapy, surgery, radiotherapy and the like, and the chemotherapy is one of the main means of cancer treatment. During chemotherapy, the development of drug resistance by tumor cells is the leading cause of chemotherapy failure. Therefore, the search for a reversal agent with low degree and good activity is the most fundamental way to solve the tumor drug resistance, and has main research value.
P-glycoprotein (P-gp) is one of the most representative proteins of the ABC transporter family, has a molecular weight of 170kD and consists of 1280 amino acid residues. Research shows that P-gp can transport medicine with diverse chemical properties and structures, including part of anticancer medicines, such as adriamycin, taxanes and the like, to cause multidrug resistance (MDR) phenomenon, thereby causing failure of cancer treatment. Thus, the study of P-gp inhibitors and substrates is of great interest for cancer therapy, and the co-administration of P-gp inhibitors with chemotherapeutic agents is an effective strategy to overcome MDR. Currently, several generations of P-gp inhibitors have been developed, the first generation reversal agents including tamoxifen, cyclosporin a, etc., of which verapamil and cyclosporin are typical representatives. However, such drugs often lack the specificity of P-glycoprotein and can cause serious side effects, and the first generation reversal agents are also limited to a large extent clinically (Sato w.et al.1991). Second generation reversal agents stavasporidide (valspodar, PSC833), dexverapamil (dexverapamil), etc., of which dexamethasone is representative, however, the development of second generation reversal agents is limited due to a series of side effects resulting from high toxicity and drug interactions (Rowinsky E.K. et al 1998; Hyafil F.et al 1993; Keller R.P.et al 1992). The main representative drugs of the third-generation P-glycoprotein inhibitors are Tariquidar (XR9576), Zosuquidar (LY335979), S9788, etc., wherein Tariquidar (XR9576) and WK-X-34 are taken as representatives (Massey P.R. et al.2014). The development of P-gp inhibitors from natural products and their derivatives has become a new direction and focus for the development of fourth generation inhibitors.
The natural products from the microorganisms are always important sources for developing innovative drugs, and provide a material basis for developing new drugs. Meanwhile, the microorganism has the advantages of short growth cycle, easy regulation and control of metabolism, easy breeding of strains, realization of industrial production through large-scale fermentation and the like, and further lays an important position in the research and development of new drugs. There are specific reports of the discovery of P-gp inhibitors from natural products of microbial origin, but it is not clear what is specifically the case.
To this end, the applicant has made extensive studies to find a series of novel compounds of long-chain fatty acid glycerol disclosed in CN113773216A, CN114014898A and CN114057811A, and found applications in reversing the activity of drug-resistant tumor cells, and continued studies to find more compounds having applications in reversing the activity of drug-resistant tumor cells.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a rare symmetrical heterocyclic new compound derived from microorganisms, a preparation method thereof and application thereof in preparing a medicine for reversing drug-resistant tumor cell activity.
One of the objects of the present invention is to provide a symmetric heterocyclic compound, which has the following structure:
another object of the present invention is to provide a method for preparing a symmetric heterocyclic compound, wherein the compound is obtained by fermenting and extracting phaeophycus rubra, the phaeophycus rubra is named as phaeophycus rubra tubufia PF02-2, and the preservation unit is: china center for type culture Collection, the preservation number is CCTCC NO: m2019957.
The invention relates to a Tubeufia rubra PF02-2 of Erythrocarpium rubicun, which is obtained by separation from Biochemical engineering center of Guizhou university, and the preservation unit is as follows: china center for type culture Collection, addresses: wuhan university, storage day: 2019.11.20, with a preservation registration number of CCTCC NO: m2019957.
The source of Tubeufia rubra PF02-2 is as follows:
sampling time: 2016, 5 months, 14 days;
sampling site: a natural protection area of Guangxi Zhuang autonomous region for preventing rain forests at urban shelters and harbors;
the sampling mode is as follows: collecting rotten wood in natural protection area of urban harbor-preventing city screen rain forest in Guangxi Zhuang autonomous region, and taking plastic sealing bag back to laboratory.
The strain of the phaeophycus erythraea rubella PF02-2 has the following properties:
the morphological characteristics of the bacterial colony are as follows: on a natural rotten wood substrate, bacterial colonies are flat, are in a net shape and a point shape, are connected into a sheet shape when the bacterial colonies are large in quantity, are colorless, transparent and white on fresh PF02-2 pure bacterial colonies obtained through separation, and are reddish brown after the PF02-2 pure bacterial colonies obtained through separation are naturally dried. Part of the mycelium is buried under the substrate, but mostly is epibiotic, and the mycelium is composed of membrane-branched mycelium and is colorless to dark brown. The conidiophores are cylindrical, are single-grown, grow in a curved manner, have membranes, are 50-150 microns long, 4.5-6 microns wide, have tapered tops, are dark brown at the bottoms, are transparent to light brown at the tops, and have smooth surfaces. The spore-forming cells grow singly or multiply, are cylindrical, have cylindrical small odontoid processes, grow coaxially from the middle part to the top part of a molecular spore stalk, are 10-19 microns long and 3-4 microns wide, are colorless, transparent, light brown and have smooth surfaces. The molecular spore is of a spiral type, is single-grown, is top-lateral-grown, is transparent, has a round top end, is curled for 2-3.5 times in a tight spiral manner, has a diameter of 35-50 micrometers, is 3-5 micrometers thick (the average diameter is 45 micrometers, and the thickness is 4.5 micrometers), gradually loosens in water, has an unclear multi-diaphragm, is colorless to light brown, and has a smooth surface. Conidia started to germinate and grow after 12h in water-agar medium. The colony grows in PDA culture medium at 25-28 deg.C for 2 weeks and has a diameter of 16mm, brown color, round shape, rough surface, obvious protrusions, pulse-like wrinkles, and complete colony edge.
Specifically, the preparation method comprises the following steps: carrying out liquid or solid fermentation culture on the phaeophycus rubellus rubella PF02-2 to obtain a fermented product; and extracting the fermentation product, and separating and purifying the obtained extract to obtain the symmetric heterocyclic compound.
The preparation method specifically comprises the following steps:
s1, strain activation: taking out the preserved strains, inoculating the strains on a basal medium plate, performing static culture for passage to the third generation, and performing amplification culture;
s2, fermentation culture: inoculating the activated strain obtained in the step S1 into a solid culture medium, and standing, fermenting and culturing for a period of time at the temperature of 26-30 ℃;
s3, extraction: taking the thalli and a culture medium, adding ethyl acetate for extraction, and concentrating the extract to obtain a fermentation product;
s4, fermentation product pretreatment: dissolving a fermentation product by using a solvent of methyl 1:1, uniformly mixing the fermentation product with silica gel according to a mass ratio of 1: 1-2, volatilizing the solvent to obtain a primary column sample, then loading the primary column sample on silica gel powder and a petroleum ether separation column, sequentially carrying out gradient elution by using petroleum ether, chloroform, ethyl acetate and methanol, carrying out reduced pressure recovery on an elution solvent by using a rotary evaporator, dissolving the elution solvent by using chloroform, acetone or methanol, then using a thin-layer chromatography dot plate, developing by using a developing agent, selecting a liquid with fluorescence at 254nm or 365nm under an ultraviolet visible light analyzer, and developing by using an 8% ethanol sulfate vanillin developer; mixing the methanol solvent eluates, and recovering methanol solvent to obtain methanol layer extract;
s5, purification and separation: a. dissolving the methanol layer extract with a methanol solvent, uniformly mixing the methanol layer extract with silica gel according to a mass ratio of 1: 1-3, loading the mixture on a pre-column after the solvent volatilizes, carrying out equilibrium reversed-phase medium-pressure column by adopting 10% methanol water, adding a pre-column containing a sample, sequentially carrying out 10 gradient elutions by adopting the methanol water, recovering the solvent from an eluent by a rotary evaporator, dissolving the methanol, then using a thin-layer chromatography dot plate, developing by using a developing agent, selecting a liquid with fluorescence at 254nm or 365nm under an ultraviolet visible light analyzer, and combining 8% ethanol sulfate vanillin color developing agent yellow-green components to obtain a Fr.10 component;
b. dissolving the component Fr.10 in a methanol solvent, uniformly mixing with silica gel according to the mass ratio of 1: 1-3, and taking a sample on a column after the solvent is volatilized; uniformly mixing silica gel powder and a solvent B, A, 150:1, loading the mixture into a separation column, adding a sample on the column, performing gradient elution by adopting a system B, A, B and A, 150:1 and 100:1, recovering the solvent from an eluent through a rotary evaporator, dissolving the solvent in methanol, spreading the solvent on a thin layer chromatography dot plate by using a developing agent, selecting a liquid with fluorescence at 254nm or 365nm under an ultraviolet visible light analyzer, and combining components with 8 percent ethanol sulfate vanillin developer which shows yellow green to obtain a Fr.10-4 component;
c. dissolving Fr.10-4 with methanol, uniformly mixing with silica gel according to a mass ratio of 1: 1-3, volatilizing the solvent to obtain a column sample, weighing silica gel powder, uniformly mixing with a chlorine-methyl formic acid (200: 30: 1) solvent, loading into a separation column, adding the column sample, performing gradient elution by adopting chlorine-methyl formic acid (200: 30: 1) and the like, carrying out thin-layer chromatography on an eluent, developing by using a developing agent, selecting a liquid with fluorescence at 254nm or 365nm under an ultraviolet visible light analyzer, and combining 8% ethanol sulfate vanillin developer to develop a grayish yellow green component to obtain the symmetric heterocyclic compound.
Wherein the solid culture medium of step S2 is oat culture medium, and is obtained by mixing 200g oat and 150mL double distilled water.
The invention also aims to provide the application of the compound and the medicinal carrier in preparing a tumor drug resistance reversal agent or a tumor drug sensitizer.
Furthermore, the drug resistant or tumor drug is adriamycin.
Further, the tumor includes breast cancer, lung cancer or leukemia.
Furthermore, the tumor drug resistance reversal agent is a transport pump inhibitor, and the transport pump inhibitor has an inhibiting effect on one or more of drug-resistant protein P-glycoprotein and multidrug-resistant protein.
The fourth purpose of the invention is to provide the application of the compound and the medicinal carrier in preparing the anti-tumor cell medicament, wherein the tumor cells comprise adriamycin-resistant breast cancer cells, adriamycin-resistant lung cancer cells or adriamycin-resistant leukemia.
Drawings
FIG. 1 is a scheme for the isolation and purification of compound Dirubracin A;
FIG. 2 is an optical rotation spectrum of Dirubicin A compound of the present invention;
FIG. 3 is an infrared spectrum of compound Dirubracin A of the present invention;
FIG. 4 is a UV spectrum of compound Dirubracin A of the present invention;
FIG. 5 is a low resolution mass spectrum of compound Dirubracin A of the present invention;
FIG. 6 is a high resolution mass spectrum of compound Dirubracin A of the present invention;
FIG. 7 shows the preparation of Dirubracin A, a compound of the present invention 1 H-NMR chart;
FIG. 8 shows the preparation of Dirubracin A, a compound of the present invention 13 C-NMR and DEPT plots;
FIG. 9 is an HSQC spectrum of compound Dirubracin A of the present invention;
FIG. 10 shows the preparation of Dirubracin A, a compound of the present invention 1 H- 1 H COSY spectrogram;
FIG. 11 is an HMBC spectrum of Dirubicin A of the present invention;
FIG. 12 is a graph of EI-MST of compound Dirubracin A of the present invention;
FIG. 13 is a graph showing the cytotoxic activity of Dirubracin A against drug-resistant tumor cells MCF-7/ADM;
FIG. 14 is a graph showing the cytotoxic activity of Dirubracin A against drug-resistant tumor cells A549/ADM;
FIG. 15 is a graph showing the cytotoxic activity of Dirubracin A against drug-resistant tumor cells K562/ADM;
FIG. 16 is a graph of the effect of Dirubracin A in combination with doxorubicin on MCF-7/ADM IC50 values;
FIG. 17 is a graph of the effect of Dirubracin A in combination with doxorubicin on the A549/ADM IC50 values;
FIG. 18 is a graph of the effect of Dirubicin A in combination with doxorubicin on K562/ADM IC50 values.
Detailed Description
The following is further detailed by way of specific embodiments:
1. preparation method of compound Dirubracin A
As shown in figure 1 of the drawings, in which,
s1, strain activation
Taking out the strain preserved on the glycerol slant from a refrigerator at minus 80 ℃, digging a strain of a 1-ring strain Tubeufia rubra by using a sterile inoculating loop, cross-streaking and inoculating the strain to a basal medium plate with the diameter of 11cm, standing and culturing at 28 ℃ for 17d, and subculturing to a third generation for amplified culture.
S2 fermentation culture
Oat solid fermentation (1L triangle flask subpackaged with 200g of oat and 150mL of double distilled water), wherein the inoculation amount of each bottle is 1 multiplied by 1cm of the area on a culture plate 2 The amount of the activated strain of (4) was cultured by standing at 28 ℃ for 105 days.
S3, extraction
Adding ethyl acetate into the thallus and the oat culture medium, extracting for three times, performing oscillation extraction for 24h at 160rpm each time, combining the extract, performing reduced pressure concentration at 40 ℃ to obtain a fermentation product, repeating the above operations, and combining the fermentation products to obtain 2027.17 g.
S4 pretreatment of fermentation product
Dissolving 2027.17g of fermentation product by using a methyl 1:1 solvent, uniformly mixing the solution with silica gel according to a mass ratio of about 1:1.5 (namely, 2027.17g of fermentation product is added with 200-mesh silica gel powder 3041g), and volatilizing the solvent to obtain a river sand-shaped sample which is used as a primary column sample; weighing 6000g of 200-mesh silica gel powder with 300 meshes and petroleum ether solvent, uniformly mixing (no bubbles can be generated in the process) the silica gel powder with the petroleum ether solvent, putting the mixture into a separation column with the length of 1.5m and the inner diameter of 200mm, slowly sinking the silica gel powder until the silica gel powder does not sink, adding an upper column sample, sequentially carrying out 4 gradient elution by adopting petroleum ether, chloroform, ethyl acetate and methanol, carrying out 2-3 gradient elution (about 36L-54L elution solvent per column volume) column volumes, collecting one elution solvent per 1000mL, carrying out reduced pressure recovery on each elution sample by a rotary evaporator, dissolving the elution sample by using 10 or 15mL of chloroform, acetone or methanol, transferring the elution sample into a penicillin bottle with the specification of 20mL, carrying out Thin Layer Chromatography (TLC) on a point plate, and using the petroleum ether: 1:1 of chloroform, petroleum ether: acetone 10:1, chloroform: acetone-5: 1, chloroform: methanol 10: 1. ethyl acetate: developing with 5:1 methanol developing agent, observing whether fluorescence is present at 254nm or 365nm under conventional ultraviolet visible light analyzer, and developing with 8% ethanol sulfate vanillin developer; the methanol layer solvent eluents were combined, and the methanol solvent was recovered to obtain 35.77g of methanol layer extract.
S5 purification and separation
a. Dissolving the methanol layer extract (35.77g) with methanol solvent, mixing with silica gel at a mass ratio of about 1:2 (i.e. adding medium pressure RP-18 reverse phase silica gel into 35.77g fermentation product), volatilizing the solvent to obtain river sand-like sample, and taking the sample as column sample; adding a pre-column with the length of 10cm and the diameter of 49mm to the upper column sample; the method comprises the steps of balancing a reversed-phase medium-pressure column (with the column length of 460mm and the diameter of 49mm) by using 10% methanol water, balancing about 5-6 column volumes (about 5-6L of elution), adding a pre-column containing a sample, performing gradient elution by using methanol water (10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%), sequentially performing 10 gradient elution, performing 4-5 column volumes of each gradient elution, receiving the eluent by using a triangular flask with the specification of 500mL, recovering a solvent from each eluent by using a rotary evaporator, dissolving and transferring the eluent into a penicillin bottle with the specification of 20mL by using 10mL of methanol, performing TLC (thin layer chromatography) spotting, and using petroleum ether: acetone ═ 2:1, chloroform: acetone-5: 1, chloroform: methanol 10: 1. ethyl acetate: developing with methanol 2:1 developing agent, observing whether fluorescence is present at 254nm or 365nm under a conventional ultraviolet visible light analyzer, developing with 8% ethanol sulfate vanillin developer, and mixing the components with yellow-green color of 8% ethanol sulfate vanillin, (i.e. components eluted with 70% methanol water), to obtain 10 th component (Fr.101.2g).
b. After the component Fr.10(1.2g) is dissolved by a methanol solvent, the mixture is uniformly mixed with 200-mesh 300-mesh silica gel according to the mass ratio of about 1:1.5 (namely 1.8g is added in 1.2g of the component), and a river sand-shaped sample is obtained after the solvent is volatilized and is used as a column sample; weighing 60g of 200-mesh 300-mesh silica gel powder and a solvent B150: 1, uniformly mixing (no bubble can be generated in the process) the silica gel powder and the solvent A150: 1, loading the silica gel powder into a separation column with the length of 240mm and the inner diameter of 24mm, slowly sinking the silica gel powder until the silica gel powder does not sink, adding a column sample, performing gradient elution by a system B and A (150:1 and 100:1) with each gradient elution being 4-5 column volumes (about 480mL-600mL), collecting eluent by a triangular conical flask with the specification of 50mL, dissolving and transferring the eluent by about 5mL of methanol into a penicillin bottle with the specification of 20mL after each eluent is recovered by a rotary evaporator, performing TLC point plate, using chloroform acetone 2:1, chloroform methanol: 10:1 drop, ethyl acetate: methanol: 3:1 developing agent, observing whether the silica gel has fluorescence at 254nm or 365nm under a conventional ultraviolet visible light analyzer, then developing with 8% ethanol sulfate vanillin developer, and mixing 8% ethanol sulfate vanillin components with yellow-green color to obtain component 4 (Fr.10-462 mg).
c. Fr.10-4(62mg) is dissolved by methanol, and is uniformly mixed with 200-mesh 300-mesh silica gel according to the mass ratio of about 1:1.5 (namely 120mg is added into 62mg components), and a river sand-shaped sample is obtained after the solvent is volatilized and is used as an upper column sample; weighing 12g of 200-mesh 300-mesh silica gel powder and 200:30:1 drops of chlorine-methyl formate (bubbles cannot be generated in the process) and uniformly mixing the silica gel powder and the solvent, putting the mixture into a separation column with the length of 240mm and the inner diameter of 15mm, slowly sinking the silica gel powder until the silica gel powder does not sink, adding a column sample once, performing gradient elution by adopting the chlorine-methyl formate (200: 30: 1) and the like, collecting eluent by using a penicillin bottle with the specification of 20mL, performing TLC (thin layer chromatography) point plate counting, developing by using a developing agent with the chlorine-methyl formate (50: 1: 1), observing whether the fluorescence is generated at 254nm or 365nm under a conventional ultraviolet visible light analyzer, developing by using 8% ethanol sulfate vanillin developer, and combining the component of the 8% ethanol sulfate vanillin developer which is gray and yellow-green to obtain a first group Dirubicin A9.1mg.
2. Structural identification of compound Dirubracin A
Dirubracin A is white oil and is easily dissolved in solvents such as methanol, acetone, DMSO, etc. [ alpha ] to]2 D 8.9 ═ 7.2(c 0.1, MeOH) details see figure 2; IR spectrum (see attached figure 3 for details) at 3422cm -1 、1725cm -1 、1646cm -1 Has absorption peaks, which shows that the compound contains hydroxyl groups and ester groups. UV (MeOH) lambda max (log ε):196(4.01), demonstrating that the compound has a double bond (see FIG. 4 for details); ESI low resolution mass spectrum shows [ M + Na ]]Is 785, [2M + Na]1548, see attached figure 5 for details. High resolution mass spectrometry (HRESI) shows that the molecular weight of the compound is 785.4927[ M + Na ]] + Molecular formula is C 42 H 70 N 2 O 10 Na, calculated unsaturation of 9, detailed in figure 6; 1D NMR in combination with HSQC (see FIGS. 7, 8, 9 for details) signals [ delta ] C 14.4(q),23.6(t),26.0(t),26.5(t),27.0(t),28.2(t),30.2～30.8(t),32.7(t),34.9(t),37.0(t),129.0(d),129.1(d),130.9(d),175.3,176.3](ii) a Bonding with 1 Hydrogen signal delta on H NMR spectrum H [0.89(6H,t,J＝6.8Hz),2.76(4H,t,J＝6.7Hz),28.2(8H,m),5.34(4H,m),5.30(4H,m)]The compound can be deduced to be composed of two long-chain fatty acid segments, and each long-chain fatty acid contains two double bonds; in addition to the two fatty acid structural fragments, the compounds also contain 8 methylene groups [41.3 (CH) 2 ×2),3.38(4H,t,J＝5.3Hz)；65.1(CH 2 ×2),3.89(4H,m)；66.2(CH 2 ×2),4.15(2H,dd,J＝11.4,4.5Hz),4.08(2H,dd,J＝11.4,6.0Hz)；67.7(CH 2 ×2),3.86(4H,m)]And two methines [69.9 (CH. times.2), 3.94(2H, m)]The compound is deduced to have a repetitive structural fragment, and detailed one-dimensional nuclear magnetic data are shown in table 1.
TABLE 1D NMR data for Dirubicin A compound
1 H- 1 The connection of the compound H-1 and H-2 can be deduced on an H COSY spectrogram (detailed in figure 10);
h-11 → H-3 → H-4; h-12 → H-6 → H-5; h-7 and H-8 are connected;
h-2 "→ H-3" → H-4 "→ H-5" → H-6 "→ H-7" → H-8 "→ H-9" → H-10 "→ H-11" → H-12 "linkage; h-2 'and H-3' are connected; h-8 ' → H-9 ' → H-10 ' → H-11 ' → H-12 ' → H-13 ' → H-14 ' linkage; h-17 '→ H-18' linkage.
On HMBC (see FIG. 11 for details), the hydrogen proton signal delta H [3.38(2H,t,J＝5.3Hz,H-1)]And delta C 176.3(s, C-10),65.1(t, C-2) showed that C-1 was linked to C-10. Delta H [3.89(2H,m,H-2)]And delta C 41.3(t, C-1),69.9(d, C-3) demonstrate that C-2 is attached to C-3 via an oxygen atom; hydrogen proton signal delta H [3.94(1H,m,H-3)]And delta C 67.6(t, C-4),66.2(t, C-11) demonstrate that C-3 is linked to C-11 in addition to C-4; hydrogen proton signal delta H [4.15(1H,dd,J＝11.4,4.5Hz,H-11),4.08(1H,dd,J＝11.4,6.0Hz,H-11)]And delta C 175.3(s, C-1 '), 67.6(t, C-2),69.9(d, C-4), demonstrating that C-11 is attached to the ester group C-1'; binding delta H [2.33(2H,t,J＝7.5Hz,H-2′)]And delta C 175.3(s, C-1 '), 26.0(t, C-3'), demonstrating that C-1 'is linked to C-2'; hydrogen proton signal delta H [4.15(1H,dd,J＝11.4,4.5Hz,H-12),4.08(1H,dd,J＝11.4,6.0Hz,H-12)]And delta C 176.3(s, C-1'), 67.6(t, C-5),69.9(d, C-6); binding delta H [3.86(2H,t,m,H-4)]And delta C 66.2(t, C-11),69.9(t, C-3,5) correlation and hydrogen proton signals [2.19(2H, t, J ═ 7.4Hz, H-2')]And delta C 176.3(s, C-1 '), 26.5(t, C-2'); it was demonstrated that C-12 is attached to another ester group C-1 ', C-2 ' is attached to C-1 ', and C-5 is attached to C-4 through an oxygen atom. In the same way, delta H [3.89(2H,m,H-7)]And delta C 41.3(t, C-8),69.9(d, C-6) demonstrate that C-6 is attached to C-7 via an oxygen atom; hydrogen proton signal delta H [3.38(2H,t,J＝5.3Hz,H-8)]And delta C 176.3(s, C-9),65.1(t, C-8), demonstrates the association of C-8 with C-9. Careful analysis of HMBC, 1 H- 1 H COSY, combined with the molecular formula given by high resolution mass spectrometry and the calculated unsaturation, can deduce that C-9 is attached to C-10 through an oxygen atom.
From the above information, it can be seen that the compound Dirubracin a contains a symmetrical cyclic heterocycle consisting of 16 atoms, and two long-chain fatty acid structural fragments consisting of 12 carbon atoms and 18 carbon atoms, and the structure is shown in the formula.
EI-MS (see FIG. 12 for details) showed a series of fragment ion peaks M/z 662([ M-C) 3 H 3 NO 3 ] + )，323([M-C 12 H 18 O-C 18 H 30 O] + )，313([M-C 6 H 10 N 2 O 5 -C 18 H 30 O] + ) The detailed fragment ion characteristics of the compound are shown in the following formula. The compound fragment ion rationality demonstrated that dirubicin a structure is correct.
3. Screening for cytotoxic Activity of Compound Dirubracin A
3.1 test cell lines: MCF-7/ADR, A549/ADR, K562/ADR
3.2RPMI1640+ 10% fetal bovine serum
3.3 cell culture
3.3.1 cell Resuscitation
Taking out the cells from the liquid nitrogen tube, quickly putting the freezing tube into a water bath kettle preheated to 37 ℃ for quick thawing, and continuously shaking to quickly melt the liquid in the tube. After about 1mL of the liquid in the vial was completely dissolved, the cells were taken out under aseptic conditions and inoculated into a cell culture dish (RPMI1640+ 10% fetal bovine serum), and placed in a 37 ℃ CO atmosphere 2 Culturing in an incubator, replacing the culture solution the next day, continuously culturing, and observing the growth condition.
3.3.2 cell passages
After the cells grow to 80-90%, sucking out the cell culture solution by using a plastic straw with the specification of 3mL under the aseptic operation condition, adding 1-2mL of PBS (without calcium and magnesium ions) for washing for 1 time, adding 1mL of digestive juice (0.25% Trypsin-0.53mM EDTA) into a culture bottle, observing the cell digestion condition under an inverted microscope, if most of the cells become round, quickly taking back the operation table, tapping several times of the culture bottle, and adding 2mL of complete culture medium to stop digestion. In the new culture flask, 4mL of the complete culture medium was added, and 1mL of the complete culture medium containing the cells was added.
3.4CCK-8 assay for cytotoxic Activity
3.4.1 setting up the concentration gradient MCF-7/ADR (0, 6.25, 12.5, 25, 50, 100, 200. mu.g/mL), K562/ADR and A549/ADR (0, 1.6, 3.125, 6.25, 12.5, 25, 50, 100), 3 replicates, doxorubicin as a positive control, DMSO as a negative control, all data calculated using SPSS software.
3.4.2 Experimental procedures
(1) Digesting the cells, counting the cells, adjusting the cell concentration to 2X 10 4 one/mL.
(2) 100 μ L of cell suspension was seeded in 96-well plates. Plates were incubated at 5% CO 2 Culturing in an incubator at 37 ℃ for 24 h.
(3) According to the grouping, compounds with different concentrations and adriamycin are respectively added, and the mixture is continuously placed in an incubator to be incubated for 48 hours at the temperature of 37 ℃.
(4) After the culture, washing with PBS (without calcium and magnesium ions) for 1 time, adding 10. mu.L of CCK-8 reagent into each well, and placing in an incubator for incubation for 3 h.
(5) Absorbance at 490nm was measured with a microplate reader
3.4 results of the experiment
The result shows that the compound dirubicin A has no cytotoxic activity on 3 drug-resistant tumor cells in an effective concentration range (below 25 mu g/mL), and can be used for continuously screening the reversed tumor cells, and the result is shown in detail in figures 13-15.
4. Application of compound Dirubracin A in activity of reversing drug resistance of drug-resistant tumor cells
4.1 test cell lines: MCF-7/ADR, A549/ADR, K562/ADR
4.2RPMI1640+ 10% fetal bovine serum
4.3 cell culture
4.3.1 cell Resuscitation
Taking out the cells from the liquid nitrogen tube, quickly putting the freezing tube into a water bath kettle preheated to 37 ℃ for quick thawing, and continuously shaking to quickly melt the liquid in the tube. After about 1mL of the liquid in the vial was completely dissolved, the cells were taken out under aseptic conditions and inoculated into a cell culture dish (RPMI1640+ 10% fetal bovine serum), and placed in a 37 ℃ CO atmosphere 2 Culturing in an incubator, replacing the culture solution the next day, continuously culturing, and observing the growth condition.
4.3.2 cell passages
After the cells grow to 80-90%, sucking out the cell culture solution by using a plastic straw with the specification of 3mL under the aseptic operation condition, adding 1-2mL of PBS (without calcium and magnesium ions) for washing for 1 time, adding 1mL of digestive juice (0.25% of Trypsin-0.53mM EDTA) into a culture bottle, observing the cell digestion condition under an inverted microscope, if most of the cells become round, quickly taking back the operation table, tapping several times of the culture bottle, and adding 2mL of complete culture medium to stop digestion. In the new culture flask, 4mL of the complete culture medium was added, and 1mL of the complete culture medium containing the cells was added.
4.4CCK-8 test to reverse tumor cell Activity
4.4.1 setting concentration gradient of MCF-7/ADR (0, 6.25, 12.5, 25, 50, 100, 200 mug/mL), K562/ADR and A549/ADR (0, 1.6, 3.125, 6.25, 12.5, 25, 50, 100) for 3 repeats; the concentration of the Dirubicin A is 5, 10 and 20 mu g/mL; verapamil was the positive control and DMSO was the negative control, all data calculated using SPSS software.
4.4.2 Experimental procedures
(1) Digesting the cells, counting the cells, adjusting the cell concentration to 2X 10 4 one/mL.
(2) 100uL of cell suspension was seeded in 96-well plates. Plates were incubated at 5% CO 2 Culturing in an incubator at 37 ℃ for 24 h.
(3) According to the grouping, compounds with different concentrations and adriamycin are added respectively, and the mixture is further placed in an incubator to be incubated for 48 hours at 37 ℃.
(4) After the culture, washing with PBS (without calcium and magnesium ions) for 1 time, adding 10 μ L of CCK-8 reagent into each well, and incubating for 3h in an incubator.
(5) Absorbance at 490nm was measured with a microplate reader
4.4.3 results of the experiment
The results show that: the combination of the compound Dirubracin A and adriamycin at the concentrations of 5 mug/mL, 10 mug/mL and 20 mug/mL respectively obviously has the activity of reversing drug-resistant tumor cells MCF-7/ADM, A549/ADM and K562/ADM (detailed figures 16, 17, 18 and table 2); particularly, the compound dirubicin A has obvious activity of reversing drug-resistant tumor cells MCF-7/ADM when being combined with adriamycin at the concentration of 20 mu g/mL (detailed shown in figure 16 and table 1), and the activity of the compound is superior to that of a positive control verapamil, and the IC of the compound is 50 The values are detailed in table 2.
TABLE 2 IC of the compound Dirubracin A in combination with ADM on three cells 50 Value of
Remarking: A. b, C shows the concentration of the compound at 5. mu.g/mL, 10. mu.g/mL and 20. mu.g/mL, respectively
The foregoing is merely an example of the present invention and common general knowledge of known specific structures and features of the embodiments is not described herein in any greater detail. It should be noted that, for those skilled in the art, without departing from the structure of the present invention, several changes and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.
Claims (10)
1. A symmetric heterocyclic compound characterized by: the structure is shown as the following formula:
2. a process for the preparation of a compound according to claim 1, characterized in that: the compound is obtained by fermenting and extracting the phaeosphaeria rubra, the phaeosphaeria rubra is named as phaeosphaeria rubra Tubeufia PF02-2, and the preservation unit is as follows: china center for type culture Collection, the preservation number is CCTCC NO: m2019957.
3. A process for the preparation of a compound according to claim 2, characterized in that: the method comprises the following steps: carrying out liquid or solid fermentation culture on the phaeophycus rubellus rubella PF02-2 to obtain a fermented product; and extracting the fermentation product, and separating and purifying the obtained extract to obtain the long-chain fatty acid glycerol alcohol compound.
4. A process for the preparation of a compound according to claim 3, characterized in that: the method specifically comprises the following steps:
s1, strain activation: taking out the preserved strains, inoculating the strains on a basal medium plate, performing static culture for passage to the third generation, and performing amplification culture;
s2, fermentation culture: inoculating the activated strain obtained in the step S1 into a solid culture medium, and standing, fermenting and culturing for a period of time at the temperature of 26-30 ℃;
s3, extraction: taking the thalli and a culture medium, adding ethyl acetate for extraction, and concentrating the extract to obtain a fermentation product;
s4, fermentation product pretreatment: dissolving a fermentation product by using a solvent of methyl 1:1, uniformly mixing the fermentation product with silica gel according to a mass ratio of 1: 1-2, volatilizing the solvent to obtain a primary column sample, then loading the primary column sample on silica gel powder and a petroleum ether separation column, sequentially carrying out gradient elution by using petroleum ether, chloroform, ethyl acetate and methanol, carrying out reduced pressure recovery on an elution solvent by using a rotary evaporator, dissolving the elution solvent by using chloroform, acetone or methanol, then using a thin-layer chromatography dot plate, developing by using a developing agent, selecting a liquid with fluorescence at 254nm or 365nm under an ultraviolet visible light analyzer, and developing by using an 8% ethanol sulfate vanillin developer; mixing the methanol solvent eluates, and recovering methanol solvent to obtain methanol layer extract;
s5, purification and separation: a. dissolving the methanol layer extract with a methanol solvent, uniformly mixing the methanol layer extract with silica gel according to the mass ratio of 1: 1-3, loading the methanol layer extract on a pre-column when the solvent volatilizes, carrying out balanced reversed-phase medium-pressure column by adopting 10% methanol water, adding a pre-column containing a sample, sequentially carrying out 10 gradient elutions by adopting the methanol water, recovering the solvent from an eluent by a rotary evaporator, dissolving the methanol, spreading the eluent by using a thin-layer chromatography dot plate by using a developing agent, selecting a liquid with fluorescence at 254nm or 365nm under an ultraviolet visible light analyzer, and combining components with 8% ethanol sulfate vanillin developer which develops yellow green to obtain a Fr.10 component;
b. dissolving the component Fr.10 in a methanol solvent, uniformly mixing with silica gel according to the mass ratio of 1: 1-3, and taking a sample on a column after the solvent is volatilized; uniformly mixing silica gel powder and a solvent B, A, 150:1, loading the mixture into a separation column, adding a sample on the column, performing gradient elution by adopting a system B, A, B and A, 150:1 and 100:1, recovering the solvent from an eluent through a rotary evaporator, dissolving the solvent in methanol, spreading the solvent on a thin layer chromatography dot plate by using a developing agent, selecting a liquid with fluorescence at 254nm or 365nm under an ultraviolet visible light analyzer, and combining components with 8 percent ethanol sulfate vanillin developer which shows yellow green to obtain a Fr.10-4 component;
c. dissolving Fr.10-4 with methanol, uniformly mixing with silica gel according to a mass ratio of 1: 1-3, volatilizing the solvent to obtain a column sample, weighing silica gel powder, uniformly mixing with a chlorine-methyl formic acid (200: 30: 1) solvent, loading into a separation column, adding the column sample, performing gradient elution by adopting chlorine-methyl formic acid (200: 30: 1) and the like, carrying out thin-layer chromatography on an eluent, developing by using a developing agent, selecting a liquid with fluorescence at 254nm or 365nm under an ultraviolet visible light analyzer, and combining 8% ethanol sulfate vanillin developer to develop a grayish yellow green component to obtain the symmetric heterocyclic compound.
5. The process for the preparation of a compound according to claim 4, wherein: the solid culture medium of the step S2 is an oat culture medium, and is obtained by mixing 200g of oat and 150mL of double distilled water.
6. The use of a compound of claim 1 and a pharmaceutically acceptable carrier in the preparation of a tumor drug resistance reversal agent or a tumor drug sensitizer.
7. Use according to claim 6, characterized in that: the drug resistant or tumor drug is adriamycin.
8. Use according to claim 7, characterized in that: the tumor comprises breast cancer, lung cancer or leukemia.
9. Use according to claim 6, characterized in that: the tumor drug resistance reversal agent is a transport pump inhibitor, and the transport pump inhibitor has an inhibiting effect on one or more of drug-resistant protein P-glycoprotein and multidrug-resistant protein.
10. The use of a compound of claim 1 and a pharmaceutically acceptable carrier for the preparation of an anti-neoplastic agent, wherein: the tumor cells comprise adriamycin-resistant breast cancer cells, adriamycin-resistant lung cancer cells or adriamycin-resistant leukemia.
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