CN104744547A - Pregnane alkaloid derivative with effect of resisting breast cancer metastasis and medical application of pregnane alkaloid derivative - Google Patents

Pregnane alkaloid derivative with effect of resisting breast cancer metastasis and medical application of pregnane alkaloid derivative Download PDF

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CN104744547A
CN104744547A CN201310749522.7A CN201310749522A CN104744547A CN 104744547 A CN104744547 A CN 104744547A CN 201310749522 A CN201310749522 A CN 201310749522A CN 104744547 A CN104744547 A CN 104744547A
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acid
cdcl
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CN104744547B (en
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段宏泉
秦楠
靳美娜
寿晓媛
甘椿椿
刘倩
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Tianjin Medical University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
    • C07J41/0005—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring the nitrogen atom being directly linked to the cyclopenta(a)hydro phenanthrene skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
    • C07J41/0005—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring the nitrogen atom being directly linked to the cyclopenta(a)hydro phenanthrene skeleton
    • C07J41/0011—Unsubstituted amino radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J43/00—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton
    • C07J43/003—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton not condensed

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Abstract

The invention provides a pregnane alkaloid derivative with an effect of resisting breast cancer cell metastasis or a pharmaceutically acceptable salt thereof. The structure of the pregnane alkaloid derivative is shown by a general formula I in the specification, wherein R1 and R2 are independently selected from hydrogen, straight-chain or branched alkyl with the carbon atom number of 1-8, naphthenic base, unsaturated alkyl, benzyl and benzene ring substituted benzyl respectively; or R1R2N is piperidyl or pyrrolyl; R3 and R4 are independently selected from hydrogen and hydroxyl respectively; or R3R4 together represents carbonyl oxygen atoms; R5 and R6 are different substituting groups and are independently selected from hydrogen or methyl respectively; and the pharmaceutically acceptable salt is a salt formed with inorganic acid or organic acid, wherein the inorganic acid or organic acid is selected from hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methane sulfonic acid, benzene methane sulfonic acid, oxalic acid, tartaric acid, maleic acid, citric acid or ascorbic acid. The invention also provides an application of the pregnane alkaloid derivative in preparation of a medicament for resisting breast cancer metastasis.

Description

A kind of pregnane alcaloid-derivatives of anti-breast cancer transferance and medicinal use
Technical field
The present invention relates to a kind of pregnane alcaloid-derivatives, preparation method and the application as anti-breast cancer diversion medicaments thereof, belong to field of pharmaceutical chemistry research.
Background technology
In recent years, tumor incidence is more and more higher, has become the number one killer that harm people are healthy.And metastases is the major obstacle of oncotherapy, according to incompletely statistics, in the clinical tumor patient made a definite diagnosis, there is transfer in the patient of about 60%.Tumour has become one of major disease of current serious threat human health.Along with the progress of life science and the development of medical science, the mankind have been deep into molecular cell aspect to the understanding of tumour, and various new operative treatment means and chemotherapeutics constantly come out.But according to american cancer institute statistics, over more than 50 years, cancer therapy is not taken on a new look, and cancer mortality still remains high, and major cause is metastases, does not have effective treatment means and the medicine of metastases at all.Current universally acknowledged transfer is malignant tumor patient main causes of death, and the malignant tumor patient of about 90% dies from metastases.Therefore, controlling transfer is the key factor determining tumour patient prognosis.
Chemotherapy is the critical treatment means of primary tumo(u)r treatment and Post operation prevention transfer.The clinical practice of decades shows, the toxic side effect that the generation of chemotherapeutics multidrug resistance and chemotherapeutics are very serious, not only seriously undermine chemotherapy effect, and the immunity system of chemotherapeutics grievous injury patient, substantially the first barrier of opposing tumor cell invasion migration is destroyed, tumor recurrence rate in half a year is up to 69%, and this is chemotherapeutical dilemma.Up to now, medicine for anti transfer of tumor is not still had to come out in the world.
Metastases is expanded and diffusion directly to surrounding tissue by tumour original site, or settle in remote organization the process that development forms nascent tumor by blood road or lymphatic metastasis oncocyte.The tumour cell transfer in vivo in various source has its fixing target organ as a rule, and namely these have metastatic tumour ubcellular group and are controlling well can form neoplasm metastasis according to certain process in specific region under experiment condition.Although in different neoplastic processes, can there is different changes in the gene of tumour cell, roughly the same in the transfer process of tumour.Calendar year 2001, ZlotnikA group study finds, the Chemotaxis of cell plays a part key in the metastasis and extension of tumour cell.Chemokine CXCL12 can inducing mammary cancer cells generation Chemotaxis.If use the antibody of the receptor CXCR 4 activity of chemokine inhibiting CXCL12, can weaken the chemotactic ability of breast cancer cell, and suppress it to spread, the Chemotaxis of inhibition tumor cell can the metastasis and extension of inhibition tumor cell effectively.
Therefore, in interference or block in the process sent out in host of malignant tumour, the Chemotaxis of inhibition tumor cell is most important link in medicine for anti transfer of tumor research, is to extend lifetime, improve life quality and reduce the most effective means of mortality ratio.On the other hand, because the medicine for anti transfer of tumor treatment after conventional primary tumor resection is lasting, long term administration process, desirable medicine for anti transfer of tumor not only blocks tumour cell Invasion and Metastasis in vivo, and should be that non-cytotoxic drugs or toxic side effect are very low, can not cause damage to immunity system.In view of limitation and the toxic side effect thereof of current anti metastasis chemotherapeutical medicine curative effect.Security is found high and can the medicine of effective prevention and corntrol metastases more and more come into one's own from Chinese medicine.
This study group patent of invention discloses a kind of Pachysandra terminalis alkaloid compound (application number: 201010114809.9) of inhibiting effect on tumor metastasis, on this invention basis, this study group is based on activeconstituents Salignone, has synthesized a series of derivative, and has carried out the research of anti-breast cancer transfer activity, wherein majority of compounds shows stronger anti-breast cancer transfer activity, compound 5,17a, 18a, 21d, 22c, 23f, 26b and 26c are particularly outstanding.
Summary of the invention
The object of the present invention is to provide a kind of there is anti-breast cancer transferance pregnane alcaloid-derivatives and described compound for the preparation of the purposes in anti-breast cancer diversion medicaments.
The present invention is realized by following technical proposals:
On the one hand, the invention provides a kind of pregnane alcaloid-derivatives or its pharmacy acceptable salt, its structure as shown in Equation 1:
Wherein, R 1with R 2separately be selected from hydrogen, the straight or branched alkyl of carbonatoms 1-8, cycloalkyl, unsaturated alkyl, benzyl, the cyclosubstituted benzyl of benzene; Or R 1r 2n is piperidyl or pyrryl;
R 3with R 4separately be selected from hydrogen, hydroxyl or R 3r 4represent carbonylic oxygen atom;
R 5with R 6for different substituting groups is separately selected from hydrogen or methyl;
Described pharmacy acceptable salt refers to and the salt that mineral acid or organic acid are formed, and wherein mineral acid or organic acid are selected from hydrochloric acid, Hydrogen bromide, sulfuric acid, phosphoric acid, methylsulfonic acid, benzene methanesulfonic acid, oxalic acid, tartrate, toxilic acid, citric acid or xitix.
The preparation method of a kind of pregnane alcaloid-derivatives provided by the invention or its pharmacy acceptable salt, its key step is: with androsterone or epiandrosterone for raw material introduces double bond through Wittig reaction, further introduce amino through Mitsunobu reaction and hydrazinolysis reaction, further introduce hydroxyl and carbonyl through Riley oxidizing reaction and Dess-Martin oxidation.Wherein, phthaloyl and tertiary fourth oxygen carbonic acyl radical are amino oxidized to avoid as amino protecting group.Part target derivative through halogenating reaction or reductive amination process or reduction of amide reaction obtained, another part target derivative then removes obtained through protecting group.
Concrete synthetic route of the present invention is as shown in Equation 2:
The synthetic route of the pregnant steroidal alkaloid derivative of formula 2
On the other hand, the invention provides a kind of pregnane alcaloid-derivatives or its pharmacy acceptable salt is preparing the purposes in anti-breast cancer diversion medicaments.
First, the present invention to synthesis pregnane alcaloid-derivatives carried out Cytotoxic mensuration, further with non-cytotoxicity dosage carried out anti-breast cancer transfer activity screening, the derivatives chemical structure of screening as shown in Equation 3:
The chemical structure of formula 3 pregnane alcaloid-derivatives
Each derivative is to the toxic action of breast cancer cell
MTT colorimetric estimation ratio juris: this analytical procedure, to reduce based on 3-(4,5-dimethylthiazole-2-base)-2,5-diphenyltetrazolium bromide (MTT), is a kind of common method detecting cell survival and upgrowth situation.MTT is a kind of general stain for cell, the succinodehydrogenase relevant to NADP is there is in the plastosome of viable cell, exogenous yellow MTT can be reduced to insoluble bluish voilet crystallisate formazan (Formazane), and be deposited in cell, this enzyme of dead cell disappears, and MTT is not reduced.Detect the change size of optical density(OD) after dissolving Formazane with DMSO by microplate reader at 550nm wavelength place, weigh the growth-inhibiting effect of testing drug to cell, and then evaluate cytotoxicity.
Experimental procedure: by cultured cell with 0.25% trysinization, after sucking-off pancreatin, digestion is stopped with the nutrient solution containing 10%FBS, mixing cell suspension, counting regulating density is 1x104, the cell suspension mixed up is added on 96 orifice plates, every hole 180ul, be placed in 37 DEG C, cultivate 24 hours in the incubator of 5%CO2, dosing after 24 hours, the multiple hole of each concentration 5, continue after having added medicine to be placed in 37 DEG C, cultivate 48 hours in the incubator of 5%CO2, every hole adds the MTT20ul of 5mg/ml afterwards, continue to be placed in 37 DEG C, cultivate 4 hours in the incubator of 5%CO2, 96 plates are taken out after 4 hours, by supernatant sucking-off, every hole adds the DMSO of 100ul, under 570nm wavelength, absorbancy is measured by microplate reader.Inhibiting rate=(1-dosing group OD value/blank group OD value) x100%
Anti-breast cancer transfer activity evaluates Transwell chemotaxis Method And Principle:
Directly 8 μm of aperture filter membranes are placed in up and down between chamber of chamber of invasion and attack at Transwell cell epicoele, tumour cell by amoeboid movement through filter membrane, can the size of analysis of cells motor capacity with this model.Chemotactic inductor (chemokine EGF) is added when drug screening, by weighing with the tumour cell of testing drug Dual culture and blankly organize the cell quantity that tumour cell chemotactic passes filter membrane and change, express the size of the migration ability of testing drug inhibition tumor cell, and then the activity of evaluation test drugs against tumor transfer is strong and weak.Positive control is reagent LY294002 (a kind of P13K inhibitor with inhibiting effect on tumor metastasis).
Experimental procedure:
1. the screening of sample non-cytotoxicity dosage: utilize mtt assay to filter out the sample concentration of non-cytotoxicity effect.
2. sample and co-culture of cells: cell is laid on 6 orifice plates, is placed in 37 DEG C, 5%CO 2incubator in cultivate within 24 hours, make it adherent, with the concentration determined, sample is added in 6 orifice plates after 24 hours, 37 DEG C of 5%CO 224 hours are hatched altogether in incubator.
3. Chemotaxis test: after the cell crossed by sample preparation being used respectively the trysinization of 0.25%, stops digestion, centrifugal 5 minutes of this cell suspension 1300rpm with the nutrient solution containing 10%FBS.Poured out by supernatant, recentrifuge after cell mixing after 5 minutes, counts, by cell density furnishing 5x10 by the BM adding 0.1% 5, be placed in incubator for subsequent use.In configuring chemokine EGF on ice, be added on the lower room of chemotactic cell, every hole 30ul, bag is laid on lower room by good film, put rubber cushion well, fixing upper room, room will be added to by ready cell suspension before, every hole 50ul, the multiple hole of each sample concentration 3, adds and is placed on CO 2take out after cultivating 3.5h in incubator, take out lower room, scrape off the cell not having chemotactic to come, be then fixed with three step staining reagents and dye, after dyeing, film paraffin oil is fixed, in counted under microscope.
4. statistical calculation method: during counting, each hole is chosen 5 visuals field and is counted respectively, averages, and three multiple holes are averaged the chemotactic cell count be under this sample concentration again.Data SPSS11.5 process, calculates half inhibiting rate IC 50.
Calculation formula:
Experimental result
Preferred compound is as shown in table 1 to MB-MDA-231 (human breast cancer cell) chemotactic inhibiting rate:
Table 1 preferred compound is to MB-MDA-231 chemotactic inhibiting rate
The evaluation of anti-breast cancer transfer activity shows, preferred compound all demonstrates anti-breast cancer transfer activity under non-cytotoxicity dosage, wherein compound 5,17a, 18a, 21d, 22c, 23f, 26b and 26c activity particularly outstanding.
Embodiment
Embodiment 1.
The synthetic method of compound 1 and 2
Take 4 equivalent potassium tert.-butoxides and 4 equivalent ethyltriphenylphosphonium bromides in reaction flask, add appropriate anhydrous THF, in stirred at ambient temperature 1 hour.In reaction solution, add epiandrosterone or the androsterone of 1 equivalent, reflux under nitrogen protection, TLC monitoring reacts completely.Add saturated aqueous ammonium chloride termination reaction, dichloromethane extraction, wash 3 times, merge organic phase, anhydrous magnesium sulfate drying, filter, concentrate to obtain crude product.Crude product obtains sterling through methylene chloride-methanol recrystallization.
(Z) synthesis (1) of the pregnant steroid-17 of-3 beta-hydroxy (20)-alkene
Be raw material with epiandrosterone, prepare as stated above, crude product obtains white solid through recrystallizing methanol, productive rate 97.8%. 1HNMR(400MHz，CDCl 3)δ5.11(q，J＝6.9Hz，1H)，3.59(m，1H)，1.65(d，J＝6.9Hz，3H)，0.87(s，3H)，0.82(s，3H). 13C NMR(100MHz，CDCl 3)δ150.5，113.2，71.3，56.3，54.4，44.8，44.4，38.2，37.2，37.0，35.5，31.9，31.4，28.7，24.4，21.4，16.9.ESI-MS m/z：301[M-H] -.
(Z) synthesis (2) of the pregnant steroid-17 of-3 Alpha-hydroxy (20)-alkene
Be raw material with androsterone, prepare as stated above, crude product obtains white solid through recrystallizing methanol, productive rate 84.0%. 1HNMR(400MHz，CDCl 3)δ5.12(qt，J1＝2.0，J2＝7.1Hz，1H)，4.06(s，1H)，1.67(dt，J＝2.0，7.1Hz，3H)，0.88(s，3H)，0.81(s，3H). 13C NMR(100MHz，CDCl 3)δ150.5，113.2，66.6，56.3，54.3，44.4，39.1，37.2，36.2，35.9，35.0，32.2，31.9，31.4，29.0，28.5，24.3，21.0，16.9，13.1，11.2.ESI-MS m/z：301[M-H]-.
Embodiment 2.
The synthetic method of compound 3 and 4
Take 1.13 equivalent triphenylphosphines, 1.1 equivalent phthalic imidines and 1 equivalents of compound 1 or 2, in reaction flask, add appropriate anhydrous tetrahydro furan and stir 1 hour under ice baths.In reaction solution, add 2 equivalent diisopropyl azodiformates, stir in room temperature lower seal, TLC monitoring is to reacting end.The cancellation that adds water is reacted, and concentrating under reduced pressure removing THF, dichloromethane extraction, washes three times, merges organic phase, anhydrous magnesium sulfate drying, filters, and concentrates to obtain crude product.Crude product obtains sterling through recrystallization.
(Z) synthesis (3) of the pregnant steroid-17 of-3 β-phthaloyl imino (20)-alkene
With 9.66g compound 2 for raw material, prepare as stated above, crude product methyl alcohol and methylene dichloride mixed solvent recrystallization obtain white solid 5.07g, productive rate 36.3%. 1H NMR(400MHz，CDCl 3)δ7.82-7.79(m，2H)，7.70-7.68(m，2H)，5.13(qt，J 11.9，7.2Hz，1H)，4.22-4.13(m，1H)，1.66(dt，J＝1.9，7.2Hz，3H)，0.97(s，3H)，0.88(s，3H). 13C NMR(100MHz，CDCl 3)δ168.5，150.5，133.8，132.1，123.0，113.2，56.2，54.1，50.6，46.1，44.4，37.8，37.2，35.6，35.1，31.8，31.7，31.4，28.6，25.1，24.4，21.3，16.9，13.1，12.2.ESI-MS m/z：430[M-H]-.
(Z) synthesis (4) of the pregnant steroid-17 of-3 α-phthaloyl imino (20)-alkene
With 12.50g compound 1 for raw material, prepare as stated above, crude product methyl alcohol and methylene dichloride mixed solvent recrystallization obtain white solid 4.34g, productive rate 21.3%. 1H NMR(400MHz，CDCl 3)δ7.81-7.82(m，2H)，7.71-7.69(m，2H)，5.12(q，J＝6.8Hz，1H)，4.51(br s，1H)，1.66(d，J＝6.8Hz，3H)，0.89(s，3H)，0.87(s，3H). 13CNMR(100MHz，CDCl 3)δ169.2，155.6，133.8，132.1，122.9，119.3，74.4，54.5，52.4，47.5，44.5，40.4，37.4，35.1，35.0，34.4，32.7，31.6，31.6，28.5，24.6，20.9，17.6，13.2，12.4.ESI-MS m/z：430[M-H]-.
Embodiment 3.
The synthetic method of compound 5,6,11 and 12
Take 1 when content of starting materials is in reaction flask, add proper amount of methanol and the backflow of 40 equivalent 80% hydrazine hydrates.TLC monitoring reaction terminates.Be cooled to room temperature, hydro-oxidation sodium water solution (4 ~ 6mol/L) in right amount to solution be white opacity.Suction filtration, washing, obtain crude product after drying, crude product obtains sterling through HW-40 gel column chromatography purifying.
(Z) synthesis (5) of the pregnant steroid-17 of-3 beta-amino (20)-alkene
With compound 3 for raw material, prepare as stated above, crude product obtains white solid through HW-40 gel column chromatography (DCM: MeOH=2: 1, V/V) purifying, and productive rate is 68.3%. 1H NMR(400MHz，CDCl 3)δ5.10(qt，J＝2.0，6.8Hz，1H)，2.67(br s，1H)，1.64(dt，J＝2.0，6.8Hz，3H)，0.86(s，3H)，0.79(s，3H). 13C NMR(100MHz，CDCl 3)δ150.5，113.2，56.3，54.5，51.1，45.5，44.4，37.6，37.2，35.6，35.1，31.9，31.4，31.3，28.7，28.6，24.4，21.3，16.9，13.1，12.3.ESI-MS m/z：300[M-H]-.
(Z) synthesis (6) of the pregnant steroid-17 of-3 alpha-amino group (20)-alkene
With compound 4 for raw material, prepare as stated above, crude product obtains white solid through HW-40 gel column chromatography (DCM: MeOH=2: 1, V/V) purifying, and productive rate is 80.3%. 1H NMR(400MHz，CDCl 3)δ5.10(q，J＝6.8Hz，1H)，3.16(br s，1H)，1.64(d，J＝6.8Hz，3H)，0.86(s，3H)，0.79(s，3H). 13C NMR(100MHz，CDCl 3)：δ150.2，113.1，56.2，54.4，49.4，45.6，44.3，39.1，37.1，36.3，34.9，32.0，31.8，31.3，28.9，28.6，24.3，20.9，16.8，13.0，11.2.ESI-MS m/z：300[M-H]-.
(E) synthesis (11) of the pregnant steroid-17 of-3 beta-amino-16 Alpha-hydroxy (20)-alkene
With compound 9 for raw material, prepare as stated above, crude product obtains white solid through HW-40 gel column chromatography (DCM: MeOH=2: 1, V/V) purifying, and productive rate is 83.2%. 1H NMR(400MHz，CDCl 3)δ5.57(q，J＝6.8Hz，1H)，4.42(d，J＝4.4Hz，1H)，2.66(br s，1H)，1.73(d，J＝6.8Hz，3H)，0.86(s，3H)，0.80(s，3H). 13C NMR(100MHz，CDCl 3)δ155.6，119.3，74.454.5，52.5，51.1，45.5，44.5，39.4，37.6，37.4，35.6，35.1，34.4，32.6，31.9，28.7，21.2，17.6，13.2，12.3.ESI-MS m/z：316[M-H]-.
(E) synthesis (12) of the pregnant steroid-17 of-3 alpha-amino group-16 Alpha-hydroxy (20)-alkene
With compound 10 for raw material, prepare as stated above, crude product obtains white solid through HW-40 gel column chromatography (DCM: MeOH=2: 1, V/V) purifying, and productive rate is 62.3%. 1H NMR(400MHz，CDCl 3)δ5.58(q，J＝7.2Hz，1H)，4.43(d，J＝4.9Hz，1H)，3.21(br s，1H)，1.74(dt，J＝7.2Hz，3H)，0.87(s，3H)，0.81(s，3H). 13C NMR(100MHz，CDCl 3)δ155.5，119.3，74.3，54.4，52.5，45.8，44.6，39.1，37.4，36.4，36.0，35.1，32.0，31.8，28.9，28.6，20.8，17.6，13.2，11.3.ESI-MS m/z：316[M-H]-.
Embodiment 4.
The synthetic method of compound 9 and 10
By 0.5 equivalent tin anhydride and 7.9 equivalent peroxy tert-butyl alcohols, under ice bath, stir 1h, drip the dichloromethane solution that 1 works as content of starting materials, continue ice bath reaction.TLC monitoring reaction terminates.With 10% sodium sulfite solution cancellation, stir 15min, with dichloromethane extraction, merge organic phase, concentrating under reduced pressure, dry crude product, crude product obtains sterling through flash chromatography on silica gel purifying.
(E) synthesis (9) of the pregnant steroid-17 of-3 β-phthaloyl imino-16 Alpha-hydroxy (20)-alkene
With 1000mg compound 3 for raw material, prepare as stated above, after 3.5h, reaction terminates.Crude product obtains white solid 896.8mg through flash chromatography on silica gel (PE: EA=8: 1-3: 1, V/V) purifying, productive rate 86.5%. 1HNMR(400MHz，CDCl 3)δ7.82-7.80(m，2H)，7.70-7.68(m，2H)，5.58(q，J＝7.2Hz，1H)，4.43(d，J＝5.2Hz，1H)，4.22-4.13(m，1H)，1.74(d，J＝7.2Hz，3H)，0.99(s，3H)，0.89(s，3H). 13C NMR(100MHz，CDCl 3)δ168.5，155.6，133.8，132.1，123.0，119.4，74.4，54.1，52.5，50.5，46.1，44.5，37.7，37.4，35.6，35.1,34.4，31.8，31.7，28.5，25.1，21.2，17.6，13.2，12.2.ESI-MS m/z：446[M-H]-.
(E) synthesis (10) of-3 α-phthaloyl imino-16-monohydric pregnant-17 (20)-alkene
With 504.0mg compound 4 for raw material, prepare as stated above, after 3h, reaction terminates.Crude product obtains white solid 323.2mg through flash chromatography on silica gel (PE: EA=8: 1-4: 1, V/V) purifying, productive rate 61.8%. 1H NMR(400MHz，CDCl 3)δ7.82-2.80(m，2H)，7.71-7.69(m，2H)，5.56(q，J＝7.2Hz，1H)，4.49(br s，1H)，4.41(d，J＝5.4Hz，1H)，1.72(d，J＝7.2Hz，3H)，0.87(s，3H)，0.86(s，3H). 13C NMR(100MHz，CDCl 3)δ169.2，155.6，133.8，132.1，122.9，119.4，74.4，54.5，52.4，47.5，44.5，40.4，37.4，35.1，34.9，34.5，32.7，31.6，28.5，24.6，20.9，17.6，13.2，12.4.ESI-MS m/z：446.6[M-H]-.
Embodiment 5.
Compound 7a, the synthetic method of 8a, 13a and 14a
By 1 when content of starting materials claims in reaction flask, add q. s. methylene chloride and dissolve, drip 1.5 equivalent Acetyl Chloride 98Min.s under ice bath, drip room temperature reaction after finishing, TLC monitoring is to reacting end.Add saturated sodium bicarbonate aqueous solution cancellation reaction, dichloromethane extraction, merge organic phase, concentrating under reduced pressure, dry crude product, crude product obtains sterling through flash chromatography on silica gel purifying.
(Z) synthesis (7a) of the pregnant steroid-17 of-3 β-kharophen (20)-alkene
With 327mg compound 5 and Acetyl Chloride 98Min. for raw material, prepare as stated above, crude product obtains white solid 238mg through flash chromatography on silica gel (PE: EA=2: 1, V/V) purifying, productive rate 67.44%. 1H NMR(400MHz，CDCl 3)δ5.67(d，J＝8.0Hz，1H)，5.10(qt，J＝2.0，7.2Hz，1H)，3.78-3.70(m，1H)，1.95(s，3H)，1.64(dt，J＝2.0，7.2Hz，3H)，0.86(s，3H)，0.80(s，3H). 13C NMR(100MHz，CDCl 3)δ169.3，150.3，113.2，56.2，54.3，49.0，45.3，44.3，37.3，37.2，35.5，35.4，35.0，31.8，31.4，28.8，28.5，24.4，23.5，21.3，16.9，13.1，12.2.ESI-MSm/z：342[M-H] -.
(Z) synthesis (8a) of the pregnant steroid-17 of-3 alpha-acetamido-(20)-alkene
With 600mg compound 6 and Acetyl Chloride 98Min. for raw material, prepare as stated above, crude product obtains white solid 647.51mg through flash chromatography on silica gel (PE: EA=3: 1-1: 2, V/V) purifying, productive rate 90.0%. 1H NMR(400MHz，CDCl 3)δ5.84(brs，1H)，5.10(qt，J＝1.9，7.2Hz，1H)，4.10(d，J＝7.0Hz，1H)，1.98(s，3H)，1.64(dt，J＝1.9，7.2Hz，3H). 13C NMR(100MHz，CDCl 3)δ169.2，150.4，113.3，56.4，54.6，44.8，44.4，41.0，37.2，36.1，35.0，33.2，32.8，31.8，31.4，28.5，28.4，26.0，24.3，23.7，21.0，16.9，13.1，11.4.ESI-MS m/z：342[M-H] -.
(E) synthesis (13a) of the pregnant steroid-17 of-3 β-acetylaminohydroxyphenylarsonic acid 16 Alpha-hydroxy (20)-alkene
With compound 11 for raw material, prepare as stated above, after 2h, reaction terminates.Crude product is through flash chromatography on silica gel (PE :eA=1: 4, V/V) purifying obtains white solid, productive rate 44%. 1H NMR(400MHz，CDCl 3)δ5.58(dd，J＝1.1，7.2Hz，1H)，5.35(d，J＝7.5Hz，1H)，4.43(d，J＝5.2Hz，1H)，3.77(m，1H)，1.96(s，3H)，1.74(dd，J＝1.1，7.2Hz，3H)，0.87(s，3H)，0.82(s，3H). 13C NMR(100MHz，CDCl 3)δ169.2，155.5，119.4，74.4，54.3，52.4，48.9，45.3，44.5，37.4，37.3，35.5，35.4，35.0，34.4，31.7，28.9，28.4，23.6，21.2，17.6，13.2，12.2.ESI-MS m/z：358.6[M-H]-.
(E) synthesis (14a) of-3 α-acetamido-16 Alpha-hydroxy-pregnant steroid-17 (20)-alkene
With compound 12 for raw material, prepare as stated above, after 1h, reaction terminates.Crude product obtains white solid through flash chromatography on silica gel (PE: EA=1: 3-1: 6, V/V) purifying, productive rate 45.2%. 1H NMR(400MHz，CDCl 3)δ5.73(br s，1H)，5.35(qd，J＝1.6，7.2Hz，1H)，4.79(d，J＝5.2Hz，1H)，4.13(s，1H)，1.99(s，3H)，1.78(d，J＝7.2Hz，3H)，0.82(s，3H)，0.73(s，3H). 13C NMR(100MHz，CDCl 3)δ169.2，156.2，117.5，71.1，55.0，51.7，44.7，44.1，41.1，36.7，36.2，35.9，34.6，33.2，32.8，31.9，28.4，26.0，23.7，20.8，20.6，14.6，11.4.ESI-MS m/z：358[M-H]-.
Embodiment 6.
Compound 7g, the synthetic method of 8g, 13g and 14g
By 1 when a small amount of tetrahydrofuran (THF) of content of starting materials dissolves, add 2 equivalent sodium bicarbonates under ice bath, drip the tetrahydrofuran solution of 2 equivalent tert-Butyl dicarbonates, continue stirred at ambient temperature, TLC monitoring reacts completely, after solvent evaporated, dichloromethane extraction, washing twice, then wash once with saturated sodium-chloride water solution, merge organic phase, anhydrous magnesium sulfate drying, filter, concentrate to obtain crude product, crude product obtains sterling through flash chromatography on silica gel purifying.
(Z) synthesis (7g) of-3 β-pregnant steroid-17 of tertiary fourth oxygen formamido group (20)-alkene
With compound 5 for raw material, prepare as stated above, after 20h, reaction terminates.Crude product obtains white solid through flash chromatography on silica gel purifying (PE: EA=20: 1-14: 1, V/V), productive rate 47.7%. 1H NMR(400MHz，CDCl 3)δ5.11(qt，J＝1.9，7.1Hz，1H)，4.37(s，1H)，3.41(br s，1H)，1.64(dt，J＝1.9，7.1Hz，3H)，1.44(s，9H)，0.86(s，3H)，0.78(s，3H). 13C NMR(100MHz，CDCl 3)δ155.3，150.4，113.2，79.0，56.2，54.3，50.1，45.4，44.3，37.4，37.2，35.8，35.5，35.0，31.8，31.4，29.2，28.5，28.4，24.4，21.3，16.9，13.1，12.2.ESI-MSm/z：401[M-H]-.
(Z) synthesis (8g) of-3 α-pregnant steroid-17 of tertiary fourth oxygen formamido group (20)-alkene
With compound 6 for raw material, prepare as stated above, after 15h, reaction terminates.Crude product obtains white solid through flash chromatography on silica gel purifying (PE: EA=12: 1, V/V), productive rate 12.6%. 1H NMR(400MHz，CDCl 3)δ5.11(qt，J＝2.0，7.1Hz，1H)，3.84(s，1H)，1.65(dt，J 1＝2.0Hz，J 2＝7.1Hz，3H)，1.45(s，9H)，0.86(s，3H)，0.80(s，3H). 13C NMR(100MHz，CDCl 3)δ150.4，113.2，56.4，54.5，44.4，37.2，36.1，35.0，33.3，31.8，31.4，28.5，26.4，24.3，21.0，16.9，13.1，11.4.ESI-MS m/z：400.6[M-H]-.
(E) synthesis (13g) of-3 β-pregnant steroid-17 of tertiary fourth oxygen formamido group-16 Alpha-hydroxy (20)-alkene
With compound 11 for raw material, prepare as stated above, after 17h, reaction terminates.Crude product obtains white solid through flash chromatography on silica gel (PE: EA=8: 1-4: 1, V/V), productive rate 80.1%. 1H NMR(400MHz，CDCl 3)δ5.57(q，J＝6.8Hz，1H)，4.41(d，J＝4.8Hz，1H)，3.41(br s，1H)，1.73(d，J＝6.8Hz，3H)，1.73(d，J＝6.8Hz，3H)，1.44(s，9H)，0.85(s，3H)，0.78(s，3H). 13C NMR(100MHz，CDCl 3)δ155.5，119.4，74.4，54.3，52.5，45.4，44.5，37.4，35.8，35.5，35.1，34.4，31.7，29.2，28.5，21.2，17.6，13.2，12.2.ESI-MS m/z：416[M-H]-.
(E) synthesis (14g) of-3 α-pregnant steroid-17 of tertiary fourth oxygen formamido group-16 Alpha-hydroxy (20)-alkene
With compound 12 for raw material, prepare as stated above, after 19h, reaction terminates.Crude product obtains white solid through flash chromatography on silica gel (PE: EA=12: 1, V/V) purifying, productive rate 16.6%. 1H NMR(400MHz，CDCl 3)δ5.58(q，J＝7.1Hz，1H)，4.42(d，J＝4.9Hz，1H)，3.84(br s，1H)，1.45(s，9H)，0.85(s，3H)，0.81(s，3H). 13C NMR(100MHz，CDCl 3)δ155.5，155.3，119.4，74.3，54.5，52.5，44.5，40.7，37.4，36.1，35.1，34.4，33.3，33.0，31.8，28.5，28.4，26.4，20.8，17.6，13.2，11.4.ESI-MS m/z：416[M-H]-.
Embodiment 7.
The synthetic method of compound 23-26e and 23-26f
Take 1 when content of starting materials is in reaction flask, add anhydrous THF and dissolve, add 6 equivalent LiAlH 4anhydrous THF solution, at 60 DEG C, stir 16h, TLC detects to reacting end, drips NaOH solution to alkaline cancellation reaction, add suitable quantity of water, methyl alcohol and methylene dichloride, diatomite filtration, concentrated, dichloromethane extraction, merge organic phase, drying, concentrate to obtain crude product, crude product obtains sterling through flash chromatography on silica gel purifying.
(Z)-3 β-pregnant steroid of methylamino--17-alkene (23e)
With compound 7g for raw material, prepare as stated above, crude product, through silica column purification (PE: EA=1: 2 → EA+1%TEA, V/V), obtains white solid, productive rate 51.0%. 1H NMR(400MHz，CDCl 3)：δ5.11(qt，J＝2.0，7.2Hz，1H)，2.42(s，3H)，1.65(dt，J＝1.9，7.1Hz，3H)，0.86(s，3H)，0.79(s，3H). 13C NMR(100MHz，CDCl 3)：δ150.7，113.3，59.3，56.5，54.7，45.5，44.6，37.6，37.5，36.3，35.8，35.2，33.8，32.2，31.6，29.2，29.0，24.6，21.5，17.1，13.3，12.5.ESI-MS m/z：316[M+H] +.
(Z)-3 α-pregnant steroid of methylamino--17-alkene (24e)
With compound 8g for raw material, prepare as stated above, crude product, through silica column purification (PE: EA=1: 1 → EA+1%TEA, V/V), obtains white solid, productive rate 16.0%. 1H NMR(400MHz，CDCl 3)：δ5.09(qt，1H，J＝1.9，7.1Hz)，2.70(d，1H，J＝2.8Hz)，2.38(s，3H)，1.64(dd，3H，J＝2.0，7.1Hz)，0.86(s，3H)，0.81(s，3H). 13CNMR(100MHz，CDCl 3)：δ150.7，113.3，56.5，54.7，54.6，44.6，39.8，37.4，36.5，35.2，34.5，33.2，32.8，32.0，31.6，28.9，25.9，24.5，21.1，17.6，13.2，11.7.ESI-MS m/z：316[M+H] +.
(Z)-3 β-pregnant steroid of ethylamino-17-alkene (23f)
With compound 7a for raw material, prepare as stated above, crude product, through silica column purification (PE: EA=1: 2 → EA+1%TEA, V/V), obtains 63.9mg white solid, productive rate 72.7%. 1H NMR(400MHz，CDCl 3)：δ5.10(qt，1H，J 1＝2.0Hz，J 2＝7.2Hz)，2.69(dq，J＝1.0，7.2Hz，2H)，1.64(dt，J＝1.9Hz，7.1Hz，3H)，1.09(t，J＝7.1Hz，6H)，0.85(s，3H)，0.78(s，3H). 13C NMR(100MHz，CDCl 3)：δ150.7，113.3，57.6，56.5，54.7，45.6，44.6，41.4，37.7，37.4，36.3，36.2，35.3，32.2，31.6，29.6，29.0，24.6，21.5，17.1，15.8，13.3，12.5.ESI-MS m/z：330[M+H] +.
(Z)-3 α-pregnant steroid of ethylamino-17-alkene (24f)
With compound 8a for raw material, prepare as stated above, crude product, through silica column purification (PE: EA=1: 1 → PE: EA=1: 1+1%TEA, V/V), obtains white solid, productive rate 31.2%. 1HNMR(400MHz，CDCl 3)：δ5.11(q，J＝6.8Hz，1H)，2.85(s，1H)，2.61(q，J＝6.9Hz，2H)，1.65(d，J＝7.4Hz，3H)，0.86(s，3H)，0.80(s，3H). 13CNMR(100MHz，CDCl 3)：δ150.8，113.3，56.5，54.5，52.4，44.6，41.8，39.9，37.4，36.5，35.2，33.5，32.8，32.0，31.6，31.5，28.9，26.1，24.5，21.1，17.0，15.7，13.2，11.7.ESI-MS m/z：330[M+H] +.
(E) the pregnant steroid of-3 β-methylamino--16 Alpha-hydroxy-17-alkene (25e)
With compound 13g for raw material, prepare as stated above, crude product, through silica column purification (PE: EA=1: 4 → PE: EA=1: 4+1%TEA, V/V), obtains white solid, productive rate 17.9%. 1H NMR(400MHz，CDCl 3)：δ5.56(qd，J＝1.2，7.2Hz，1H)，4.40(d，J＝5.0Hz，1H)，2.40(s，3H)，1.71(dd，3H，J＝0.84，7.1Hz)，0.84(s，3H)，0.79(s，3H). 13C NMR(100MHz，CDCl 3)：δ155.5，119.2，74.2，59.1，54.5，52.5，45.3，44.5，37.4，37.3，36.1，35.6，35.1，34.4，33.7，31.9，29.0，28.8，21.2，17.6，13.2，12.3.ESI-MS m/z：332[M+H] +.
(E) the pregnant steroid of-3 α-methylamino--16 Alpha-hydroxy-17-alkene (26e)
With compound 14g for raw material, prepare as stated above, crude product, through silica column purification (PE: EA=1: 2 → EA+1%TEA, V/V), obtains white solid, productive rate 21.8%. 1H NMR(400MHz，CDCl 3)：δ5.58(qd，1H，J＝1.1，7.2Hz)，4.42(d，J＝4.7Hz，1H)，2.74(br s，1H)，2.40(s，3H)，1.73(dd，J＝0.76，7.2Hz，3H)，0.85(s，3H)，0.81(s，3H). 13C NMR(100MHz，CDCl 3)：δ155.6，119.2，74.3，54.6，54.2，52.5，44.6，39.6，37.4，36.3，35.1，34.5，34.2，32.8，32.5，31.7，28.7，25.5，20.8，17.6，13.2，11.5.ESI-MS m/z：332[M+H] +.
(E) the pregnant steroid of-3 β-ethylamino-16 Alpha-hydroxy-17-alkene (25f)
With compound 13a for raw material, prepare as stated above, crude product obtains white solid through silica column purification (PE: EA=1: 2 → PE: EA=1: 2+1%TEA, V/V), productive rate 54.7%. 1H NMR(400MHz，CDCl 3)：δ5.55(qd，J＝1.1，7.2Hz，1H).4.40(d，J＝5.0Hz，1H)，2.65(qd，J＝1.4，7.2Hz，2H)，2.46(m，1H)，2.23(m，1H)，1.73(dd，J＝0.80，7.2Hz，3H)，1.09(qd，J＝7.1Hz，3H)，0.85(s，3H)，0.80(s，3H). 13C NMR(100MHz，CDCl 3)：δ155.6，119.3，74.3，57.4，54.5，52.5，45.4，44.5，41.2，37.4，36.1，36.0，35.1，34.4，31.9，29.4，28.8，21.2，17.6，15.6，13.2，12.3.ESI-MS m/z：346[M+H] +.
(E) the pregnant steroid of-3 α-ethylamino-16 Alpha-hydroxy-17-alkene (26f)
With compound 14a for raw material, prepare as stated above, crude product obtains white solid through silica column purification (PE: EA=1: 2 → PE: EA=1: 2+1%TEA, V/V), productive rate 53.5%. 1H NMR(400MHz，CDCl 3)：δ5.56(q，J＝6.8Hz，1H)，4.40(d，J＝4.4Hz，1H)，2.85(s，1H)，2.61(d，J＝6.8Hz，2H)，2.22(m，1H)，1.73(d，J＝6.8Hz，3H)，1.12(t，J＝6.8Hz，3H)，0.85(s，3H)，0.80(s，3H). 13C NMR(100MHz，CDCl 3)：155.6，119.2，74.3，54.3，52.5，52.2，44.6，41.6，39.7，37.4，36.3，35.1，34.4，33.2，32.5，31.8，28.7，25.8，20.8，17.6，15.4，13.2，11.5.ESI-MS m/z：346[M+H] +.
Embodiment 8.
The synthetic method of compound 15a-c and 16a-c
By 1 when content of starting materials and 1.5 equivalent Dess-Martin oxygenants are placed in reaction flask, add q. s. methylene chloride room temperature reaction, TLC monitoring is to reacting end.Add saturated sodium bicarbonate aqueous solution in reaction solution to stir, with dichloromethane extraction, the organic phase of merging washes twice with saturated sodium-chloride water solution, anhydrous magnesium sulfate drying, filters, and concentrate to obtain crude product, crude product obtains sterling through flash chromatography on silica gel purifying.
(E) synthesis (15a) of-3 β-pregnant steroid-17 of tertiary fourth oxygen formamido group (20)-alkene-16-ketone
With compound 22 for raw material, prepare as stated above, after 1h, reaction terminates.Crude product obtains white solid through flash chromatography on silica gel (PE: EA=6: 1-4: 1, V/V) purifying, productive rate 44.0%. 1H NMR(400MHz，CDCl 3)δ6.49(q，J＝7.2Hz，1H)，4.40(br s，1H)，3.42(br s，1H)，1.84(d，J＝7.2Hz，3H)，1.44(s，9H)，1.01(s，3H)，0.82(s，3H). 13C NMR(100MHz，CDCl 3)δ206.6，148.0，129.0，54.0，50.0，45.4，43.4，37.9，37.1，36.3，35.7，35.6，34.2，31.9，29.1，28.4，28.3，20.8，17.6，13.2，12.2.ESI-MS m/z：414[M-H] -.
(E) synthesis (16a) of-3 α-pregnant steroid-17 of tertiary fourth oxygen formamido group (20)-alkene-16-ketone
With compound 10 for raw material, prepare as stated above, after 2h, reaction terminates.Crude product obtains white solid through flash chromatography on silica gel (PE: EA=6: 1-4: 1, V/V) purifying, productive rate 55.6%. 1H NMR(400MHz，CDCl 3)δ6.49(q，J＝7.2Hz，1H)，4.83(brs，1H)，3.85(br s，1H)，1.84(d，J＝7.2Hz，3H)，1.45(s，9H)，1.01(s，3H)，0.84(s，3H). 13C NMR(100MHz，CDCl 3)δ206.5，155.2，147.9，129.0，79.0，54.2，50.1，45.7，43.4，40.6，37.9，36.3，36.2，34.1，33.2，32.7，31.9，28.5，28.2，26.3，20.5，17.6，13.2，11.4.ESI-MS m/z：414[M-H]-.
(E) synthesis (15b) of the pregnant steroid-17 of-3 β-N-methyl-tert fourth oxygen formamido group (20)-alkene-16-ketone
With compound 13h for raw material, prepare as stated above, crude product, through flash chromatography on silica gel purifying (PE: EA=10: 1), obtains white solid, productive rate 36%. 1H NMR(400MHz，CDCl 3)：δ6.48(q，J＝7.6Hz，1H)，1.84(d，J＝7.6Hz，3H)，1.46(s，9H)，1.01(s，3H)，0.84(s，3H). 13C NMR(100MHz，CDCl 3)：δ206.5，155.7，148.0，128.9，79.1，54.0，50.0，45.8，43.4，37.9，37.4，36.3，35.6，34.2，31.9，28.5，25.2，20.9，17.6，13.1，12.3.ESI-MS m/z：430[M+H] +.
(E) synthesis (16b) of the pregnant steroid-17 of-3 α-N-methyl-tert fourth oxygen formamido group (20)-alkene-16-ketone
With compound 14h for raw material, prepare as stated above, crude product, through flash chromatography on silica gel purifying (PE: EA=10: 1-8: 1), obtains white solid, productive rate 35%. 1H NMR(400MHz，CDCl 3)：δ6.48(q，J＝7.6Hz，1H).2.74(s，3H)，1.84(d，J＝7.6Hz，1H)，1.46(s，9H)，1.01(s，3H)，0.84(s，3H) 13C NMR(150Hz，CDCl 3)：δ206.5，155.7，148.0，128.9，79.1，54.0，50.0，45.8，43.4，37.9，37.4，36.3，35.6，34.2，32.0，28.6，28.5，25.2，20.9，17.6，13.1，12.3.ESI-MS m/z：430[M+H] +.
(E) synthesis (15c) of the pregnant steroid-17 of-3 β-N-ethyl tertiary fourth oxygen formamido group (20)-alkene-16-ketone
With compound 13i for raw material, prepare as stated above, crude product, through flash chromatography on silica gel purifying (PE: EA=18: 1), obtains white solid, productive rate 64%. 1H NMR(400MHz，CDCl 3)：δ H0.85(s，3H)，1.01(s，3H)，1.10(t，J＝6.8Hz，3H)，1.46(s，9H)，1.84(d，J＝7.2Hz，3H)，3.14(s，2H)，6.48(q，J＝7.6Hz，1H). 13C NMR(150Hz，CDCl 3)：δ206.8，155.4，148.0，128.9，79.0，54.0，50.0，46.0，43.4，37.9，37.6，36.3，35.7，34.0，33.0，31.9，28.6，28.5，26.3，20.9，17.7，13.1，12.4ESI-MS m/z：444[M+H] +.
(E) synthesis (16c) of the pregnant steroid-17 of-3 α-N-ethyl tertiary fourth oxygen formamido group (20)-alkene-16-ketone
With compound 14i for raw material, prepare as stated above, crude product, through flash chromatography on silica gel purifying (PE: EA=15: 1) wash-out, obtains white solid, productive rate 64%. 1H NMR(400MHz，CDCl 3)：δ6.49(q，J＝7.2Hz，1H)，3.13(s，2H)，1.84(d，J＝7.6Hz，1H)，1.46(s，9H)，1.11(t，J＝7.0Hz，3H)，1.01(s，3H)，0.85(s，3H). 13C NMR(100Hz，CDCl 3)：δ206.5，155.4，148.0，128.9，79.0，54.0，50.8，50.0，46.0，43.4，37.9，37.6，36.3，35.7，34.2，33.0，31.9，28.6，20.9，17.6，13.1，12.4.ESI-MS m/z：444[M+H] +.
Embodiment 9.
The synthetic method of compound 17a-c and 18a-c
Take 1 when content of starting materials is in reaction flask, add q. s. methylene chloride and stir under ice bath, then add the stirring of appropriate trifluoroacetic acid, TLC monitoring is to reacting end.Evaporated under reduced pressure solvent, adds sodium bicarbonate aqueous solution to pH=9 ~ 10.Dichloromethane extraction 3 times, merges organic phase, anhydrous magnesium sulfate drying, and filter, concentrating under reduced pressure obtains crude product.Crude product obtains sterling through HW-40 gel column chromatography purifying.
(E) synthesis (17a) of the pregnant steroid-17 of-3 beta-amino (20)-alkene-16-ketone
With compound 23 for raw material, prepare as stated above, after 1h, reaction terminates.Crude product obtains yellow oil through HW-40 gel column chromatography (DCM: MeOH=2: 1, V/V) purifying, and productive rate is 92.6%. 1H NMR(400MHz，CDCl 3)δ6.48(q，J＝7.6Hz，1H)，2.67(br s，1H)，1.84(d，J＝7.6Hz，3H)，1.01(s，3H)，0.83(s，3H). 13C NMR(100MHz，CDCl 3)δ206.6，148.0，129.0，54.2，51.1，50.1，45.5，43.4，39.3，38.0，37.3，36.4，35.7，34.2，32.5，32.0，28.5，20.9，17.7，13.2，12.3.ESI-MS m/z：316[M+H] +.
(E) synthesis (18a) of the pregnant steroid-17 of-3 alpha-amino group (20)-alkene-16-ketone
With compound 11 for raw material, prepare as stated above, after 1h, reaction terminates.Crude product obtains yellow oil through HW-40 gel column chromatography (DCM: MeOH=2: 1, V/V) purifying, and productive rate is 96.1%. 1H NMR(400MHz，CDCl 3)δ6.48(q，J＝7.2Hz，1H)，3.24(br s，1H)，1.84(d，J＝7.2Hz，3H)，1.01(s，3H)，0.83(s，3H). 13C NMR(100MHz，CDCl 3)δ206.6，148.0，128.9，54.1，50.1，45.8，43.4，39.0，37.9，36.5，36.3，35.8，34.2，31.9，31.8，28.7，28.4，20.5，17.7，13.2，12.3.ESI-MS m/z：316[M+H] +.
(E) synthesis (17b) of the pregnant steroid-17 of-3 β-methylamino-(20)-alkene-16-ketone
With compound 15b for raw material, prepare as stated above, reaction product is without the need to purifying, and yield is 93.9%. 1H NMR(400MHz，CDCl 3)：δ6.48(q，J＝7.5Hz，1H)，4.13(s，1H)，2.48(s，3H)，1.83(d，J＝7.5Hz，3H)，1.00(s，3H)，0.83(s，3H). 13C NMR(100MHz，CDCl 3)：δ206.4，148.0，128.9，58.8，54.1，50.0，45.1，43.4，37.5，36.8，36.3，36.1，34.2，34.0，32.4，31.9，28.5，27.5，20.9，17.6，13.1，12.1.ESI-MS m/z：330[M+H] +.
(E) synthesis (18b) of the pregnant steroid-17 of-3 α-methylamino-(20)-alkene-16-ketone
With compound 16b for raw material, prepare as stated above, reaction product is without the need to purifying, and yield is 65.6%. 1H NMR(400MHz，CDCl 3)：δ7.81(m，2H)，7.69(m，2H)，4.18(m，1H)，0.99(s，3H)，0.88(s，3H). 13C NMR(100MHz，CDCl 3)：δ221.3，168.5，133.8，132.1，123.0，54.2，51.4，50.4，47.8，46.1，37.8，35.9，35.7，35.1，31.6，30.8，28.3，25.0，21.8，20.4，13.8，12.2.ESI-MS m/z：330[M+H] +.
(E) synthesis (17c) of the pregnant steroid-17 of-3 β-methylamino-(20)-alkene-16-ketone
With compound 15c for raw material, prepare as stated above, reaction product is without the need to purifying, and yield is 67.2%. 1H NMR(400MHz，CDCl 3)：δ6.47(q，J＝7.5Hz，1H)，2.80(q，J＝7.2Hz，2H)，2.65(m，1H)，1.83(d，J＝7.2Hz，3H)，1.17，(t，J＝7.2Hz，3H)，0.10(s，3H)，0.82(s，3H). 13C NMR(100MHz，CDCl 3)：δ206.4，148.0，128.9，57.1，54.1，50.1，45.1，43.4，40.6，37.9，36.9，36.3，36.0，34.2，34.1，31.9，28.4，27.5，20.8，17.6，14.0，13.1，12.1.ESI-MS m/z：344[M+H] +.
(E) synthesis (18c) of the pregnant steroid-17 of-3 α-ethylamino (20)-alkene-16-ketone
With compound 16c for raw material, prepare as stated above, reaction product is without the need to purifying, and yield is 75.6%. 1H NMR(400MHz，CDCl 3)：δ7.81(m，2H)，7.69(m，2H)，4.18(m，1H)，1.00(s，3H)，0.84(s，3H). 13C NMR(100MHz，CDCl 3)：δ221.3，168.5，133.8，132.1，123.0，54.2，51.4，50.4，47.8，46.1，37.8，35.9，35.7，35.1，31.6，30.8，28.3，25.0，21.8，20.4，13.8，12.2.ESI-MS m/z：344[M+H] +.
Embodiment 10.
The synthetic method of compound 21-26a
Take about 100mg raw material in reaction flask, add q. s. methylene chloride and dissolve, then add about 0.4g paraformaldehyde, at 50 DEG C, after stirring and refluxing 1h, add 3 equivalent NaBH (OAc) 3, continue to stir.TLC monitoring reaction terminates.Saturated NaHCO 3cancellation is reacted, dichloromethane extraction 3 times, and the organic phase of merging is to wash twice, and anhydrous magnesium sulfate drying, filters, and concentrates to obtain crude product.Crude product obtains sterling through flash chromatography on silica gel purifying.
(Z) synthesis (23a) of-3 β-pregnant steroid of dimethylamino-17-alkene
With compound 5 for raw material, prepare as stated above, crude product, through flash chromatography on silica gel purifying (PE: EA=1: 1 → 1: 1+1%TEA, V/V), obtains white solid, productive rate 55.7%. 1HNMR(400MHz，CDCl 3)：δ5.09(qt，J＝1.8，7.1Hz)，2.26(s，6H)，1.65(dt，3H，J＝1.7，7.1Hz)，0.85(s，3H)，0.76(s，3H). 13C NMR(100MHz，CDCl 3)：δ150.6，113.3，64.3，56.5，54.7，45.9，44.6，42.0，37.9，37.4，36.0，35.2，32.2，31.6，31.3，29.2，24.9，24.5，21.5，17.0，13.3，12.4.ESI-MS m/z：330[M+H] +.
(Z) synthesis (24a) of the amino pregnant steroid-17-alkene of-3 alpha, alpha-dimethyls
With compound 6 for raw material, prepare as stated above, crude product, through flash chromatography on silica gel purifying (PE: EA=1: 1 → 1: 1+1%TEA, V/V), obtains white solid, productive rate 11.9%. 1H NMR(400MHz，CDCl 3)：δ5.11(qt，J＝2.0，7.2Hz，1H)，2.23(s，6H)，1.65(dt，J＝1.9，7.2Hz，3H)，0.86(s，3H)，0.82(s，3H). 13C NMR(100MHz，CDCl 3)：δ150.9，113.3，62.0，56.5，54.3，44.6，44.1，39.7，37.4，36.4，35.3，33.0，32.0，31.6，29.0，25.1，24.6，21.2，17.1，13.3，12.3.ESI-MS m/z：330[M+H] +.
(E) the pregnant steroid of-3 β-dimethylamino-16 Alpha-hydroxy-17-alkene (25a)
With compound 11 for raw material, prepare as stated above, crude product, through silica column purification (PE: EA=1: 2 → 1: 2+1%TEA, V/V), obtains white solid, productive rate 18.6%. 1H NMR(400MHz，CDCl 3)：δ5.57(qd，J＝1.2，7.2Hz，1H)，4.41(d，J＝4.8Hz，1H)，2.28(s，6H)，1.73(dd，J＝0.8，7.2Hz，3H)，0.86(s，3H)，0.78(s，3H). 13C NMR(100MHz，CDCl 3)：δ155.8，119.5，74.5，64.3，54.7，52.7，45.9，44.7，42.0，37.9，37.6，36.0，35.3，34.6，32.1，31.2，29.1，24.9，21.2，17.8，13.4，12.6.ESI-MS m/z：346[M+H] +.
(E) amino-16 Alpha-hydroxy pregnant steroid-17-alkene (26a) of-3 alpha, alpha-dimethyls
With compound 12 for raw material, prepare as stated above, crude product, through silica column purification (PE: EA=1: 2 → 1: 2+1%TEA, V/V), obtains white solid, productive rate 75.3%. 1H NMR(400MHz，CDCl 3)：δ5.55(qd，J＝1.2，7.2Hz，1H)，4.38(d，J＝4.8Hz，1H)，2.20(s，6H)，1.71(d，J＝0.96，7.2Hz，3H)，0.83(s，3H)，0.81(s，3H). 13C NMR(100MHz，CDCl 3)：δ155.7，119.2，74.4，61.8，54.2，52.6，44.8，44.1，39.7，37.6，36.4，35.4，34.7，33.1，32.0，31.9，28.8，25.1，21.0，17.7，13.2，12.2.ESI-MS m/z：346[M+H] +.
(E)-3 β-pregnant steroid of dimethylamino-17-alkene-16-ketone (21a)
With compound 17a for raw material, prepare as stated above, crude product, through silica column purification (PE: EA=1: 1 → 1: 1+1%TEA, V/V), obtains white solid, productive rate 63.0%. 1H NMR(400MHz，CDCl 3)：δ6.48(q，J＝7.5Hz，1H)，2.28(s，6H)，1.83(d，J＝7.5Hz，3H)，1.00(s，3H)，0.81(s，3H). 13C DEPT135(100MHz，CDCl 3)：δ128.9，64.1，54.2，50.6，45.6，41.8，37.9，37.4，36.3，34.2，32.0，31.0，28.8，24.7，20.9，17.7，13.2，12.3.ESI-MS m/z：344[M+H] +.
(E) the amino pregnant steroid-17-alkene-16-ketone (22a) of-3 alpha, alpha-dimethyls
With compound 18a for raw material, prepare as stated above, crude product, through silica column purification (PE: EA=1: 1 → 1: 1+1%TEA, V/V), obtains white solid, productive rate 50.5%. 1H NMR(400MHz，CDCl 3)：δ6.47(q，J＝7.5Hz，1H)，2.22(s，6H)，1.83(d，J＝7.5Hz，3H)，1.00(s，3H)，0.85(s，3H). 13C NMR(100MHz，CDCl 3)：δ206.7，148.1，128.8，61.6，53.9，50.1，43.9，43.5，39.5，38.0，36.4，36.3，34.2，32.7，31.9，31.8，28.5，24.9，20.6，17.7，13.1，12.1.ESI-MS m/z：344[M+H] +.
Embodiment 11.
The synthetic method of compound 21-26d, 24m and 26g
Take about 1 when content of starting materials and 1.2 equivalents to cyanobenzaldehyde in reaction flask, add q. s. methylene chloride and dissolve, at 40 DEG C, after stirring and refluxing 4h, add 3 equivalent NaBH (OAc) 3, continue to stir.TLC monitoring is to reacting end.Saturated NaHCO 3cancellation react, dichloromethane extraction 3 times, the organic phase of merging to wash twice.Anhydrous magnesium sulfate drying, filters, and concentrate to obtain crude product, crude product obtains sterling through flash chromatography on silica gel purifying.
(Z) synthesis (23d) of-3 β-to the amino pregnant steroid-17-alkene of cyano group benzyl
With compound 5 for raw material, prepare as stated above, through flash chromatography on silica gel purifying, (moving phase PE: diethylamine=20: 1), obtains Tan solid to crude product, yield 27.4%. 1H NMR(400MHz，CDCl 3)δ7.60(d，2H，J＝8.0Hz)，7.44(d，2H，J＝8.0Hz)，5.10(qt，1H，J＝7.2，2.0Hz,)，3.88(s，2H)，1.65(dt，3H，J＝7.2，2.0Hz)，0.86(s，3H)，0.80(s，3H). 13C NMR(100MHz，CDCl 3)δ150.5，132.2，128.6，119.0，113.2，110.6，57.0，56.3，54.5，50.9，50.6，45.3，44.4，37.4，37.2，36.1，35.1，31.9，31.4，29.4，28.8，24.4，21.3，16.9，13.1，12.4ESI-MS m/z：417[M+H] +.
(Z) synthesis (24d) of-3 α-to the amino pregnant steroid-17-alkene of cyano group benzyl
With compound 6 for raw material, prepare as stated above, through flash chromatography on silica gel purifying, (PE: diethylamine=20: 1), obtains red brown solid to crude product, yield 28.6%. 1H NMR(400MHz，CDCl 3)δ7.60(d，2H，J＝8.0Hz)，7.47(d，2H，J＝8.0Hz)，5.11(qt，1H，J＝7.2，3.2Hz，)，3.82(s，2H)，2.84(s，1H)，1.65(d，3H，J＝7.2Hz)，0.86(s，3H)，0.80(s，3H). 13C NMR(100MHz，CDCl 3)δ150.5，132.1，128.6，119.0，113.1，110.6，56.3，54.5，51.9，51.1，，44.4，39.7，37.3，36.4，35.1，33.4，32.7，31.9，31.4，28.7，26.0，24.3，21.0，16.9，13.1，11.5.ESI-MS m/z：417[M+H] +.
(Z)-3 β-to amino-16 Alpha-hydroxy pregnant steroid-17-alkene (25d) of cyano group benzyl
With compound 11 for raw material, prepare as stated above, crude product, through flash chromatography on silica gel purifying (moving phase PE: EA=3: 1-1: 1), obtains yellow solid, yield 25.7%. 1H NMR(400MHz，CDCl 3)δ7.60(d，2H，J＝7.6Hz)，7.46(d，2H，J＝7.6Hz)，5.56(q，1H，J＝7.2Hz)，4.41(d，1H，J＝4.0Hz)，3.88(s，2H)，2.45(s，1H)，1.72(d，3H，J＝7.6Hz)，0.87(s，3H)，0.81(s，3H). 13C NMR(100MHz，CDCl 3)δ155.6，132.2，128.8，119.4，118.9，110.8，74.4，60.4，56.9，54.5，52.5，50.4，45.3，44.5，37.4，37.3，36.1，35.1，34.4，31.8，28.7，21.2，21.0，17.6，14.2，13.2,12.3.ESI-MS m/z：433[M+H] +.
(E)-3 α-to amino-16 Alpha-hydroxy pregnant steroid-17-alkene (26d) of cyano group benzyl
With compound 12 for raw material, prepare as stated above, through flash chromatography on silica gel purifying, (PE: diethylamine=10: 1), obtains yellow oil to crude product, yield 29.8%. 1H NMR(400MHz，CDCl 3)δ7.60(d，2H，J＝8.4Hz)，7.46(d，2H，J＝8.4Hz)，5.57(qd，1H，J＝1.2，7.2Hz，)，4.41(d，1H，J＝3.6Hz)，3.81(s，2H)，2.84(s，1H)，1.73(dd，3H，J 1＝1.2Hz，J 2＝7.2Hz)，0.85(s，3H)，0.80(s，3H). 13C NMR(100MHz，CDCl 3)δ155.6，147.1，132.1，128.6，119.1，119.3，110.5，74.4，54.5，52.6，51.9，51.1，44.6，43.7，37.4，36.4，35.1，34.4，33.4，32.6，31.8，28.6，26.0，20.9，17.6，13.2，11.5.ESI-MS m/z：433[M+H] +.
(E) synthesis (21d) of-3 β-to the amino pregnant steroid-17-alkene-16-ketone of cyano group benzyl
With compound 17a for raw material, prepare as stated above, crude product, through flash chromatography on silica gel purifying (PE: EA=3: 1, V/V), obtains white solid, yield 25.8%. 1H NMR(400MHz，CDCl 3)δ7.51(d，2H，J＝8.0Hz)，7.39(d，2H，J＝8.0Hz)，6.41(q，1H，J＝7.6Hz，)，3.75(s，2H)，2.79(s，1H)，1.77(d，3H，J＝7.6Hz)，0.94(s，3H)，0.77(s，3H). 13C NMR(100MHz，CDCl 3)δ206.5，148.0，132.1，129.0，128.6，118.9，110.8，54.2，52.4，50.5，50.1，44.4，39.6，37.4，36.5，36.4，34.4，32.4，32.0，28.7，21.2，20.5，17.7，14.3，13.1，11.5.ESI-MS m/z：431[M+H] +.
(E) synthesis (22d) of-3 α-to the amino pregnant steroid-17-alkene-16-ketone of cyano group benzyl
With compound 18a for raw material, prepare as stated above, crude product, through flash chromatography on silica gel purifying (PE: EA=3: 1, V/V), obtains white solid, yield 23.8%. 1H NMR(400MHz，CDCl 3)δ7.51(d，2H，J＝8.0Hz)，7.39(d，2H，J＝8.0Hz)，6.41(q，1H，J＝7.6Hz,)，3.75(s，2H)，2.79(s，1H)，1.77(d，3H，J＝7.6Hz)，0.94(s，3H)，0.77(s，3H). 13C NMR(100MHz，CDCl 3)δ206.5148.0，132.1，128.9，128.7，119.0，110.6，54.2，51.9，51.1，50.1，43.4，39.6，37.9，36.5，36.4，34.2，33.3，32.4，32.0，28.4，26.0，20.5，17.7，13.1，11.5.ESI-MS m/z：431[M+H] +.
(Z) synthesis (24m) of the amino pregnant steroid-17-alkene-16-ketone of-3 α-benzyl
With compound 6 for raw material, prepare as stated above, crude product, through flash chromatography on silica gel purifying (PE: EA=4: 1), obtains white oil thing, yield 37.0%. 1H NMR(400MHz，CDCl 3)δ7.24(q，4H，J＝7.2Hz)，7.16(d，1H，J＝7.2Hz)，5.03(q，1H，J＝6.8Hz)，3.69(s，2H)，2.81(s，1H)，1.57(d，3H，J＝6.8Hz)，0.79(s，3H)，0.73(s，3H). 13C NMR(100MHz，CDCl 3)δ150.6，128.3，128.1，126.8，113.1，56.4，54.5，51.6，51.4，，44.4，39.6，37.3，36.4，35.1，33.4，32.7，31.9，31.5，28.8，26.0，24.4，21.0，16.9，13.1，11.6.ESI-MS m/z：392[M+H] +.
Embodiment 12.
Compound 21-26b, the synthetic method of 21-26c, 23g and 24g-1
Take 1 and work as the salt of wormwood (or sodium bicarbonate) of content of starting materials and 2-3 equivalent in reaction flask, after adding q. s. toluene, add the corresponding bromoalkane of 5-10 equivalent, the lower reaction of backflow.TLC monitoring, to reacting end, is cooled to room temperature, obtains sterling through flash chromatography on silica gel purifying.
(Z)-3 beta-tetrahydro pyrryl pregnant steroid-17-alkene (23b)
With compound 5 for raw material, prepare as stated above, reaction mixture, through flash chromatography on silica gel purifying (PE: EA=1: 1 → 1: 4+1%TEA, V/V), obtains white solid, productive rate 21.74%. 1H NMR(400MHz，CDCl 3)：δ5.10(qt，J＝2.0，7.2Hz，1H)，2.58(s，4H)，1.64(dt，J＝1.9，7.2Hz，3H)，0.85(s，3H)，0.79(s，3H). 13C NMR(100MHz，CDCl 3)：δ150.7，113.3，64.6，56.5，54.7，52.0，45.4，44.6，37.6，37.4，36.0，35.2，34.8，32.2，31.6，29.0，28.0，24.5，23.4，21.4，17.1，13.3，12.5.ESI-MS m/z：356[M+H] +.
(Z)-3 α-pregnant steroid of Pyrrolidine base-17-alkene (24b)
With compound 6 for raw material, prepare as stated above, reaction mixture, through flash chromatography on silica gel purifying (PE: EA=1: 1 → 1: 4+2%TEA, V/V), obtains white solid, productive rate 15.3%. 1HNMR(400MHz，CDCl 3)：δ5.11(qt，J＝2.0，7.1Hz，1H).2.46(s，4H)，1.65(d，J＝1.8，7.1Hz，3H)，0.86(s，3H)，0.81(s，3H). 13C NMR(100MHz，CDCl 3)：δ150.7，113.1，65.6，56.3，52.4，44.4，37.2，36.1，35.1，33.4，31.8，32.9，31.5，30.6，28.8，24.4，23.7，22.6，21.0，19.2，16.9，13.7，13.1，12.0.ESI-MS m/z：356[M+H] +.
(Z)-3 β-pregnant steroid of piperidyl-17-alkene (23c)
With compound 5 for raw material, prepare as stated above, reaction mixture, through flash chromatography on silica gel purifying (PE: EA=2: 1 → 2: 1+1%TEA, V/V), obtains white solid, productive rate about 94.6%. 1H NMR(400MHz，CDCl 3)：δ5.09(qt，J＝1.8，7.1Hz，1H)，2.50(s，4H)，1.63(d，J＝1.8，7.1Hz，3H)，0.84(s，3H)，0.75(s，3H). 13C NMR(100MHz，CDCl 3)：δ150.7，113.3，64.8，56.4，54.7，50.5，46.2，44.6，38.2，37.4，36.2，35.2，32.2，31.6，30.9，29.2，26.6，25.0，24.5，24.3，21.5，17.1，13.3，12.5.ESI-MS m/z：370[M+H] +.
(Z)-3 α-pregnant steroid of piperidyl-17-alkene (24c)
With compound 6 for raw material, prepare as stated above, reaction mixture, through flash chromatography on silica gel purifying (PE: EA=1: 1 → 1: 1+1%TEA, V/V), obtains white solid, productive rate 47.2%. 1H NMR(400MHz，CDCl 3)：δ5.16(q，J＝7.1Hz，1H)，2.36(s，4H)，1.65(d，J＝70Hz，3H)，0.86(s，3H)，0.82(s，3H). 13C NMR(100MHz，CDCl 3)：δ113.3，65.8，51.6，44.6，39.7，37.4，35.2，33.5，31.7，30.8，29.9，28.9，26.5，24.7，24.6，21.2，19.4，17.1，13.9，13.5，13.3，12.4.ESI-MS m/z：370[M+H] +.
(Z)-3 β-pregnant steroid of n-propylamine base-17-alkene (23g)
With compound 5 for raw material, prepare as stated above, reaction mixture, through flash chromatography on silica gel purifying (PE: EA=20: 1 → 10: 1, V/V), obtains white solid, productive rate 14.4%. 1H NMR(400MHz，CDCl 3)：δ5.10(q，J＝7.2Hz，1H)，2.96(br s，1H)，2.63(t，J＝7.6Hz，2H)，1.63(d，J＝7.2Hz，3H)，0.85(s，3H)，0.80(s，3H). 13CNMR(100MHz，CDCl 3)：δ149.5，112.1，55.1，52.9，51.7，47.8，43.3，38.3，36.1，35.1，34.0，31.3，30.7，30.4，27.6，23.3，19.9，15.9，12.1，10.7，10.5.ESI-MS m/z：344[M+H] +.
(Z)-3 α-pregnant steroid of n-propylamine base-17-alkene (24g)
With compound 6 for raw material, prepare as stated above, reaction mixture, through flash chromatography on silica gel purifying (PE: EA=20: 1 → 10: 1, V/V), obtains white solid, productive rate 24.4%. 1HNMR(400MHz，CDCl 3)：δ5.10(q，J＝7.2Hz，1H)，2.69(t，J＝7.6Hz，2H)，1.64(d，J＝7.2Hz，3H)，0.86(s，3H)，0.81(s，3H). 13C NMR(100MHz，CDCl 3)：δ149.4，112.2，56.3，55.3，53.4，46.9，44.3，43.4，36.2，34.9，34.0，31.3，30.9，30.4，28.7，27.7，23.3，20.1，15.9，12.1，11.3，10.7.ESI-MS m/z：344[M+H] +.
(Z)-3 β-pregnant steroid of propargylamino-17-alkene (23h)
With compound 5 for raw material, prepare as stated above, reaction mixture, through flash chromatography on silica gel purifying (PE: EA=20: 1 → 10: 1, V/V), obtains white solid, productive rate 24.4%. 1H NMR(400MHz，CDCl 3)：δ5.11(qt，J＝1.9，7.2Hz，1H)，3.47(d，J＝2.4Hz，2H)，2.71(s，1H)，2.21(t，J＝2.4Hz，1H)，1.65(dt，J＝2.0，7.2Hz，6H)，0.86(s，3H)，0.79(s，3H). 13C NMR(100MHz，CDCl 3)：δ150.8，113.2，56.5，55.7，54.7，45.3，44.6，37.537.4，35.4，35.3，32.1，31.6，29.0，28.8，24.6，21.5，17.0，13.3，12.5.ESI-MS m/z：340[M+H] +.
(Z) the amino pregnant steroid-17-alkene (24h) of-3 alpha-acetylenes third
With compound 6 for raw material, prepare as stated above, reaction mixture, through flash chromatography on silica gel purifying (PE: EA=20: 1 → 10: 1, V/V), obtains white solid, productive rate 22.4%. 1H NMR(400MHz，CDCl 3)：δ5.11(qt，J＝2.0，7.1Hz，1H)，3.42(d，J＝2.4Hz，2H)，.3.07(s，1H)，2.18(t，J＝2.4Hz，1H)，1.65(dt，J＝2.0，7.2Hz，3H)，0.86(s，3H)，0.80(s，3H). 13C NMR(100MHz，CDCl 3)：δ150.8，113.3，82.8，56.5，54.5，51.2，44.6，39.7，37.4，36.5，36.0，35.2，33.2，32.8，32.0，31.6，28.8，26.1，24.5，21.1，17.1，13.3，11.7.ESI-MSm/z：340[M+H] +.
(Z)-3 β-N, the pregnant steroid of N-bis-propargylamino-17-alkene (24i)
With compound 5 for raw material, prepare as stated above, reaction mixture, through flash chromatography on silica gel purifying (PE: EA=20: 1 → 10: 1, V/V), obtains white solid, productive rate 28.4%. 1H NMR(400MHz，CDCl 3)：δ5.11(qt，J＝2.0，7.1Hz，1H)，3.57(d，J＝2.0Hz，4H)，2.51(m，1H)，2.20(t，J＝2.3Hz，2H)，1.65(dt，J＝1.9，7.2Hz，3H)，0.86(s，3H)，0.78(s，3H). 13C NMR(100MHz，CDCl 3)：δ150.7，113.4，80.1，59.9，56.4，54.6，45.7，44.6，39.3，37.8，37.4，35.9，35.2，32.9，32.1，31.6，29.1，26.2，24.5，21.4，17.1，13.3，12.4.ESI-MS m/z：378[M+H] +.
(Z)-3 α-N, the pregnant steroid of N-bis-propargylamino-17-alkene (24i)
With compound 6 for raw material, prepare as stated above, reaction mixture, through flash chromatography on silica gel purifying (PE: EA=20: 1 → 10: 1, V/V), obtains white solid, productive rate 29.5%. 1H NMR(400MHz，CDCl 3)：δ5.11(qt，J＝2.0，7.2Hz，1H).3.52(d，J＝2.0Hz，4H)，2.65(s，1H)，2.19(s，2H)，1.65(dt，J＝1.9，7.2Hz，3H)，0.86(s，3H)，0.82(s，3H). 13C NMR(100MHz，CDCl 3)：δ150.7，113.3，79.9，56.5，54.8，54.3，44.6，40.0，39.7，37.4，36.5，35.2，33.1，32.0，31.6，31.5，28.7，24.7，24.5，21.2，17.1，13.3，12.2.ESI-MS m/z：378[M+H] +.
(Z)-3 α-N-Cyclohexylamino pregnant steroid-17-alkene (24j)
With compound 6 for raw material, prepare as stated above, reaction mixture, through flash chromatography on silica gel purifying (PE: EA=20: 1 → 10: 1, V/V), obtains white solid, productive rate 27.1%. 1H NMR(400MHz，CDCl 3)：δ5.11(qt，J＝2.0，7.2Hz，)，1.64(dq，J＝1.8，7.2Hz，3H)，0.85(s，3H)，0.79(s，3H). 13C NMR(100MHz，CDCl 3)：δ150.6，113.1，56.3，54.5，53.7，48.4，44.4，39.8，37.2，36.2，35.0，34.1，34.0，33.6，32.7，31.8，31.4，28.7，26.2，26.0，25.4，25.3，24.3，21.0，16.9，13.1，11.5.ESI-MS m/z：384[M+H] +.
(23-3 α-N-cyclo-heptylamino pregnant steroid-17-alkene (24k)
With compound 6 for raw material, prepare as stated above, reaction mixture, through flash chromatography on silica gel purifying (PE: EA=20: 1 → 10: 1, V/V), obtains white solid, productive rate 21.7%. 1HNMR(400MHz，CDCl 3)：δ5.10(q，J＝7.0Hz，1H)，2.88(s，1H)，2.60(m，1H)，1.64(d，3H，J＝7.0Hz)，0.86(s，3H)，0.80(s，3H). 13C NMR(100MHz，CDCl 3)：δ150.5，113.1，56.3，56.1，54.5，48.8，44.4，40.0，37.2，36.2，35.7，35.3，35.0，33.7，32.8，31.9，31.4，28.7，28.1，27.9，25.9，24.7，24.6，24.3，21.0，16.9，13.1，11.5.ESI-MS m/z：398[M+H] +.
(Z)-3 α-pregnant steroid of just pungent amino-17-alkene (241)
With compound 6 for raw material, prepare as stated above, reaction mixture, through flash chromatography on silica gel purifying (PE: EA=20: 1 → 10: 1, V/V), obtains white solid, productive rate 21.7%. 1H NMR(400MHz，CDCl 3)：δ5.10(q，J＝7.0Hz，1H)，2.81(br s，1H)，2.53(t，J＝6.8Hz，2H)，1.64(d，J＝7.0Hz，3H)，0.86(s，3H)，0.79(s，3H). 13C NMR(100MHz，CDCl 3)：δ150.6，113.1，56.3，54.4，52.3，47.6，44.4，39.7，37.2，36.3，35.1，33.4，32.7，31.9，31.8，31.4，30.4，29.5，29.3，28.8，27.5，25.9，24.3，22.7，21.0，16.9，14.1，13.1，11.5.ESI-MSm/z：414[M+H] +.
(E)-3 beta-tetrahydro pyrryl-16 Alpha-hydroxy pregnant steroid-17-alkene (25b)
With compound 11 for raw material, prepare as stated above, reaction mixture, through flash chromatography on silica gel purifying (PE: EA=1: 1 → PE: EA=1: 1+1%TEA, V/V), obtains white solid, productive rate 9.4%. 1H NMR(400MHz，CDCl 3)：δ5.57(q，J＝7.2Hz，1H)，4.41(d，J＝3.8Hz，1H)，2.63(br s，4H)，1.81(s，4H)，1.73(d，J＝7.2Hz，3H)，0.85(s，3H)，0.81(s，3H). 13C NMR(100MHz，CDCl 3)：δ155.8，119.5，74.6，64.6，54.7，52.7，51.9，45.5，44.7，37.6，37.5，36.0，35.3，34.6，32.1，29.0，27.9，23.4，21.4，17.8，13.4，12.5.ESI-MS m/z：372[M+H] +.
(E)-3 α-pregnant steroid of Pyrrolidine base-16 Alpha-hydroxy-17-alkene (26b)
With compound 12 for raw material, prepare as stated above, reaction mixture, through flash chromatography on silica gel purifying (PE: EA=1: 1 → PE: EA=1: 1+1%TEA, V/V), obtains white solid, productive rate 10.3%. 1H NMR(400MHz，CDCl 3)：δ5.57(qd，J＝1.2，7.2Hz，1H)，4.40(d，J＝4.8，1H)，2.46(br s，4H)，1.76(s，4H)，1.72(d，J＝7.2Hz，3H)，0.84(s，3H)，0.80(s，3H). 13C NMR(100MHz，CDCl 3)：155.7，119.2，74.4，61.2，53.9，52.5，52.4，44.6，39.3，37.4，36.2，35.2，34.5，33.3，32.8，31.8，28.7，26.3，23.7，20.9，17.6，13.2，12.0.ESI-MSm/z：372[M+H] +.
(E) the pregnant steroid of-3 β-piperidyl-16 Alpha-hydroxy-17-alkene (25c)
With compound 11 for raw material, prepare as stated above, reaction mixture, through flash chromatography on silica gel purifying (PE: EA=1: 1 → 1: 1+1%TEA, V/V), obtains white solid, productive rate 60.2%. 1H NMR(400MHz，CDCl 3)：δ5.57(qd，J＝1.2，7.2Hz，1H)，4.41(s，1H)，2.51(s，4H)，1.72(dd，J＝1.2，7.2Hz，3H)，0.85(s，3H)，0.77(s，3H). 13C NMR(100MHz，CDCl 3)：δ155.5，119.4，74.4，64.5，54.5，52.5，50.4，46.0，44.5，38.0，37.4，35.9，35.1，34.4，31.9，30.7，29.0，26.5，24.9，24.2，21.2，17.6，13.2，12.4.ESI-MS m/z：386[M+H] +.
(E) amino-16 Alpha-hydroxy pregnant steroid-17-alkene (26c) of-3 α-piperidines
With compound 12 for raw material, prepare as stated above, reaction mixture, through flash chromatography on silica gel purifying (PE: EA=1: 1 → 1: 1+1%TEA, V/V), obtains white solid, productive rate 73.9%. 1H NMR(400MHz，CDCl 3)：δ5.57(qd，J＝1.1，7.1Hz，1H)，4.41(br s，1H)，2.36(br s，4H)，2.11(br s，1H)，1.74(dd，3H，J 1＝0.68Hz，J 2＝7.2Hz)，0.85(s，3H)，0.82(s，3H). 13C NMR(100MHz，CDCl 3)：δ155.5，119.3，77.2，74.4，59.3，54.4，52.5，51.4，39.6，37.4，35.1，34.4，33.2，31.8，31.3，28.6，26.4，24.7，24.4，20.9，17.6，13.2，12.3.ESI-MSm/z：386[M+H] +.
(E)-3 beta-tetrahydro pyrryl pregnant steroid-17-alkene-16-ketone (21b)
With compound 17a for raw material, prepare as stated above, reaction mixture, through flash chromatography on silica gel purifying (PE: EA=1: 1 → 1: 1+1%TEA, V/V), obtains white solid, productive rate 24.2%. 1H NMR(400MHz，CDCl 3)：δ6.49(qd，J＝2.0，7.5Hz，1H).4.30(m，1H)，2.74(br s，4H)，1.85(dd，J＝2.0，7.5Hz，3H)，1.01(s，3H)，0.87(s，3H). 13C NMR(100MHz，CDCl 3)：δ206.5.148.0，128.8，64.4，54.1，51.6，50.1，45.2，43.4，37.9，36.3，35.9，34.2，32.0，30.6，28.5，23.2，20.8，19.2，17.7，13.2，12.3.ESI-MS m/z：370[M+H] +.
(E)-3 α-pregnant steroid of Pyrrolidine base-17-alkene-16-ketone (22b)
With compound 18a for raw material, prepare as stated above, reaction mixture, through flash chromatography on silica gel purifying (PE: EA=1: 1 → 1: 1+1%TEA, V/V), obtains white solid, productive rate 3.8%. 1HNMR(400MHz，CDCl 3)：δ5.69(q，J＝7.2Hz，1H)，2.47(s，4H)，1.84(d，J＝7.2Hz，1H)，1.00(s，3H)，0.85(s，3H). 13C NMR(100MHz，CDCl 3)206.5.148.1，128.8，65.6，64.4，54.1，52.3，50.1，43.4，37.9，36.3，35.9，34.2，32.0，30.6，28.5，23.2，20.8，19.2，17.7，13.2，11.9.ESI-MS m/z：370[M+H] +.
(E)-3 β-pregnant steroid of piperidyl-17-alkene-16-ketone (21c)
With compound 17a for raw material, prepare as stated above, reaction mixture, through flash chromatography on silica gel purifying (PE: EA=1: 1 → 1: 1+1%TEA, V/V), obtains white solid, productive rate 31.0%. 1H NMR(400MHz，CDCl 3)：δ5.69(q，J＝7.2Hz，1H)，2.59(s，4H)，2.07(d，J＝7.2Hz，1H)，0.90(s，3H)，0.82(s，3H). 13C NMR(100MHz，CDCl 3)：δ205.4，147.1，127.8，63.6，49.4，49.1，45.0，42.4，36.9，36.7，35.7，35.4，35.1，33.2，31.0，29.5，28.7，27.7，25.2，23.7，23.0，19.9，16.6，12.1，11.4.ESI-MS m/z：384[M+H] +.
(E)-3 α-pregnant steroid of piperidyl-17-alkene-16-ketone (22c)
With compound 18a for raw material, prepare as stated above, reaction mixture, through flash chromatography on silica gel purifying (PE: EA=1: 1 → PE: EA=1: 1+1%TEA, V/V), obtains white solid, productive rate 23.9%. 1H NMR(400MHz，CDCl 3)δ6.48(q，1H，J＝7.5Hz)，2.36(br s，4H)，1.84(d，3H，J＝7.5Hz)，1.01(s，3H)，0.85(s，3H). 13C NMR(100MHz，CDCl 3)δ206.5，148.1，128.8，59.3，54.1，51.4，50.1，43.5，39.5，38.0，36.3，34.3，33.0，32.0，30.6，28.5，26.4，24.7，24.4，20.6，19.2，17.7，13.7，13.2，12.3.ESI-MS m/z：384[M+H] +.
Embodiment 13.
The anti-breast cancer transfer activity of each derivative is evaluated
As mentioned before, experimental result is in table 2 for specific experiment method and step:
Table 2 compound is to MB-MDA-231 (human breast cancer cell) chemotactic inhibiting rate
*showed cell toxicity under proof load.
Contriver thinks, researchist is finding the compound with activity of resisting tumor metastasis always, often obtains a class and has the compound structure that can accept activity level and all have great importance for preparing medicine for anti transfer of tumor.The natural lead compound Salignone that the present invention finds according to this seminar early stage, 3,16 and 20 structural modification sites as derivative synthesis on pregnane parent nucleus, carried out the derivative Design and synthesis of a large amount of different substituents, different steric configuration (α and β), 52 synthesized derivatives are new compound that this seminar synthesizes first, that have no bibliographical information.Further by cytotoxicity and anti-breast cancer transfer activity screening experiment, therefrom preferred 22 reactive derivatives, and preferred 8 strong reactive derivatives further, its anti-breast cancer transfer activity is suitable with positive control LY294002.
Those skilled in the art are difficult to the alkaloidal structure of common pregnane disclosed in prior art; through so a large amount of structural modifications; then preferred substituents be limited in less scope; thus obtain the compound of application claims protection, namely compound structure of the present invention is not apparent to those skilled in the art.
Further, because first the present invention screens through cytotoxicity, rejecting has Cytotoxic derivative, derivative through determination of activity is under non-cytotoxicity dosage, demonstrates anti-breast cancer transferance, the activity of 8 wherein representative derivatives and positive control LY294002 are quite (in existing anti metastasis marketed drug, not yet having the small molecules synthetic drugs of anti metastasis to go on the market).This do not have Cytotoxic anti-breast cancer transfer activity compound and can meet the long term administration demand of control required for cancer metastasis clinically, and visible the present invention has been provided with significant progress.Compound of the present invention has unforeseeable technique effect, and relative to activity of resisting tumor metastasis compound in general sense, the present invention has more importantly using value.

Claims (5)

1. pregnane alcaloid-derivatives or its pharmacy acceptable salt, its structure is as shown in general formula I:
Wherein, R 1with R 2independently be selected from hydrogen, the straight or branched alkyl of carbonatoms 1-8, cycloalkyl, undersaturated alkyl, benzyl, the cyclosubstituted benzyl of benzene; Or R 1r 2n is piperidyl or pyrryl;
R 3with R 4independently be selected from hydrogen, hydroxyl; Or R 3r 4common designation carbonylic oxygen atom;
R 5with R 6for different substituting groups independently be selected from hydrogen or methyl;
Described pharmacy acceptable salt refers to and the salt that mineral acid or organic acid are formed, and wherein mineral acid or organic acid are selected from hydrochloric acid, Hydrogen bromide, sulfuric acid, phosphoric acid, methylsulfonic acid, benzene methanesulfonic acid, oxalic acid, tartrate, toxilic acid, citric acid or xitix.
2. pregnane alcaloid-derivatives as claimed in claim 1 or its pharmacy acceptable salt, it has following structure:
3. pregnane alcaloid-derivatives as claimed in claim 2 or its pharmacy acceptable salt, it is selected from compound 5,6,12,17a, 18a, 21a, 21d, 22a, 22c, 22d, 23a, 23b, 23e, 23f, 23h, 24a, 24c, 24h, 24m, 25e, 26b and 26c.
4. pregnane alcaloid-derivatives as claimed in claim 2 or its pharmacy acceptable salt, it is selected from compound 5,17a, 18a, 21d, 22c, 23f, 26b and 26c.
5. the pregnane alcaloid-derivatives as described in claim 1-4 any one or its pharmacy acceptable salt are preparing the purposes in anti-breast cancer diversion medicaments.
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CN105294465A (en) * 2015-12-08 2016-02-03 天津医科大学 Chiral ionone alkaloid derivative with breast cancer metastasis prevention function and medical application of chiral ionone alkaloid derivative
CN109091481A (en) * 2018-09-29 2018-12-28 贵阳中医学院 A kind of application of pregnane type open country fan peanut alkaloids
CN116549459A (en) * 2023-07-05 2023-08-08 天津医科大学眼科医院 Application of pregnane alkaloid derivative in preparation of drugs for treating fundus diseases

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CN105294465A (en) * 2015-12-08 2016-02-03 天津医科大学 Chiral ionone alkaloid derivative with breast cancer metastasis prevention function and medical application of chiral ionone alkaloid derivative
CN109091481A (en) * 2018-09-29 2018-12-28 贵阳中医学院 A kind of application of pregnane type open country fan peanut alkaloids
CN116549459A (en) * 2023-07-05 2023-08-08 天津医科大学眼科医院 Application of pregnane alkaloid derivative in preparation of drugs for treating fundus diseases
CN116549459B (en) * 2023-07-05 2023-09-29 天津医科大学眼科医院 Application of pregnane alkaloid derivative in preparation of drugs for treating fundus diseases

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