CN108191937B - Polyene androsterone compound and application thereof - Google Patents
Polyene androsterone compound and application thereof Download PDFInfo
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- CN108191937B CN108191937B CN201810000419.5A CN201810000419A CN108191937B CN 108191937 B CN108191937 B CN 108191937B CN 201810000419 A CN201810000419 A CN 201810000419A CN 108191937 B CN108191937 B CN 108191937B
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Abstract
The invention discloses a polyene androsterone compound and its application; the compound is extracted and separated from the plant of the genus Sinomenium, and the structural formula of the compound is shown as the following formula:
wherein R is1、R2、R3Are all selected from hydrogen, hydroxyl, methyl, halogen, aliphatic hydrocarbon group and aliphatic amino; experiments prove that the compound reported in the invention has strong immunosuppressive activity; the compound of the invention provides a lead compound for developing immunosuppressant and antitumor preparation, and is beneficial to developing and utilizing plant medicinal resources.
Description
Technical Field
The invention relates to a polyene androsterone compound and its application in preparing immunosuppressive and antitumor drugs.
Background
Steroid drugs are the most commonly used clinical immunosuppressants, such as prednisone, dexamethasone, methylprednisolone, and the like. It can effectively inhibit inflammatory factors and reduce various immune cells. The immunosuppressant provides an effective therapeutic drug for autoimmune diseases and rejection reactions after organ transplantation, and is usually used for inhibiting rejection reactions after organ transplantation, treating graft-versus-host diseases after bone marrow transplantation, or treating autoimmune diseases such as rheumatoid arthritis, Crohn's disease and the like.
At present, more than 100 malignant tumors are solved, and the number of dead cancer accounts for about 13 percent of the death number of diseases in the world; cancer deaths are expected to continue to increase worldwide, with an estimated 1200 million people dying from cancer in 2030; the occurrence and development of tumors are closely related to the decline of the immune function of the whole body, and once the tumors occur, the inhibition of the immune function of the body is deepened, thereby promoting the development of the tumors. The metastasis of tumor cells from the primary site to other sites continues to grow, which is a significant life-threatening cause of tumors. Among a plurality of factors of tumor pathogenesis, various types of immunosuppressive molecules play a key role in the formation and development process of tumors, can be used as a new target of antitumor drugs, and provide a new idea for the biological treatment of tumors.
The autoimmune diseases and cancers mostly are chronic or progressive diseases and need long-term administration, but the existing long-term administration of glucocorticoids (such as dexamethasone) and immunosuppressants generally has the defects of large toxic and side effects, inconvenient use and the like. As a natural product, the traditional Chinese medicine has the advantages of good compatibility with human tissues, small side effect and the like, and is increasingly paid more attention to the research of immunosuppressants and antitumor preparations. Therefore, the compound with the functions of immunosuppression and malignant tumor cell proliferation inhibition is searched from natural products, and the application value of the compound for developing novel immunosuppressive agents and antitumor preparations with high efficiency and low toxicity is achieved. The polyene androsterone compound and the application thereof as an immunosuppressive drug are not reported at present.
Cistus genus (Epigynum) Is a genus of the family Cistus belonging to the family Apocynaceae, and according to the results of our earlier studies, plants of the genus Cistus contain primarily androstane, pregnane, and glycosides thereof, under the drive of interest in finding new lead compounds, we have conducted a number of biological activity screening experiments on these new compounds, and found that the polyenoic androsterone compound (12 β -hydroxyandrosta-3, 5,14(15) -trien-16-one) has immunosuppressive activity at a concentration of 12.5. mu.M and has a significant difference (at a concentration of 25, 50. mu.M) compared with the model group (see the description of the examples below) (see the contents of 12, 5, 14-trien-16-one)P<0.05), has better immunosuppressive activity. In an anti-tumor test, a cell proliferation inhibition laboratory is developed aiming at human acute lymphocytic leukemia cells, chronic myelocytic leukemia cells, malignant melanoma cells, astrocytoma cells, adenocarcinoma cells, malignant epithelial tumor cells, glioblastoma multiforme cells, lung squamous carcinoma cells, ovarian gland cells, breast cancer cells, lung cancer cells and the like, and the result shows that the activity of the cell proliferation inhibition laboratory is equivalent to that of positive control dexamethasone when the concentration is 25 mu M, and 5 strains of cells show drug resistance to dexamethasone.
Disclosure of Invention
The invention aims to provide a polyene androsterone compound with a structural formula shown as a formula I:
Ⅰ
wherein R is1、R2、R3Are all selected from hydrogen, hydroxyl, methyl, halogen, aliphatic hydrocarbon group and aliphatic amino.
The aliphatic substituent in the above aliphatic hydrocarbon group and aliphatic amino group means a saturated or unsaturated aliphatic substituent, wherein the saturated aliphatic substituent means a straight-chain or branched alkyl group, a cycloalkyl group such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, sec-butyl, cyclopropylcyclododecyl and the like, and the unsaturated aliphatic group means an alkenyl group such as allyl, isopentenyl, 1-dodecenyl, alkynyl group such as 1-octadecynyl, 2-octadecynyl, alkadienyl group such as geranyl, 1, 3-octadecenyl, 9-octadecenyl and the like, or an alkadienyl group.
Another object of the present invention is to provide a 12 β -hydroxyandrosta-3, 5,14(15) -trien-16-one having the structural formula II:
Ⅱ。
the invention also aims to apply the polyene androsterone compound in the preparation of immunosuppressive drugs.
In particular to application of immunosuppressive drugs in preparing treatment drugs for autoimmune diseases or preparing treatment drugs for organ transplantation rejection resistance.
The invention also aims to apply the compound in preparing anti-tumor drugs.
The application of the invention can also be added with one or more pharmaceutically acceptable auxiliary materials, wherein the auxiliary materials comprise conventional filling agents, diluents, adhesives, excipients, absorption enhancers, filling agents, surfactants, stabilizers and the like in the pharmaceutical field, and flavoring agents, pigments, sweeteners and the like can also be added when necessary.
The application of the invention can be prepared into various forms such as pills, powder, tablets, granules, oral liquid, injection and the like besides capsules.
After the spleen cells are prepared, inducing by ConA or LPS, adding test compounds with different concentrations into a test group, culturing for 72 h, and respectively measuring the light absorption values of the test group, an induced control group and an uninduced control group by a CCK-8 reagent method so as to evaluate the capability of the compounds for stimulating the proliferation of T cells or B cells, wherein the experimental result shows that the polyenoic androsterone compound of the invention, namely 12 β -hydroxyandrost-3, 5,14(15) -trien-16-one, has obvious inhibiting effect on the proliferation of splenic lymphocytes of Balb/c mice stimulated by ConA (concanavalin), and has obvious difference compared with the control group without the compound (the compound is prepared by using healthy Balb/c mice) (thep<0.05)。
Inoculating cell suspension with certain concentration adjusted to human tumor cells in logarithmic growth phase to a 96-well culture plate, culturing for 24 hr, adding compounds with different concentrations after adherence, 10 μ l/well with 3 multiple wells for each concentration, counting cells, and determining cell survival rate by using a tray blue staining method, wherein the survival rate is × 100% of uncolored cells/total number of cells, the survival rate is more than 95% for test, and the cell suspension is adjusted to 1 × 106one/mL. Setting cell blank control group, positive control group (dexamethasone) and sample group (concentration is 25 μmol. L according to the result of preliminary test)-1All containing equal concentrations of DMSO). The negative control is equal volume of culture medium, and the corresponding DMSO concentration of compound 25. mu.g/mL is used as vehicle control to eliminate the influence of DMSO on cell growth. Adding the medicine, continuously placing at 37 ℃ and 5% CO2Culturing in incubator, adding 20 μ L MTT (5 mg/mL) per well after 1, 3, 6, 12, 18, and 24 hr of drug action, culturing for 4 hr, adding triple solution [ 10% SDS-5% isobutanol-0.012 mol/L HCl (W/V/V)]Formazan is dissolved in 100 mu l. Measuring OD value of each well at 570nm wavelength by enzyme labeling instrument to estimate cell proliferation inhibition rate。
Detailed Description
The present invention will be described in further detail with reference to the following examples, but the present invention is not limited thereto, and the method in the examples is carried out in a conventional manner unless otherwise specified, and reagents used therein are, for example, conventional commercially available reagents or reagents prepared in a conventional manner without specifically specified.
Example 1: preparation of 12 beta-hydroxyandrosta-3, 5,14(15) -trien-16-one
Taking 11.0kg of dry powder of overground parts (stems and leaves) of a Laos small-flower Sinomenii plant sample, carrying out reflux extraction for 3 times by using 75% ethanol, 48 hours each time, filtering to remove filter residues, combining filtrate obtained by three times of extraction, carrying out vacuum concentration, then carrying out extraction for 3 times by using ethyl acetate, and weighing a concentrated ethyl acetate extraction layer to obtain 352 g; the ethyl acetate layer extract was roughly separated by C18 column, and eluted with 20%, 40%, 50%, 60%, 80%, 100% by volume of aqueous methanol solution to give 6 fractions (fr.a, fr.b, fr.c, fr.d, fr.e) in total. Fr.d four fractions fr.b1-B4 were obtained by preparing the liquid phase and performing gradient elution with 40-80% by volume aqueous methanol. And then the Fr.B2 passes through a 200-mesh 300-mesh silica gel column, and chloroform-acetone (the volume ratio is 20:1-3: 1) is used as eluent for elution, so that four parts Fr.B2a-Fr.B2d are obtained in total. B2b was purified by semi-preparative high performance liquid chromatography (acetonitrile-water, 70: 30) to give the compound of formula ii (20 mg). The polyene androsterone compound in the formula II is a new compound through identification; the identification results are as follows:
the compound 12 β -hydroxyandrosta-3, 5,14(15) -trien-16-one of formula II is yellowish amorphous powder, and is easily soluble in chloroform-methanol mixture (chloroform: methanol =1: 1), pyridine, etcα]26 D - 98.5 (c0.04, MeOH); UV (MeOH)λ max(log): 203 nm (3.5); IR (KBr)ν max3473 2902, 2831, 1733, 1717, 1458,1375, 1329, 1047, 962 cm–1;HR-ESI-MSm/z284.1846 [M+H]+Bonding of13CNMR spectra to infer its molecular formula C19H24O2。1H NMR (CDCl3500Mz) and13C NMR(CDCl3125Mz) is shown in Table 1; to be provided withThe above data combined with 2D NMR analysis confirm that the chemical structural formula of the compound is shown as formula II, and the compound is a novel natural organic compound.
TABLE 1 preparation of 12 β -hydroxyandrosta-3, 5,14(15) -trien-16-one1H NMR and13c NMR data
Example 2: preparation of 12 beta-hydroxyandrosta-3, 5,14(15) -trien-16-one
Taking an air-dried 11 kg of Yunnan sipunculus stems sample, crushing, performing reflux extraction for 3 times by using methanol, recovering a solvent, concentrating to a small volume, extracting for 3 times by using ethyl acetate, concentrating an ethyl acetate layer, and weighing to obtain 1.2 kg; the ethyl acetate layer was roughly separated with macroporous adsorbent resin D101, and eluted with 40%, 60%, 80%, and 100% by volume of aqueous methanol to give a total of 5 fractions (Fr.A, Fr.B, Fr. C, Fr.D, Fr.E). Fr.E (280g) was stirred into 1.5 times the amount of silica gel, dried conventionally with silica gel, packed into a column, gradient-eluted with a chloroform-acetone (0: 100-95: 5) two-phase system as the mobile phase, and combined according to TLC inspection to give three fractions Fr.E 1-E3. Fr.e2 was eluted on a reverse phase C18 column methanol-water gradient using a C18 packing to give two fractions, fr.e2a-E2 b. E2a eluted through silica gel column with petroleum ether-acetone system, then through gel eluted with chloromethyl (1: 1, V/V) system and further through semi-preparative high performance liquid purification to give compound of formula ii (7 mg). The androstane compound in the formula II is identified as a novel natural organic compound.
Example 3: immunosuppressive detection assay
(1) Preparation of spleen lymphocyte suspension
Taking 18-22 g of healthy BABL/c mice to exsanguinate and kill, placing the mice in 75% alcohol for soaking and disinfecting for 5 minutes, taking out the mice, placing the mice in a sterile tray with the left side facing upwards in a super clean bench, clamping the skin hair in the middle of the abdomen with disinfected tweezers to form an incision, shearing off all layers of the abdominal wall with another set of instruments, taking out the spleen with a third set of instruments, removing fat and connective tissues, and placing PBS (phosphate buffered saline) (PBS)Buffer), floating blood was washed off. Then, the spleen tissue is moved to a plate containing RPMI 1640 incomplete culture solution, cut into small pieces by using scissors, the spleen is ground in a 200-mesh stainless steel screen mesh by using a sterile syringe core, the ground spleen is washed by PBS for a few times, and the suspension is transferred to a 15mL centrifuge tube by using a pipettor; centrifuging at 1000r/min for 5 min; the supernatant was aspirated off, and 3mL of erythrocyte lysate (Tris-NH) was added4Cl), standing for 2min, adding 10mL PBS to stop reaction, centrifuging (1200rpm, 5min), removing supernatant, washing precipitate with 5mL PBS twice, centrifuging under the same condition, suspending precipitate with 5mL RPMI 1640 complete culture solution containing 10% fetal calf serum, counting with 0.8% trypan blue viable cell exclusion method, adding RPMI 1640 complete culture solution to dilute, and adjusting cell concentration to 1 × 106About one/mL.
(2) Preparation of test solutions
2mg of monomeric compound is precisely weighed, dissolved by adding DMSO, and diluted to the required concentration by PBS before loading, and the final concentration of DMSO after loading is not more than 0.1%.
(3) Experiment grouping
Normal control group: 100 μ L spleen cell suspension +10 μ L RPMI 1640 complete Medium +10 μ L LPBS
Model group: mu.L spleen cell suspension + 10. mu.L of LCoA (final concentration 10. mu.g/mL) + 10. mu.L of LPBS
Sample group: mu.L spleen cell suspension + 10. mu.L LCoA (final concentration 10. mu.g/mL) + 10. mu.L sample
In 96-well plates, lymphocyte suspensions (1 × 10) were added to each well6one/mL) 100. mu.L, 10. mu.L of ConA (final concentration of 10. mu.g/mL), 10. mu.L of reagent compound dilutions (final concentrations of 12.5, 25, 50. mu.g/mL, respectively), and 4 replicates of dexamethasone were also prepared in three corresponding concentration groups, and wells of the normal control group were filled with 10. mu.L of 1640 complete medium (containing 10% fetal bovine serum) and 10. mu.L of PBS, respectively.
(4) Culturing: standing at 37 deg.C for 5% CO2Culturing in an incubator for 72 hours.
(5) CCK-8 method for determining OD value of cells
After 72 hours of incubation, in each wellAdding 10 μ L of CCK-8 reagent (Biyunyan), standing at 37 deg.C and 5% CO2After further culturing in the incubator for 4 hours, the absorbance at 450nm was measured per well to calculate the cell proliferation, and the Stimulation Index (SI) was calculated as follows:
SI (stimulation index) = OD value of mitogen-added culture/OD value of non-mitogen-added culture;
(6) data processing
The OD value of the experimental data is expressed by means of 'mean +/-standard deviation', and the mathematical statistics and the analysis of variance work are completed by Origin software.
(7) Results of the experiment
Table 2: stimulation index of ConA-stimulated Balb/c mouse spleen lymphocyte proliferation by Compounds of formula II
The 12 β -hydroxyandrosta-3, 5,14(15) -trien-16-one in the formula II has stimulation indexes of 2.90, 2.52 and 1.79 at the concentrations of 12.5, 25 and 50 mu M respectively, and the significance analysis shows that the middle concentration group and the high concentration group have significant difference compared with the model group (the significant difference is shown in the formula II) (the significant analysis shows that the stimulation indexes of the middle concentration group and the high concentration group are respectively 2.90, 2.52 and 1.79)P<0.05）。
The positive control results were:
table 3: stimulation index of dexamethasone to ConA-stimulated splenic lymphocyte proliferation in Balb/c mice
The stimulation indexes of dexamethasone at the concentrations of 12.5, 25 and 50 mu M are respectively 1.62, 1.41 and 1.21; the significance analysis shows that each concentration group has significant difference compared with the model group (P<0.05）。
The experimental result shows that 12 β -hydroxyandrosta-3, 5,14(15) -trien-16-one has immunosuppressive activity at the concentration of 12.5 mu M and has significant difference compared with the model group at the concentrations of 25 and 50 mu M (theP<0.05), has better immunosuppressive activity.
Example 4: in vitro antitumor Activity test
(1) Material
DMSO (Sigma, USA), fetal bovine serum (HyClone, USA), RPMI-1640 culture medium (HyClone, USA), phosphate buffer (Beyotime, Shanghai), double antibody (HyClone, USA), CCK-8 (Token chemical technology, Inc.), human tumor cells (CCRF-CEM, MOLT-4, K-562, MALME-3M, UACC-62, SNB-75, OVCAR8, EKVX, U0-31, SF-295, NCI-H226, SK-OV3, MDA _ MB-468, Hop 92), the compound of the invention and dexamethasone were all formulated with DMSO.
(2) Method of producing a composite material
Inoculating cell suspension with certain concentration adjusted to human tumor cells in logarithmic growth phase to a 96-well culture plate, culturing for 24 hr, adding compounds with different concentrations after adherence, 10 ml/well with 3 multiple wells for each concentration, counting cells, and determining cell survival rate by using a tray blue staining method, wherein the survival rate is × 100% of uncolored cells/total cells, the survival rate is more than 95% for test, and the cell suspension is adjusted to 1 × 106one/mL. Setting cell blank control group, positive control group (dexamethasone) and sample group (concentration is 25 μmol. L according to the result of preliminary test)-1All containing equal concentrations of DMSO). The negative control is equal volume of culture medium, and the corresponding DMSO concentration at 25mg/ml of compound is used as a solvent control to eliminate the influence of DMSO on cell growth.
(3) Culturing
Adding the medicine, continuously placing at 37 ℃ and 5% CO2Culturing in an incubator.
(4) Determination of OD value by MTT method
After the drugs respectively act for 1, 3, 6, 12, 18 and 24 hours, 20ml of MTT (5 mg/ml) is added into each hole, the culture is continued for 4 hours, and 100ml of triple liquid [ 10% SDS-5% isobutanol-0.012 mol/L HCl (W/V/V) ] is added into each hole to dissolve the formazan, and under the normal condition, the formazan generation amount is in direct proportion to the number of viable cells, so that the number of the viable cells can be presumed according to the OD value of the optical density, and the cell inhibition ratio is calculated according to the following formula:
inhibition ratio (Percent) to (OD value)Control wellOD valueSample adding hole) OD valueControl well×100％；
(5) Data processing
The OD value of the experimental data is expressed by means of 'mean +/-standard deviation', and the mathematical statistics and the analysis of variance work are completed by Origin software.
(6) Results of the experiment
The compound of the formula II has better inhibition effect on 14 human tumor cell strains; the result shows that 5 strains of human tumor cells (MOLT-4, K-562, SNB-75, SF-295 and NCI-H226) have drug resistance to dexamethasone, and the compound of the formula II has better cytotoxic activity; in addition, the compound of the formula II has the inhibiting effect on other 9 tumor cells (SK-OV 3, MDA _ MB-468, Hop92, EKVX, U0-31, OVCAR8, UACC-62 and MALME-3M, CCRF-CEM) and has the activity equivalent to that of a positive control.
Table 4: effect of Compounds of formula II on the survival of 5 human tumor cells
Claims (2)
1. A polyene androsterone compound having the formula:
the polyene androsterone compound has the name of 12 beta-hydroxyandrosta-3, 5,14(15) -triene-16-one.
2. Use of a polyene androsterone compound of claim 1 in the manufacture of an immunosuppressive drug.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103073607A (en) * | 2013-02-26 | 2013-05-01 | 昆明理工大学 | 12[beta]-hydroxyandrostane-4,6,8(9),13(14)-tetraene-3,11,16-triketone and application thereof |
| CN103073608A (en) * | 2013-02-26 | 2013-05-01 | 昆明理工大学 | Androstane-4, 6, 8 (9), 13 (14)-tetraene-3, 11, 16-triketone and application thereof |
| CN103142621A (en) * | 2013-03-28 | 2013-06-12 | 昆明理工大学 | Medical applications of four buxus alkaloids compounds |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103073607A (en) * | 2013-02-26 | 2013-05-01 | 昆明理工大学 | 12[beta]-hydroxyandrostane-4,6,8(9),13(14)-tetraene-3,11,16-triketone and application thereof |
| CN103073608A (en) * | 2013-02-26 | 2013-05-01 | 昆明理工大学 | Androstane-4, 6, 8 (9), 13 (14)-tetraene-3, 11, 16-triketone and application thereof |
| CN103142621A (en) * | 2013-03-28 | 2013-06-12 | 昆明理工大学 | Medical applications of four buxus alkaloids compounds |
Non-Patent Citations (1)
| Title |
|---|
| 《Cytotoxicity and Synergistic Effect of the Constituents from Roots of Aglaia odorata (Meliaceae)》;Liu, Bing;《Natural Product Research》;20161231;第30卷(第4期);全文 * |
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