CN103360358B - 15-oxospiramilactone derivatives, and preparation method and applications thereof - Google Patents

15-oxospiramilactone derivatives, and preparation method and applications thereof Download PDF

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CN103360358B
CN103360358B CN201310300116.2A CN201310300116A CN103360358B CN 103360358 B CN103360358 B CN 103360358B CN 201310300116 A CN201310300116 A CN 201310300116A CN 103360358 B CN103360358 B CN 103360358B
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cdcl
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CN103360358A (en
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郝小江
晏晨
刘海洋
何红平
李林
何小丽
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Kunming Institute of Botany of CAS
Key Laboratory of Natural Product Chemistry of Guizhou Academy of Sciences
Shanghai Institute of Nutrition and Health of CAS
Center for Excellence in Molecular Cell Science of CAS
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Shanghai Institutes for Biological Sciences SIBS of CAS
Kunming Institute of Botany of CAS
Key Laboratory of Natural Product Chemistry of Guizhou Academy of Sciences
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Abstract

The invention provides various type derivatives of 15-oxospiramilactone, a pharmaceutical composition by taking the derivatives as an active component, a preparation method of the derivatives, and applications in preparation of antitumor drugs. During research on antitumor natural products, it is discovered that 15-oxospiramilactone possesses activities of resisting multiple tumor cell strains, and most of the derivatives are stronger than the primer 15-oxospiramilactone in activity. The above compounds are subjected to a test of inhibiting activity of Wnt signaling pathway and a test of inhibiting activity of various tumor cell strains, and it is discovered that antineoplastic activity of the above compounds is positively correlated to the activity of inhibiting Wnt signaling pathway.

Description

15-oxospiramilactone derivative and its preparation method and application
Technical field:
The invention belongs to technical field of pharmaceuticals, specifically, relate to diterpene salicylate 15-oxospiramilactone (S-3) derivative, its preparation method, the pharmaceutical composition being activeconstituents with this compounds, and it prepares the application in antitumor drug.
Background technology:
Although tumour science has been made significant headway, up to now, cancer has been still public health problem the most serious in the world.Worldwide, estimate new cancer number by by 2008 1,270 ten thousand, rise to more than 2,000 ten thousand of the year two thousand thirty, tumour has become the number one killer of human health." poor cancer " sickness rate remains high, and " rich cancer " also increases rapidly, and China is in the period from developing country's distribution to developed country's distribution transition.The reason of China cancer patients rejuvenation is caused to have environmental pollution, bad life style and stress excessive.Meanwhile, another factor that " cancer feelings " are surging is the quick aging of population.At present, the anti-cancer therapies of clinical application comprises operation, radiation and chemotherapy, although these means have some curative effects, and the toxic side effect larger due to it and bring the huge human body and spiritual misery to patient.The major issue simultaneously affecting chemotherapy effect there occurs the resistance to cell toxicity medicament, some cancer cells produce resistance to a kind of antitumor drug, also develop immunity to drugs to other non-similar drugs, be the major cause causing tumour chemotherapy (chemotherapy) failed simultaneously.The antitumor drug of many natural origins is as alkaloid anticarcinogen (colchicine, vinealeucoblastine(VLB), harringtonine and taxol etc.), anthracycline antitumor antibiotic (Zorubicin and daunorubicin), all very easily there is MDR in epipodophyllotoxin class (Vp-16 and VM-26) and synthetic drug (mitoxantrone and the pyridine of amine benzene bifurcation).Newfound medicine is as taxol and the STI-571 treating chronic myelogenous leukemia, and be all just just found that there is resistance for clinical, this makes problem more serious.But signal transduction pathway also exists greatest differences between normal cell and tumour cell, this otherness just in time gives the antitumor drug with the feature such as targeting, potent low toxicity new Historic Opportunities for th e Development.So far, existing in the art there are no the report of 15-oxospiramilactone of the present invention (S-3) derivative.
Summary of the invention:
The object of this invention is to provide 15-oxospiramilactone (S-3) derivative that one has structure formula I, (II), its preparation method, the pharmaceutical composition being activeconstituents with this compounds, and it is preparing the application in antitumor drug.
The object of the invention is to be realized by following technological method:
15-oxospiramilactone (S-3) derivative of following structural formula (I), (II):
Wherein: R in structure formula I 1, R 6for-OH or=O, R 2for-H or=O or=CH 2or-Cl or-Br or-SCH 3or-CH 2oH or-CH 2n (CH 3) 2or-CH 2n (CH 2) 4o or-CH 2oCOCH 3or-CH 2oSO 2cH 3, R 3for-H or R 2with R 3between be double bond; X 1, X 3for-CH 2or-CH or carbonyl; X 2for Sauerstoffatom or nitrogen-atoms or-SO 2;
Work as X 1during for CH, R 5for-OCH 3or R 1with R 5form oxo bridge;
Work as X 2during for nitrogen-atoms, R 4for-H or-CH 2cH 2oH or-CH 2cH 2oTBDMS or R 4, R 5, X 1, X 2form oxazole ring or R 4, X 2, X 3form oxazole ring.
In structure formula II, R 1, R 5for-OH, or=O; R 2for-H or=O or=CH 2or-CH 2oH or-CH 2n (CH 2) 4o or-CH 2oCOCH 3or-CH 2oSO 2cH 3; R 3, R 4for-0H or-COCH 3or-COCH 2cl or-CON (CH 2) 4o or-Ms or-CH 2cN or 2-Thenoyl; X 1for-CH 2or-CO.
Particularly, 15-oxospiramilactone of the present invention (S-3) derivative is:
The present invention provides a kind of pharmaceutical composition with anti-tumor activity simultaneously, and it comprises one or more above-mentioned 15-oxospiramilactone derivatives for the treatment of significant quantity and pharmaceutically acceptable auxiliary material.
The preparation method of above-claimed cpd of the present invention, comprises the steps:
(1) compound 1-9,25, the preparation method of 26:
(2) preparation method of compound 10:
(3) preparation method of compound 11-13:
(4) preparation method of compound 14-20:
(5) preparation method of compound 21:
(6) preparation method of compound 22,27:
(7) compound 23,24, the preparation method of 28-36:
(8) preparation method of compound 37-41:
(9) syntheti c route of compound 42,43,44:
(10) preparation method of compound 45,46,47,48,49:
(11) preparation method of compound 50-55:
(12) preparation method of compound 56-58:
The above-mentioned compound of the present invention is preparing the application in antitumor drug.
The application of compound in preparation Wnt signal path inhibitor medicine that the present invention is above-mentioned.
When the compounds of this invention is used as medicine, directly can uses, or use with the form of pharmaceutical composition.This pharmaceutical composition contains 0.1-99.5%, and be preferably the compounds of this invention of 0.5-90%, all the other are acceptable on pharmacology, pharmaceutically acceptable carrier of and inertia nontoxic to humans and animals and/or vehicle.
Described pharmaceutical carrier or vehicle are one or more solids, semisolid and liquid diluent, filler and pharmaceutical preparation assistant agent.Pharmaceutical composition of the present invention is used with the form of per weight dose.The method that the composition of phenanthridines analog derivative of the present invention adopts pharmacy and field of food to generally acknowledge is prepared into various formulation, as liquid preparation (injection, suspensoid, emulsion, solution, syrup etc.), solid preparation (tablet, capsule, granule, electuary etc.), spray, aerosol etc.Medicine of the present invention can carry out anti-antineoplastic treatment through route of administration such as injection (intravenous injection, intravenous drip, intramuscular injection, abdominal injection, subcutaneous injection) and oral, sublingual administration, mucous membrane dialysis.
The present invention's research in earlier stage shows: the 15-oxospiramilactone (S-3) coming from pink blossom Ramulus et Folium Spiraeae Salicifolia has brand-new mechanism of action, the apoptosis that S-3 induces non-Bax/Bak to rely on, further investigation finds that S-3 induces the high expression level of pro apoptotic protein Bim specifically, itself and Bcl-2 have an effect at mitochondrial level, Bcl-2 conformational change is caused to be converted to short apoptosis function by anti-apoptotic, thus the release of activating cells pigment C causes the dependent apoptosis of the non-Bax/Bak of cell, this is the new mechanism of cell death inducing; Find that S-3 is tumour cell Wnt signal path optionally inhibitor, suppressed the expression of Wnt downstream target gene by the interaction weakening β-catenin and TCF4; Expressing by suppressing downstream target gene causes cell cycle arrest in the G2/M phase; SW480 transplants experiment of nude mouse and shows, the S-3 of low dosage gets final product the growth of Tumor suppression.The present invention on this basis, to the large intestine of S-3 and colon tumor strain, Normocellular cytotoxic activity, and the early stage Fast Evaluation of ADME/T has done analysis, finds that S-3 has " first pass effect ", it eliminates the transformation period is 0.43 hour, the hydrolysate anti-tumor activity completely dissolve of lactonic ring.For the defect of its druggability, structure of modification and composition optimizes are carried out to it, using the cytotoxic activity of the inhibit activities of Wnt signal path, different tumor line etc. as foundation, find patent medicine more reasonably antineoplastic compound be very important.
Embodiment:
Further illustrate essentiality content of the present invention with embodiments of the invention below, but do not limit the present invention with this.
Embodiment 1:
The preparation of the compounds of this invention:
(1) compound 1-9,25, the syntheti c route of 26 is:
Preparation method:
Compound 1: get the drying kinetics compound 15-oxospiramilactone (S-3) (2.00g, 6.06mmol) coming from pink blossom Ramulus et Folium Spiraeae Salicifolia, at N 2protection is lower dissolves with dry methylene chloride 5ml, by oxalyl chloride (7.28mmol, 692ul) be injected in the long-neck low-temp reaction bottle of the methylene dichloride that 5ml drying is housed in nitrogen protection, be placed in-78 DEG C to stir 20 minutes, then slowly DMSO(14.56mmol is dripped, 1032ul, in in5ml DCM) solution, after within 5 minutes, dripping, low temperature stirs 30 minutes, again the dichloromethane solution of compound S-3 is slowly added drop-wise in reaction flask, lower stirring is stirred after 1 hour in-78 DEG C, under fast stirring, slow dropping triethylamine (14.56mmol, 2020ul), add and be naturally warming up to normal temperature afterwards, pour in the water of 20ml, extracting and demixing, water layer continues to use 20ml dichloromethane extraction, combined dichloromethane, saturated common salt water washing, anhydrous sodium sulfate drying, after concentrated, resistates obtains compound 1 through silica gel column chromatography, colourless powder 1888.2mg, yield: 95.0%.
Compound 2: weigh up compound 1(1.0mmol, 328mg), paraformaldehyde (5mmol, 150mg), Bu 4nI (0.2mmol, 73.8mg), Anhydrous potassium carbonate (2.0mmol, 276mg) as in the dry round-bottomed flask of 50ml, N 2under protection; by dry toluene 15ml sample dissolution; reaction mixture stirs 24 hours at 50 DEG C, and TLC point plate determination raw material is cooled to room temperature and adds 20ml water after disappearing; be extracted with ethyl acetate (2 × 20ml); organic layer uses saturated common salt water washing in succession, anhydrous sodium sulfate drying, concentrated that residue by silicagel column chromatography obtains compound 2; colourless powder 204mg, yield: 60%.
Compound 3: weigh paraformaldehyde (5mmol, 150mg) and be suspended in the dry toluene solution of 10ml, N 2under protection; injection morpholine (5mmol, 436ul), stirs 1h at 40 DEG C; after solution becomes clarification; be down to stirring at room temperature, inject compound 1(1mmol, 328mg successively) toluene solution 5ml; AcOH(1.0mmol; 60ul), stirring at room temperature 3 hours, TLC shows raw material and disappears; stopped reaction; add the dilution of 20ml saturated sodium bicarbonate, with ethyl acetate (2 × 20ml) extraction, water, saturated common salt water washing; anhydrous sodium sulfate drying; silica gel column chromatography obtains compound 3, colourless powder 320mg, yield: 75%.
Compound 4: weigh paraformaldehyde (5mmol, 150mg) successively, dimethylamine hydrochloride (5mmol, 407.7mg) is suspended in the dry toluene solution of 10ml, N 2under protection, at 40 DEG C, stir 1h, after solution becomes clarification; be down to stirring at room temperature, inject compound 1(1mmol, 328mg successively) toluene solution 5ml; AcOH(1.0mmol, 60ul), stirring at room temperature 1 hour; TLC shows raw material and disappears, stopped reaction, adds the dilution of 20ml saturated sodium bicarbonate; with ethyl acetate (2 × 20ml) extraction, water, saturated common salt water washing, anhydrous sodium sulfate drying; silica gel column chromatography obtains compound 4, colourless powder 366mg, yield: 95%.
Compound 5,6: Weigh Compound 1(1mmol, 328mg), dissolve with the dry DMSO of 10ml, N 2nCS(1.1mmol, 147mg is added under protection), in stirred at ambient temperature 1h; TCL display is not reacted, and stirs 3h at heating 45 DEG C, and TLC point plate finds that raw material disappears; stopped reaction, is cooled to room temperature and adds the dilution of 20ml water, extract by ethyl acetate (2 × 20ml); water, saturated common salt water washing, anhydrous sodium sulfate drying, silica gel column chromatography obtains compound 5; colourless powder 164.7mg, yield 46%, compound 6; colorless oil 149.6mg, yield: 40%.
Compound 7: Weigh Compound 1(1mmol, 328mg), dissolve with the dry DMSO of 10ml, N 2nBS(1.1mmol, 196mg is added under protection), in stirred at ambient temperature 1h; TCL display is not reacted, and stirs 3h at heating 45 DEG C, and TLC point plate finds that raw material disappears; stopped reaction; be cooled to room temperature and add the dilution of 20ml water, with ethyl acetate (2 × 20ml) extraction, water, saturated common salt water washing; anhydrous sodium sulfate drying; silica gel column chromatography obtains compound 7, brown-red powder 374mg, yield: 92%.
Compound 8: Weigh Compound 1(1mmol, 328mg) dissolve by the dry toluene of 5ml, N 2nCS(1.1mmol, 146.8mg is added under protection), ionic liquid [(BMIM) PF 4] 1ml, stirring at room temperature 1h, raw material disappears, and stops stirring adding the dilution of 20m water, and with ethyl acetate (2 × 20ml) extraction, water, saturated common salt water washing, anhydrous sodium sulfate drying, silica gel column chromatography obtains compound 8, colourless powder 50mg, yield: 13.8%.
Compound 9: get compound S-3(1.0mmol, 330mg) be dissolved in toluene: in dimethyl sulfoxide (DMSO)=2:110ml solution, N 22-iodosobenzoic acid (IBX, 3.0mmol, 840mg) is added under protection; stir 30 hours at 70 DEG C, raw material is cooled to room temperature after disappearing; with the diluted ethyl acetate of 100ml; use saturated sodium bicarbonate solution successively, water, saturated common salt water washing; anhydrous sodium sulfate drying; concentrated that residue by silicagel column chromatography obtains compound 9, colourless powder 97.8mg, yield: 30%.
Compound 25,26: get compound 1(330mg; 1.0mmol) be dissolved in the methanol solution of 10ml; add morpholine (1.5eq, 1.5mmol) stirring at room temperature 10 hours, after raw material disappears; stopped reaction; concentrating under reduced pressure obtains resistates, obtains compound 25(187mg, yield: 45% through silica gel column chromatography; 26(179mg, yield; 43%).
(2) preparation of compound 10:
Compound 10: by oxalyl chloride (1.0mmol, 95ul) at N 2protection bet is injected in dry long-neck reaction flask, miscible with the dry DCM of 5ml, stir at being placed in-78 DEG C, slow dropping DMSO (2.0mmol, 142ulin2ml DCM), stir 30min, slowly dripping S-1(332mg, 1.0mmol in2ml DCM), stir 30min at-78 DEG C after, drip triethylamine (2.0mmol, 278ul) naturally rise to room temperature afterwards, the 20ml that adds water dilutes, extract with methylene dichloride (2*20ml), saturated common salt water washing, anhydrous sodium sulfate drying, compound S-3(165mg is obtained) through silica gel column chromatography, with compound M-1(165mg), compound M-1 is prepared compound 10 by the method similar to preparing compound 2, colourless powder 162.4mg, two step yield: 47.5%.
(3) preparation of compound 11-13:
Preparation method:
By compound S-1(3.0g, 9.04mmol) be dissolved in analytically pure acetone 250ml, N 2protection is lower stirs, and add the tosic acid (monohydrate) of (1.81mmol, 343.5mg), at room temperature stir 24 hours, in reaction process, continuous adularescent solid washes out, after raw material reaction is complete, and stopped reaction.Filtering solids, sherwood oil: acetone=20:1 solution, washing leaching cake, mother liquor continues the solution weight crystallization with sherwood oil-acetone=1:1, and air-dry, combining solid, obtains midbody compound M-2(3.30g, yield 97%).The M-2 of drying is dissolved in dry 250mlTHF solution, N 2protection, be placed in ice bath to stir, slowly add Lithium Aluminium Hydride (10.5mmol, 400mg), stir after 20 minutes, to be placed on 45 DEG C of oil bath pans heated and stirred 3 hours, after TLC shows raw material disappearance, reaction flask is placed in ice bath again and stirs, slowly add the sodium hydroxide solution 200ml of 2N, stir 10 minutes in ice bath, suction filtration, ethyl acetate washing leaching cake, filtrate layering, aqueous fraction continues to be extracted with ethyl acetate 2 times, combined ethyl acetate, use saturated common salt water washing, anhydrous sodium sulfate drying, concentrated solvent obtains midbody compound M-3, colourless powder 3.33g, yield: 100%.Get compound M-3(376.0mg, 1.0mmol) be dissolved in the DCM solution of 10ml drying, at room temperature slowly add PDC(1.0mmol, 376mg), room temperature rapid stirring 5 hours, after TLC point board raw material disappears, poured into by reactant in frozen water, layering, water layer uses 20ml dichloromethane extraction again, merge organic layer, saturated common salt water washing, anhydrous sodium sulfate drying, concentrated, midbody compound M-4(336mg is obtained, yield: 90%) through silica gel column chromatography.By compound M-4(224mg, 0.60mmol) be dissolved in the analytical pure methyl alcohol of 10ml, drip 2NHCl(3ml), stir 24 hours at 35 DEG C, after raw material disappears, stop stirring, methyl alcohol is spin-dried under decompression, thin up, extraction into ethyl acetate, uses saturated sodium bicarbonate solution, saturated common salt water washing in succession, anhydrous sodium sulfate drying, silica gel column chromatography obtains midbody compound M-5, colorless oil 198mg, yield: 95%.
Compound 11: get compound M-5(50mg, 0.144mmol) be dissolved in the dry methylene chloride of 10ml, divide 3 batches and add freshly prepd activated manganese dioxide, often criticize Manganse Dioxide amount for (0.288mmol, 25mg), interval time is 10 hours, after raw material disappears substantially, stop stirring, filter Manganse Dioxide, with methylene dichloride repetitive scrubbing Manganse Dioxide, concentrated methylene dichloride obtains grey oily matter, compound 11 is obtained, colorless oil 28mg, yield: 56.2% through silica gel column chromatography.
Compound 12: by oxalyl chloride (1.28mmol, 110ul) at N 2protection bet is injected in dry long-neck reaction flask, miscible with the dry DCM of 5ml, stir at being placed in-78 DEG C, slow dropping DMSO(2.56mmol, 182ul in2ml DCM), stir 30min, slowly dripping M-5(148mg, 0.425mmol in2mlDCM), stir 30min at-78 DEG C after, drip triethylamine (2.56mmol, 369ul) naturally rise to room temperature afterwards, the 10ml that adds water dilutes, extract with methylene dichloride (2 × 10ml), saturated common salt water washing, anhydrous sodium sulfate drying, compound 12 is obtained through silica gel column chromatography, colourless powder 146mg, yield: 100%.
Compound 13: weigh up compound 12(0.29mmol, 100mg), paraformaldehyde (1.45mmol, 44mg), Bu4NI(0.06mmol, 21mg), Anhydrous potassium carbonate (0.58mmol, 80mg) as in the dry round-bottomed flask of 25ml, N 2under protection; with in dry toluene 10ml sample dissolution; reaction mixture stirs 24 hours at 50 DEG C, and TLC point plate determination raw material is cooled to room temperature and adds 10ml water after disappearing; be extracted with ethyl acetate (2 × 15ml); organic layer uses saturated common salt water washing in succession, anhydrous sodium sulfate drying, concentrated that residue by silicagel column chromatography obtains compound 13; colourless powder 56.7mg, yield: 55%.
(4) syntheti c route of compound 14-20:
Preparation method:
By compound M-3(376.0mg, 1.0mmol) be dissolved in the THF of 10ml drying, N 2under protection; add triphenyl phosphorus (1.1mmol; 288mg); slow dropping diisopropyl azodiformate (1.1mmol, 222mg are diluted in the dry THF of 2ml); stirring at normal temperature 30 minutes; after TLC point plate determination raw material disappears, the dilute hydrochloric acid (5ml) adding 2N stirs 24 hours at 35 DEG C, and TLC tracks to after acetonylidene all takes off; thin up reaction solution; be extracted with ethyl acetate, saturated common salt water washing, anhydrous sodium sulfate drying; concentrated; resistates obtains compound M-6 through silica gel column chromatography, colorless oil 318mg, yield: 100%.
The preparation of compound 14,18 is obtained by the method similar to compound 10; Simultaneously compound 15,20 preparation by compound 12,13 similar methods and obtaining.
The preparation of compound 16,17,20: round bottom reaction flask Weigh Compound 15(100mg, the 0.318mmol successively with 25ml), salt of wormwood (0.636mmol, 87.8mg), paraformaldehyde (1.59mmol, 47.7mg), N 2under protection, by the toluene sample dissolution of 10ml drying, stirred at ambient temperature 24 hours, TLC follows the tracks of, after raw material disappears, stop stirring, add the dilution of 10ml water, with ethyl acetate (2 × 15ml), organic layer saturated common salt water washing, anhydrous sodium sulfate drying, concentrated that residue by silicagel column chromatography obtains compound 16, colourless powder 82mg, yield: 75%; The compound 16(50mg, the 0.15mmol that in succession take), DMAP (5mg) in the round bottom reaction flask of 10ml, N 2the lower methylene dichloride with 5ml drying of protection dissolves, and in succession uses injector to inject pyridine (0.18mmol, 15ul), Methanesulfonyl chloride (0.18mmol, 14ul), at room temperature stirs 3 hours, TLC point plate is followed the tracks of, and after raw material disappears, stops stirring, add the dilution of 10ml water, methylene dichloride (2 × 15ml), organic layer saturated common salt water washing, anhydrous sodium sulfate drying, concentrated that residue by silicagel column chromatography obtains compound 17, colorless oil 62mg, yield: 98%; In the round bottom reaction flask of 15ml, Weigh Compound 17(50mg, 0.118mmol), N 2under protection, dissolve by the dry tetrahydrofuran of 5ml, add DBU(0.236mmol, 35ul), stirring at room temperature 24 hours, TLC detects and finds that raw material disappears, and direct concentrating under reduced pressure, the compound of silica gel column chromatography obtains compound 20, colourless powder 27mg, yield: 70%.
Compound 19: by compound M-6(1.0mmol, 318mg) being dissolved in 20ml analyzes in acetone, the Jones reagent(2.2mmol of new configuration is slowly dripped under ice bath, 0.84ml) rapid stirring 30 minutes, after finding that raw material disappears, add Virahol (5ml) stopped reaction, concentrate and remove acetone, thin up, be extracted with ethyl acetate 2 times, saturated common salt water washing, anhydrous sodium sulfate drying, concentrated, resistates obtains compound through silica gel column chromatography and obtains compound 15(72mg, yield: 20.7%), compound M-8(45mg, yield: 13.6%), (BMIM) PF4 ionic liquid suspendible of compound M-8 5ml, in succession add morpholine (0.136mmol, 12ul), acetic acid (0.136mmol, 9ul), paraformaldehyde (0.68mmol, 20mg), 10h is stirred at 70 DEG C, after raw material disappears, react with saturated sodium bicarbonate solution 10ml cancellation, ethyl acetate (2 × 20ml) extracts, organic layer uses saturated common salt water washing in succession, anhydrous sodium sulfate drying, concentrated that residue by silicagel column chromatography obtains compound 19(32mg, yield: 70%).
(5) syntheti c route of compound 21:
Preparation method:
Compound 21: in the round-bottomed flask of 25ml, Weigh Compound M-4(112mg, 0.3mmol) be dissolved in the methylene dichloride of 10ml, N 2dMAP (5mg) is added successively, pyridine (1.0mmol, 80.6ul), Ac under protection 2o(0.45mmol, 42.5ul), room temperature stirs 3h, after raw material disappears, concentrated resistates, with 10ml dissolve with methanol resistates, add 2N HCl(5ml) stir 24 hours at 35 DEG C, TLC point plate finds, raw material disappears, produce new point, concentrate and remove methyl alcohol, add extraction into ethyl acetate 2 times, in succession use saturated sodium bicarbonate solution, saturated common salt water washing ethyl acetate layer, anhydrous sodium sulfate drying, concentrated that resistates obtains compound M-10 through silica gel column chromatography, compound M-10 obtains compound 21(79mg through a Swern oxidation, two step yield 95%).
(6) syntheti c route of compound 22,27:
Preparation method:
Compound 22, 27: by compound S-1(500mg, 1.50mmol) be placed in the round bottom reaction flask of 250ml, with 100ml analytical pure dissolve with methanol sample, add (7.5mmol, KOH 420mg), stir, heat back and heat up in a steamer 24 hours, after raw material disappears, stopped reaction, thin up, concentrating under reduced pressure removes methyl alcohol, by the dilute hydrochloric acid furnishing neutral solution of 2N, be extracted with ethyl acetate 2 times, saturated common salt water washing, anhydrous sodium sulfate drying, concentrating under reduced pressure obtains intermediate M-11 crude product 550mg, get dry crude Compound M-11(100mg, 0.28mmol) be dissolved in and be equipped with in 50ml dry DCM round bottom reaction flask, N 2under protection, add brand-new MnO in batches 2(1.42mmol, 123.5mg), stirring at normal temperature 72 hours, after raw material no longer changes, filter Manganse Dioxide, concentrated, resistates obtains compound 27 through silica gel column chromatography, colourless powder 30mg, yield: 30.2%.Get dry crude Compound M-11(200mg, 0.56mmol) be dissolved in the round bottom reaction flask that 50ml analytical pure acetone is housed, under being placed in ice bath, slow dropping Jones reagent (2.52mmol, 0.96ml), stir after 30 minutes under ice bath, TLC point plate finds that raw material disappears, react with Virahol (5ml) cancellation, concentrate and remove acetone, thin up, be extracted with ethyl acetate 2 times, saturated common salt water washing, anhydrous sodium sulfate drying, concentrated, resistates obtains compound through silica gel column chromatography and obtains compound 22, colourless powder 80mg, yield: 40.8%.
(7) compound 23,24, the syntheti c route of 28-36:
Preparation method:
By compound M-3(376.0mg, 1.0mmol) be dissolved in the methylene dichloride of 10ml drying, be placed in 0 DEG C, N 2thionyl chloride (1.1mmol is slowly injected under gas shielded, 80ul), after adding, rise to stirring at room temperature 30 minutes, raw material disappears, the cancellation that adds water is reacted, be spin-dried for DCM, add 10ml THF sample dissolution, add the dilute hydrochloric acid 5ml of 2N again, stir 24 hours at 35 DEG C, after acetonylidene is all taken off, stop stirring, add extraction into ethyl acetate 2 times, in succession use saturated sodium bicarbonate solution, saturated common salt water washing ethyl acetate layer, anhydrous sodium sulfate drying, concentrated that resistates obtains midbody compound M-12 through silica gel column chromatography, compound M-12 obtains compound 23 through manganese dioxide, M-12 has prepared compound 24 with preparing the similar method of compound 13 simultaneously.
In the round bottom reaction flask of 50ml, by compound M-3(752.0mg, 2.0mmol), DMAP (10mg) is dissolved in the methylene dichloride of 15ml drying, is placed in 0 DEG C, N 2slowly pyridine (6.0mmol is injected into successively under gas shielded, 484ul), diacetyl oxide (6.0mmol, 567ul), after adding, rise to stirring at room temperature 24 hours, after TLC shows raw material disappearance, add 20ml shrend to go out reaction, extract with methylene dichloride (2 × 20ml), saturated common salt water washing dichloromethane layer, anhydrous sodium sulfate drying, concentrate to obtain resistates, by 15ml THF sample dissolution, add the dilute hydrochloric acid 8ml of 2N again, stir 24 hours at 35 DEG C, after acetonylidene is all taken off, stop stirring, add extraction into ethyl acetate 2 times, in succession use saturated sodium bicarbonate solution, saturated common salt water washing ethyl acetate layer, anhydrous sodium sulfate drying, concentrated that resistates obtains midbody compound M-13 through silica gel column chromatography, colorless oil 773mg, yield: 92%.
Compound 28,30: Weigh Compound M-13(150mg, 0.36mmol) be dissolved in dry methylene dichloride, add freshly prepd Manganse Dioxide (2.16mmol, 188mg) in batches, stirring at room temperature 72 hours, after raw material disappears substantially, filter Manganse Dioxide, with methylene dichloride repetitive scrubbing Manganse Dioxide, filtrate concentrates to obtain resistates, compound 30 is obtained, colorless oil 113mg, yield: 75% through silica gel column chromatography.Get compound 30(80mg, 0.19mmol) be dissolved in analytically pure tetrahydrofuran (THF), add the HCl(5ml of 2N) stirring at room temperature 24 hours, after raw material disappears, add the dilution of 10ml water, by ethyl acetate (2 × 20ml) extracting twice, ethyl acetate layer uses saturated sodium bicarbonate, water, saturated common salt water washing in succession, anhydrous sodium sulfate drying is concentrated that resistates obtains compound 28 through silica gel column chromatography, colourless powder 38mg, yield: 60%.
Compound 31,32,33,34,36 through to above 15,16,17,20 the similar method of preparation and prepare; Compound 31 obtains compound 29 through hydrochloric acid hydrolysis, and it is identical that method and compound 30 prepare compound 28; Compound 35 is prepared with the similar method of compound 3.
(8) syntheti c route of compound 37-41:
Preparation method:
Compound 37,38,40 is similar with the preparation method of 30,31; Preparation method is similar with the preparation of compound 19 for compound 39,41.
(9) syntheti c route of compound 42,43,44:
Preparation method:
Compound 42,43: compound M-3(376.0mg, 1.0mmol) be placed in the round bottom reaction flask of 25ml, N 2the lower THF with 10ml drying of protection dissolves, stir under ice bath, slowly add 60%NaH(3.0mmol, 120mg), stir 20 minutes, slow injection 4-morpholine carbonyl chloride (3.0mmol, 351ul), rise to stirring at room temperature 3 hours, do not react, 12 hours are heated up in a steamer next time again at 40 DEG C, after raw material disappears, the 10ml cancellation that adds water is reacted, ethyl acetate equal-volume extracts 2 times, combined ethyl acetate layer, saturated common salt water washing, anhydrous sodium sulfate drying, concentrate to obtain resistates, intermediate M-17 and M-18 is obtained through silica gel column chromatography, be all oily compound thing, wherein M-17(269mg, yield: 55%), M-18(171mg, yield: 35%), then M-17 and M-18 passes through another hydroxymethyl sulfonylation and de-acetonylidene again, polite oxidation obtains compound 42(colorless oil 230mg respectively, two step yield: 44%), compound 43(colorless oil 143mg, two step yield: 27%).
Compound 44: weigh compound M-3(376.0mg, 1.0mmol) in the round bottom reaction flask of 25ml, N 2the lower DMF with 10ml drying of protection dissolves, stir under ice bath, slowly add 60%NaH(3.0mmol, 120mg), stir 20 minutes, slowly inject bromoacetonitrile (3.0mmol, 209ul) stir 2 hours under ice bath, TLC detects and finds that raw material disappears, and stop stirring, add water cancellation, add the extraction into ethyl acetate layering of 50ml, the water continuous washing of each 20ml 3 times, saturated common salt water washing, anhydrous sodium sulfate drying, concentrate to obtain resistates, intermediate M-16 is obtained, colorless oil 311mg, yield 75% through silica gel column chromatography; Then M-16 is dissolved in the methylene dichloride of 10ml drying, adds DMAP (5mg), N 2pyridine (2.0mmol, 161ul) is added successively, thiophene-2-formyl chloride (2.0mmol, 215ul) under protection; 12 hours are heated up in a steamer next time, after raw material disappears, then through a de-acetonylidene at 40 DEG C; polite oxidation obtains compound 44, colorless oil 264mg, two step yield: 55%.
(10) syntheti c route of compound 45,46,47,48,49:
Preparation method:
The preparation method of compound 47,48 is similar to compound 37,38 preparation method, the preparation method preparing compound 45,46,49 from compound 48 is similar to the preparation method of compound 19, wherein the yield of compound 49 is 55%, and the yield of compound 45 is 20%, and the yield of compound 46 is 10%.
(11) syntheti c route of compound 50-55:
Preparation method:
Compound 51-54: Weigh Compound spiramine C/D(1.0g, 2.80mmol), with the analytical pure dissolve with methanol of 100ml, slowly add sodium borohydride (5.0mmol under room temperature in batches, 189.5mg), add rear stirring 30min, after raw material disappears, acetone is slowly dripped to no longer emitting bubble under ice bath, add the dilution of 50ml water, concentrating under reduced pressure removes methyl alcohol, extract by ethyl acetate (2 × 100ml), washing, saturated common salt water washing, anhydrous sodium sulfate drying, concentrating under reduced pressure recycling design obtains not purified directly next step reaction of resistates, the resistates of drying is dissolved in the dry DMF of 20ml, N 2protection adds TERT-BUTYL DIMETHYL CHLORO SILANE (TBDMSCl, 2.50mmol, 376.7mg) successively under ice bath, imidazoles (imidazole, 2.50mmol, 170mg), stirring at room temperature 2h, after raw material disappears, add water (50ml) dilution, extract by ethyl acetate (2 × 100ml), use water, saturated common salt water washing successively, anhydrous sodium sulfate drying, concentrating under reduced pressure obtains resistates and obtains midbody compound M-22(950mg through silica gel column chromatography, 2.0mmol, two step yield: 71.4%).By oxalyl chloride (2.0mmol, 190ul) at N 2protection bet is injected in dry long-neck reaction flask; miscible with the dry DCM of 10ml; stir at being placed in-78 DEG C; slow dropping DMSO (4.0mmol, 283.6ul in2ml DCM), stirs 30min; slowly dripping M-22(950mg; 2.0mmol, in5ml DCM), stir 30min at-78 DEG C after; drip triethylamine (4.0mmol; 554.9ul) naturally rise to room temperature, the 20ml that adds water dilutes, and extracts with methylene dichloride (2 × 20ml); saturated common salt water washing; anhydrous sodium sulfate drying, concentrates to obtain paste (927mg, yield: 98%).Get paste (460mg, 0.97mmol) to carry out silica gel column chromatography and obtain compound M-23(220mg, 0.46mmol) and M-24(215mg, 0.45mmol).By M-23(220mg, 0.46mmol), paraformaldehyde (69mg, 2.3mmol), Bu 4nI (34mg, 0.09mmol), dry salt of wormwood (127mg, 92mmol) as in the dry round-bottomed flask of 50ml, N 2under protection, with in dry toluene 10ml sample dissolution, reaction mixture stirs 12 hours at 50 DEG C, after TLC point plate determination raw material disappears, be cooled to room temperature and add water 20ml, be extracted with ethyl acetate (2 × 20ml), organic layer uses saturated common salt water washing in succession, anhydrous sodium sulfate drying, concentrates to obtain resistates; THF10ml dry for dried resistates is dissolved, N 2protection, stirs hemostasis TBAF (0.55mmol, 550ul; 1M TBAF in THF); stirring at room temperature 2h, after raw material disappears, adds the dilution of 20ml water; extract by ethyl acetate (2*20ml); saturated common salt water washing, anhydrous sodium sulfate drying, concentrated that residue by silicagel column chromatography obtains compound 51(colorless oil; 85mg, yield: 49.8%).M-24(215mmol, 0.45mmol) obtain compound 50(colorless oil, 105mg, 65% with TBAF deprotection).Mixture (467mg) the polite oxidation again (swern oxidation) of getting M-23 and M-24 obtains M-25 (420mg; 0.89mmol); get M-25 (100mg; 0.21mmol); compound 52(colorless oil is obtained with TBAF deprotection; 55mg, yield: 73%).Get M-25 (320mg, 0.68mmol), paraformaldehyde (102mg, 3.4mmol), Bu 4nI (50.2mg, 0.136mmol), dry salt of wormwood (188mg, 1.36mmol) as in the dry round-bottomed flask of 50ml, N 2under protection; with in dry toluene 10ml sample dissolution, reaction mixture stirs 12 hours at 50 DEG C, after TLC point plate determination raw material disappears; be cooled to room temperature and add water 20ml; be extracted with ethyl acetate (2 × 20ml), organic layer uses saturated common salt water washing in succession, anhydrous sodium sulfate drying; concentrate to obtain resistates; compound 53(colorless oil is obtained, 147.7mg, yield: 45%) through silica gel column chromatography.Get compound 53(100mg, 0.207mmol) obtain compound through TBAF deprotection and obtain compound 54(colorless oil, 49mg, yield: 65%).
Compound 55: Weigh Compound spiramine C/D (100mg, 0.28mmol) is dissolved in the methylene dichloride of 30ml, adds freshly prepd Manganse Dioxide (0.28mmol in batches under room temperature, 0.56mmol, 0.56mmol), interval time is 10h, 10h, 10h, after raw material disappears substantially, filters Manganse Dioxide, use methylene dichloride repetitive scrubbing, merging filtrate, concentrated resistates obtains compound 55(69.6mg through silica gel column chromatography, productive rate: 70%).
(12) syntheti c route of compound 56-58:
Preparation method:
Compound 56: Weigh Compound spiramine C/D (100mg, 0.28mmol) is dissolved in the analytically pure acetone of 30ml, adds freshly prepd Manganse Dioxide (0.56mmol in batches, 0.56mmol, 0.56mmol, 0.56mmol, 0.56mmol), stirred at ambient temperature 72h, after raw material disappears substantially, filters Manganse Dioxide, use acetone repetitive scrubbing, merging filtrate, concentrated resistates through the compound 48(31.6mg of silica gel column chromatography, productive rate: 30%).
Compound 57, 58: Weigh Compound spiramine C/D(500mg, 1.4mmol) be dissolved in the analytical pure methyl alcohol of 50ml, add sodium borohydride (2.8mmol in batches, 105.8mg), stirred at ambient temperature 30min, acetone termination reaction is added after raw material disappears, add the dilution of 30ml water, concentrating under reduced pressure removes methyl alcohol, extract by ethyl acetate (2 × 100ml), washing, saturated common salt water washing, anhydrous sodium sulfate drying, concentrating under reduced pressure obtains resistates without being further purified directly next step reaction, by dried residue (454mg, 1.26mmol) be dissolved in the methyl alcohol of 25ml, add K 3fe (CN) 6the aqueous solution (1.24g, 3.78mmo in10ml H 2o), then add the aqueous solution (in 750mg KOH solution 5ml water) of KOH, flow heated water 4h, after raw material disappears, filter, the resistates of concentrating under reduced pressure, obtains compound M-26(180mg through silica gel column chromatography, two step yields: 35.8%).Compound M-26 is dissolved in analytically pure acetone, adds Manganse Dioxide (880mg, 10.1mmol) in batches, stirring at room temperature 72h, after raw material disappears substantially, stops stirring, filter Manganse Dioxide, with acetone repetitive scrubbing Manganse Dioxide, merging filtrate, concentrate to obtain resistates, through the compound 57(59.8mg of silica gel column chromatography, 32%), compound 58(33mg, 18%).
The spectral data of above-claimed cpd is as follows:
Compound (1): 1h-NMR (400MHz, CDCl 3) δ: 6.05 (1H, d, J=1.2Hz), 5.34 (1H, d, J=1.2Hz), 4.24 (1H, dd, J=2.4,12.4Hz), 4.16 (1H, d, J=12.4Hz), 2.86-2.91 (2H, m), 2.78 (1H, dd, J=6.0,18.0Hz), 2.05-2.32 (5H, m), 1.66-1.95 (7H, m), 1.48-1.50 (1H, m), 1.25-1.38 (2H, m), 0.91 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 206.2,195.9,173.2,146.6,119.4,76.0,57.5,45.8,44.9,44.8,40.4,38.2,36.6,35.9,33.1,27.2,25.9,24.3,22.8,20.1.HR-EI-MS m/z328.1673 [M] +(C 20h 24n 2o 4, calcd328.1675).
Compound (2): 1h-NMR (400MHz, CDCl 3) δ: 5.98 (1H, s), 5.93 (1H, s), 5.34 (1H, s), 5.26 (1H, s), 3.93 (2H, s), 2.83 (1H, d, J=3.2Hz), 2.68 (1H, t, J=11.2Hz), (2.52 1H, d, J=1.2Hz), 2.16-2.22 (3H, m), 2.01-2.08 (1H, m), 1.87-1.97 (2H, m), 1.54-1.82 (5H, m), 1.35 (1H, ddd, J=4.4,12.4,16.8Hz), 0.94 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 202.3,195.7,173.3,146.2,144.8,125.8,119.9,75.4,57.8,53.5,44.7,42.5,39.5,36.3,36.2,35.0,27.3,25.9,24.3,20.9,20.8.HR-EI-MS m/z340.1682 [M] +(C 21h 24o 4, calcd340.1675).
Compound (3): 1h-NMR (400MHz, CDCl 3) δ: 6.02 (1H, s), 5.37 (1H, s), 4.17 (1H, d, J=12.0Hz), 4.02 (1H, d, J=12.0Hz), 3.71 (4H, t, J=4.4Hz), 2.85 (1H, d, J=2.4Hz), 2.66-2.75 (3H, m), 2.53 (4H, brs), 2.10-2.53 (5H, m), 1.89-2.01 (2H, m), 1.62-1.82 (3H, m), 1.53-1.60 (3H, m), 1.31-1.35 (1H, m), 0.92 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 208.2,197.0,173.0,146.6,119.7,75.5,68.8 (2CH 2-morpholine), 63.5,58.2,53.6 (2CH 2-morpholine), 52.5,47.4,44.6,42.5,40.4,37.1,36.3,34.4,27.6,26.0,22.5,22.3,20.6.HR-EI-MS m/z427.2366 [M] +(C 25h 33nO 5, calcd427.2359).
Compound (4): 1h-NMR (400MHz, CDCl 3) δ: 6.04 (1H, d, J=1.6Hz), 5.39 (1H, d, J=1.6Hz), 4.22 (1H, dd, J=2.4,12.0Hz), 4.04 (1H, dd, J=1.6,12.0Hz), 2.40-2.90 (6H, m), 2.38 (6H, s), 1.25-2.29 (13H, m), 0.91 (3H, s) .HR-EI-MS m/z385.2266 [M] +(C 23h 31nO 4, calcd385.2253).
Compound (5): 1h-NMR (400MHz, CDCl 3) δ: 6.10 (1H, s), 5.47 (1H, s), 4.16 (1H, d, J=12.0Hz), 4.06 (1H, d, J=12.0Hz), 3.88 (1H, m), 3.61 (1H, m), 2.88 (1H, m), 2.81 (1H, m), 2.63-2.69 (2H, m), 2.34 (1H, t, J=10.0,10.0Hz), 2.20-2.28 (2H, m), 1.92-2.12 (3H, m), 1.49-1.84 (6H, m), 1.34-1.42 (1H, m), 0.90 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 210.4,198.8,172.7,145.8,121.1,75.2,65.9,58.2,52.6,48.5,44.0,42.2,40.1,36.4,35.9,33.6,27.1,25.9,22.2,21.7,20.2.HR-EI-MS m/z358.1791 [M] +(C 21h 26o 5, calcd358.1980).
Compound (6): 1h-NMR (400MHz, CDCl 3) δ: 6.04 (1H, d, J=1.6Hz), 5.36 (1H, d, J=1.6Hz), 4.22 (1H, dd, J=2.0,12.0Hz), 4.09 (1H, dd, J=1.2,12.0Hz), 3.08 (1H, d, J=4.8Hz), 2.86 (1H, m), 2.67 (1H, m), 2.50 (1H, t, J=10.0,10.0Hz), 2.21-2.29 (2H, m), 2.17 (3H, s), 1.90-2.11 (3H, m), 1.68-1.84 (2H, m), 1.53-1.67 (4H, m), 1.30-1.38 (1H, ddd, J=4.4,11.0,15.2Hz), 1.02 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 200.4,197.1,172.8,146.6,119.5,75.5,57.4,54.8,48.9,44.5,44.0,40.3,40.2,36.8,34.0,27.6,25.8,23.6,22.2,20.5,16.2.HR-EI-MS m/z374.1536 [M] +(C 21h 26o 4s, calcd374.1552).
Compound (7): 1h-NMR (400MHz, CDCl 3) δ: 6.11 (1H, d, J=1.2Hz), 5.42 (1H, d, J=1.2Hz), 4.20 (1H, d, J=4.8Hz), 4.19 (1H, d, J=1.6Hz), 4.10 (1H, dd, J=1.6,12.4Hz), (2.90 1H, d, J=3.2Hz), 2.67 (1H, m), 2.56 (1H, m), 2.50 (1H, m), 2.21-2.29 (2H, m), 1.41-2.10 (9H, m), 1.12 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 199.8,194.8,172.1,145.7,120.3,75.3,59.1,58.4,45.0,43.3,41.0,40.3,36.9,36.0,34.1,27.5,25.8,23.3,22.4,20.2.HR-EI-MS m/z406.0786 [M] +(C 20h 23o 4br, calcd406.0780).
Compound (8): 1h-NMR (400MHz, CDCl 3) δ: 6.10 (1H, d, J=1.2Hz), (5.42 1H, d, J=1.2Hz), 4.09-4.16 (3H, m), 2.89 (1H, s), 2.67 (1H, t, J=11.6Hz), 2.54 (1H, t, J=9.6Hz), 2.22-2.99 (3H, m), 1.92-2.13 (3H, m), 1.81-1.85 (2H, m), 1.59-1.70 (4H, m), 1.42 (1H, m), 1.25 (1H, m), 1.08 (1H, s). 13c-NMR (100MHz, CDCl 3) δ: 199.9,194.9,172.1,146.0,120.4,75.6,58.9,58.8,54.6,44.5,41.3,40.3,37.0,36.2,33.9,27.5,25.9,23.2,22.5,20.3.HR-EI-MS m/z362.1287 [M] +(C 20h 23o 4cl, calcd362.1285).
Compound (9): 1h-NMR (400MHz, CDCl 3) δ: 6.13 (1H, s), 6.02 (1H, d, J=1.6Hz), 5.31 (1H, d, J=1.6Hz), 4.27 (1H, d, J=10.8Hz), 3.83 (1H, dd, J=2.4,10.8Hz), 3.26 (1H, m), 2.88 (1H, t, J=2.8Hz), 2.49-2.54 (2H, m), 2.17 (1H, m), 1.70-1.96 (7H, m), 1.56 (1H, m), 1.18 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 196.9,195.6,171.5,159.8,147.3,123.4,118.4,79.8,54.9,47.6,42.5,38.9,38.8 (C tand C s), 35.8,25.5,24.5,23.4,21.8,19.5.HR-EI-MS m/z326.1512 [M] +(C 20h 22o 4, calcd326.1518).
Compound (10): 1h-NMR (400MHz, CDCl 3) δ: 5.89 (1H, s), 5.43 (1H, s), 5.10 (1H, brs), 4.08 (1H, d, J=11.6Hz), 4.00 (1H, d, J=11.6Hz), 3.95 (1H, s), 2.70 (1H, s), 2.64 (1H, t, J=10.8Hz), 2.51 (1H, brs), 2.47 (1H, brs), 2.46 (1H, d, J=13.2Hz), 2.17 (2H, s), 2.05-2.13 (1H, m), 1.56-1.83 (8H, m), 1.32-1.38 (1H, m), 1.03 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 208.5,173.4,151.2,144.1,125.8,112.6,75.3,74.8,53.0,44.1,40.2,36.6,35.5,27.8,25.1,24.3,20.8,17.0.HR-EI-MS m/z342.1839 [M] +(C 21h 26o 4, calcd342.1831).
Compound (11): 1h-NMR (400MHz, CDCl 3) δ: 5.96 (1H, d, J=1.6Hz), 5.25 (1H, d, J=1.6Hz), 5.11 (1H, s), 4.18 (1H, dd, J=4.8, J=12.0Hz), 3.66 (1H, dd, J=2.4, J=11.2Hz), 3.55 (1H, dd, J=1.2, J=11.2Hz), 3.49 (3H, s), 2.73 (1H, brs), 2.64-2.72 (2H, m), 1.18-1.94 (14H, m), 0.78 (1H, m), 0.75 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 204.5,147.2,117.5,103.7,71.3,70.5,55.9,50.5,49.0,43.6,41.9,40.7,35.8,34.2,33.0,27.1,24.9,24.1,23.1,21.9,18.7.HR-EI-MS m/z346.2122 [M] +(C 21h 30o 4, calcd346.2144).
Compound (12): 1h-NMR (400MHz, CDCl 3) δ: 6.02 (1H, d, J=1.6Hz), 5.32 (1H, d, J=1.6Hz), 4.40 (1H, s), 3.45 (2H, m), 3.42 (3H, s), 2.77 (1H, brs), 2.48-2.64 (2H, m), 2.16-2.40 (4H, m), 1.96 (1H, t, J=10.4Hz), 1.63-1.88 (6H, m), 1.51-1.54 (1H, m), 1.41-1.43 (1H, m), 0.97 (1H, m), 0.66 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 209.9,196.7,146.5,119.3,102.7,70.4,57.7,55.5,47.9,45.7,40.4,39.7,37.5,35.7,33.6,31.2,27.2,26.8,23.5,21.6,21.4.HR-EI-MS m/z344.1998 [M] +(C 21h 28o 4, calcd344.1988).
Compound (13): 1h-NMR (400MHz, CDCl 3) δ: 5.99 (1H, d, J=1.2Hz), 5.98 (1H, dd, J=1.2,2.4Hz), 5.33 (1H, dd, J=1.2,2.4Hz), 5.32 (1H, d, J=1.2Hz), 4.39 (1H, d, J=1.2Hz), 3.46 (1H, dd, J=1.8,11.2Hz), 3.43 (3H, s), 3.27 (1H, dd, J=1.8,11.2Hz), 2.78 (1H, d, J=3.0Hz), 2.33-2.45 (4H, m), (2.15 1H, t, J=10.4Hz), (1.94 1H, t, J=10.4Hz), 1.52-1.88 (7H, m), 1.07 (1H, m), 0.84 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 203.3,196.3,146.6,145.5,126.2,119.3,103.3,71.6,57.0,55.2,54.8,44.2,41.0,39.2,35.9,35.5,30.6,27.4,26.6,24.0,22.7,21.3.HR-EI-MS m/z356.1985 [M] +(C 22h 28o 4, calcd356.1988).
Compound (14): 1h-NMR (400MHz, CDCl 3) δ: 6.00 (1H, d, J=1.6Hz), 5.29 (1H, d, J=1.6Hz), 4.13 (1H, dddd, J=2.8,5.2,11.6,12.8Hz), 4.07 (1H, dd, J=2.8,11.6Hz), 3.87 (1H, d, 11.6Hz), 3.54 (1H, dd, J=2.8,11.6Hz), 3.47 (1H, d, 11.6Hz), 2.77 (1H, brs), 2.64 (1H, d, J=3.2Hz), 2.33-2.41 (1H, m), 1.97-2.17 (2H, m), 0.78-1.83 (13H, m), 0.73 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 203.8,146.3,118.5,73.0,70.5,67.5,50.0,47.3,42.2,40.8,39.3,38.3,35.8,33.0,27.3,25.1,24.6,23.4,22.2,18.7.HR-EI-MSm/z316.2043 [M] +(C 20h 28o 3, calcd316.2038).
Compound (15): 1h-NMR (400MHz, CDCl 3) δ: 6.04 (1H, s), 5.33 (1H, s), 3.90 (1H, d, J=11.2Hz), 3.76 (1H, dd, J=2.4,11.2Hz), 3.45 (2H, s), 2.82 (1H, d, J=2.4Hz), 2.58 (1H, d, J=9.2Hz), 2.38-2.46 (1H, m), 2.08-2.18 (2H, m), 1.39-1.98 (11H, m), 1.14-1.23 (1H, m), 0.66 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 208.6,195.6,145.8,119.6,71.7,66.9,56.9,45.6,45.0,40.4,37.4,37.2,36.9,35.5,33.3,26.9,26.1,25.1,22.3,21.8.HR-EI-MS m/z314.1889 [M] +(C 20h 26o 3, calcd314.1882).
Compound (16): 1h-NMR (400MHz, CDCl 3) δ: 6.07 (1H, d, J=0.8Hz), (5.41 1H, d, J=0.8Hz), 3.82-3.89 (2H, m), 3.55 (1H, m), 3.52 (1H, d, J=10.8Hz), 3.29-3.36 (2H, m), 2.85 (1H, brs), 2.67-2.71 (2H, m), 2.36-2.47 (2H, m), 2.02-2.13 (2H, m), 1.93-1.99 (1H, m), 1.70-1.80 (3H, m), 1.56-1.63 (2H, m), 1.44-1.52 (3H, m), 1.25-1.30 (1H, m), 0.70 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 212.2,198.8,145.6,120.7,72.8,68.9,66.1,57.6,52.2,49.3,43.7,40.6,37.4,35.8,35.6,34.2,26.8,25.8,24.1,21.8,21.6.HR-EI-MS m/z344.1989 [M] +(C 21h 28o 4, calcd344.1988).
Compound (17): 1h-NMR (400MHz, CDCl 3) δ: 6.00 (1H, s), 5.34 (1H, s), 4.32 (1H, dd, J=3.6, J=9.6Hz), 4.25 (1H, dd, J=5.6, J=9.6Hz), 3.91 (1H, d, J=11.2Hz), 3.49 (1H, d, J=12.0Hz), 3.30-3.37 (2H, m), 3.12 (3H, s), 2.77-2.82 (2H, m), 2.34-2.45 (2H, m), 2.04-2.17 (2H, m), 1.96-1.97 (1H, m), 1.46-1.81 (8H, m), 1.25-1.31 (1H, m), 0.73 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 209.0,195.6,145.8,119.7,72.5,72.0,68.8,57.8,48.5,47.5,42.8,40.6,37.3,36.9,35.8,35.5,34.3,26.6,26.1,25.3,22.1,21.6.HR-EI-MSm/z422.1759 [M] +(C 22h 30o 6s, calcd422.1763).
Compound (18): 1h-NMR (400MHz, CDCl 3) δ: 5.94 (1H, s), 5.46 (1H, s), 5.08 (2H, s), 3.93 (1H, s), 3.91 (1H, d, J=12.0Hz), 3.58 (1H, dd, J=2.8, J=12.0Hz), (3.44 1H, d, J=11.2Hz), 3.25 (1H, dd, J=2.8, J=11.2Hz), 2.34-2.49 (4H, m), 2.03 (1H, m), 1.21-2.06 (11H, m), 0.86 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 209.4,150.0,144.8,126.8,113.1,74.5,73.7,69.6,53.2,48.6,44.9,41.6,36.7,35.7,35.3,35.2,26.6,25.6,24.4,21.8,20.3.HR-EI-MS m/z328.2040 [M] +(C 21h 28o 3, calcd328.2038).
Compound (19): 1h-NMR (400MHz, CDCl 3) δ: 5.98 (1H, s), 5.35 (1H, s), 3.97 (1H, d, J=12.0Hz), 3.53 (1H, dd, J=2.8, J=12.0Hz), 3.43 (1H, dd, J=1.2, J=11.6Hz), 3.14 (1H, dd, J=3.2, J=11.6Hz), 3.12 (1H, d, J=6.0Hz), 2.87 (1H, d, J=6.0Hz), 2.42-2.57 (2H, m), 2.41 (1H, brs), 2.07-2.18 (3H, m), 1.52-1.99 (9H, m), 1.53 (1H, m), 1.34-1.36 (1H, m), 0.83 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 203.8,201.1,145.1,126.9,73.9,68.9,60.3,58.0,53.2,52.8,43.1,41.0,37.2,35.4,34.1,24.5,24.3,24.1,23.4,21.7.HR-EI-MS m/z342.1840 [M] +(C 21h 28o 4, calcd342.1831).
Compound (20): 1h-NMR (400MHz, CDCl 3) δ: 5.99 (2H, s), 5.35 (1H, s), 5.33 (1H, s), 3.93 (1H, d, J=11.6Hz), 3.53 (1H, dd, J=2.4,11.6Hz), (3.42 1H, d, J=11.6Hz), 3.13 (1H, dd, J=2.4,11.6Hz), 2.83 (1H, d, J=2.4Hz), 2.41-2.50 (3H, m), 1.47-2.06 (10H, m), 1.25-1.34 (1H, m), 0.83 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 202.7,195.8,145.9,145.7,126.4,119.3,73.9,69.2,57.0,53.2,44.0,41.1,37.1,35.7,35.5,35.4,26.8,26.1,24.3,23.7,21.7.HR-EI-MS m/z326.1879 [M] +(C 21h 26o 3, calcd326.1882).
Compound (21): 1h-NMR (400MHz, CDCl 3) δ: 5.93 (1H, d, J=1.6Hz), 5.25 (1H, d, J=2.0Hz), 5.24 (1H, d, J=1.6Hz), 4.12 (1H, d, J=5.2Hz), 3.86 (1H, dd, J=3.2,11.6Hz), 3.30 (1H, dd, J=1.6,11.6Hz), 2.81 (1H, m), 2.25-2.35 (2H, m), 2.02-2.08 (1H, m), 1.48-1.91 (10H, m), 1.17-1.27 (3H, m), 0.69 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 200.7,148.0,117.4,96.3,68.9,66.1,49.1,43.7,43.4,40.3,36.7,34.7,33.7,29.6,27.3,24.5,24.1,23.5,22.5,20.8.HR-EI-MS m/z314.1882 [M] +(C 20h 26o 3, calcd314.1882).
Compound (22): 1h-NMR (400MHz, CDCl 3) δ: 6.08 (1H, s), 5.39 (1H, s), 2.71-2.91 (3H, m), 2.41-2.22 (1H, m), 2.23 (1H, t, J=9.6Hz), 1.47-2.13 (12H, m), 1.30 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 203.5,194.9,170.5,169.8,146.1,120.2,57.2,45.8,45.5,44.8,44.0,39.1,37.3,35.7,35.4,26.8,25.9,24.9,21.7,19.9.HR-EI-MS m/z342.1456 [M] +(C 20h 22o 5, calcd342.1467).
Compound (23): 1h-NMR (400MHz, CDCl 3) δ: 6.00 (1H, d, J=1.6Hz), 5.29 (1H, d, J=1.6Hz), 4.81 (1H, d, J=14.0Hz), 4.16 (1H, dd, J=6.4, 12.4Hz), 4.02 (1H, d, J=12.4Hz), 3.95 (1H, d, J=14.0Hz), 3.61 (1H, brs), 2.82 (1H, m), 2.74 (1H, d, J=2.0Hz), 2.18-2.26 (3H, m), 1.71-1.86 (6H, m), 1.66 (1H, d, J=14.0Hz), 1.43-1.55 (3H, m), 1.35 (1H, ddd, J=4.0, 14.4Hz), 1.79 (1H, d, J=12.0Hz), 0.85-0.91 (4H, m). 13c-NMR (100MHz, CDCl 3) δ: 204.8,146.7,117.9,77.2,71.2,58.2,51.3,50.4,46.0,38.8,38.5,36.1,35.4,30.9,28.7,28.1,25.3,25.0,19.1,15.5.HR-EI-MS m/z380.1651 [M] +(C 20h 28o 5s, calcd380.1657).
Compound (24): 1h-NMR (500MHz, CDCl 3) δ: 6.14 (1H, d, J=2.4Hz), 6.04 (1H, d, J=1.2Hz), 5.80 (1H, d, J=2.4Hz), 5.36 (1H, d, J=1.2Hz), 4.86 (1H, d, J=13.6Hz), 4.02 (1H, brs), 3.87 (1H, d, J=13.6Hz), 2.87 (1H, m), 2.65 (1H, m), 2.22-2.36 (2H, m), 1.15-2.14 (15H, m). 13c-NMR (125MHz, CDCl 3) δ: 198.7,195.5,146.3,146.1,124.2,119.7,66.4,57.7,56.2,54.5,45.2,38.5,37.9,35.8,32.5,31.7,29.2,27.3,26.9,22.4,18.1.HR-EI-MS m/z390.1512 [M] +(C 21h 26o 5s, calcd390.1501).
Compound (25): 1h-NMR (400MHz, CDCl 3) δ: 4.26 (1H, dd, J=2.0,11.6Hz), 4.09 (1H, d, J=11.6Hz), 4.03 (1H, dd, J=4.4,11.6Hz), 3.69 (4H, m), 3.17-3.21 (1H, m), 2.87 (1H, m), 2.67 (1H, dd, J=4.4,12.4Hz), 2.49-2.52 (3H, m), 2.43-2.46 (1H, d, 12.4Hz), 2.31-2.38 (4H, m), (2.21-2.24 1H, d, 13.2Hz), 1.98-2.05 (1H, m), 1.15-1.80 (12H, m), 0.95 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 220.7,174.0,76.3,69.8,66.8,56.9,53.5,50.8,49.8,46.9,45.9,43.3,40.5,37.3,32.7,29.4,27.3,27.2,23.5,20.2,18.4,17.3.HR-EI-MS m/z417.2514 [M] +(C 24h 35nO 5, calcd417.2515).
Compound (26): 1h-NMR (400MHz, CDCl 3) δ: 4.25 (1H, d, J=11.6Hz), 4.03-4.10 (2H, m), 3.69-3.74 (4H, m), 3.00-3.03 (1H, m), 2.59-3.61 (4H, m), 2.36-2.43 (4H, m), 2.17-2.27 (3H, m), 1.98-2.01 (1H, m), 1.13-1.82 (12H, m), 0.94 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 220.1,174.0,76.4,69.6,66.9,56.9,53.5,51.0,49.7,47.1,45.9,44.2,40.6,37.6,32.8,29.2,27.3,24.0,23.5,21.2,20.2,16.4.HR-EI-MS m/z417.2532 [M] +(C 24h 35nO 5, calcd417.2515).
Compound (27): 1h-NMR (400MHz, CD 3oD) δ: 5.89 (1H, d, J=3.2Hz), 5.26 (1H, d, J=3.2Hz), 4.10 (1H, dd, J=4.8,12.0Hz), 3.49 (1H, d, J=11.6Hz), 3.41 (1H, d, J=11.6Hz), 2.73 (1H, brs), 2.14-2.57 (3H, m), 1.97-2.03 (1H, m), 1.42-1.90 (10H, m), 1.21-1.28 (1H, m), 0.99 (3H, s), 0.97 (1H, m), 0.80 (1H, td, J=3.6,12.8Hz). 13c-NMR (100MHz, CD 3oD) δ: 203.7,179.1,149.0,117.9,71.5,65.3,53.9,51.7,50.3,46.7,39.7,36.5,30.7,29.4,28.0,27.3,26.5,24.2,20.8,17.2.HR-EI-MS m/z348.1937 [M] +(C 20h 28o 5, calcd348.1937).
Compound (28): 1h-NMR (400MHz, CD 3oD) δ: 5.90 (1H, d, J=1.6Hz), 5.26 (1H, d, J=1.6Hz), 4.05 (1H, dd, J=4.8,12.0Hz), 3.97 (1H, d, J=12.4Hz), 3.91 (1H, d, J=12.4Hz), 3.72 (1H, d, J=11.2Hz), 3.57 (1H, d, J=11.2Hz), 2.75 (1H, brs), 2.06-2.12 (4H, m), 1.66-1.92 (8H, m), 1.53-1.63 (1H, m), 1.28-1.41 (2H, m), 0.95-1.06 (3H, m), 0.99 (3H, s), 0.70 (1H, m). 13c-NMR (100MHz, CD 3oD) δ: 204.1,149.3,117.5,71.6,65.9,62.3,54.4,51.6,43.9,39.2,37.3,37.2,36.4,29.0,28.0,27.8,26.5,20.5,19.0.HR-EI-MSm/z334.2146 [M] +(C 20h 30o 4, calcd334.2144).
Compound (29): 1h-NMR (400MHz, CDCl 3) δ: 6.04 (1H, s), 5.34 (1H, s), 4.30 (1H, d, J=12.8Hz), 4.22 (1H, d, J=12.8Hz), 3.60 (1H, d, J=11.2Hz), 3.56 (1H, d, J=11.2Hz), 2.83 (1H, brs), 2.58-2.77 (2H, m), 2.28 (1H, t, J=11.2Hz), 2.02-2.13 (2H, m), 2.00 (3H, s), 1.52-1.93 (11H, m), 1.25 (1H, brs), 1.00-1.07 (2H, m), 0.96 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 208.4,196.5,170.5,146.2,119.5,64.1,64.0,58.1,51.8,48.4,38.7,38.4,38.1,35.6,33.6,27.1,26.3,25.7,23.5,20.9,18.0.HR-EI-MSm/z374.2082 [M] +(C 22h 30o 5, calcd374.2093).
Compound (30): 1h-NMR (400MHz, CDCl 3) δ: 5.98 (1H, d, J=1.2Hz), 5.28 (1H, d, J=1.2Hz), 4.59 (1H, d, J=13.2Hz), 4.25 (1H, d, J=13.2Hz), 4.10 (1H, m), 3.99 (1H, d, J=11.2Hz), 2.79 (1H, s), 2.48 (1H, s), 2.13 (3H, s), 2.06 (3H, s), 1.43-1.97 (15H, m), 1.12-1.16 (1H, m), 1.03 (3H, s), 0.82 (1H, m). 13c-NMR (100MHz, CDCl 3) δ: 203.8,171.1,170.8,146.7,118.2,71.2,66.8,63.3,53.0,50.5,46.8,41.5,36.5,36.4,35.6,35.3,28.0,27.5,25.6,25.0,21.1,20.9,19.5,18.0.HR-EI-MS m/z418.2341 [M] +(C 24h 34o 6, calcd418.2355).
Compound (31): 1h-NMR (400MHz, CDCl 3) δ: 6.05 (1H, d, J=0.8Hz), 5.35 (1H, d, J=0.8Hz), 4.34 (1H, d, J=12.8Hz), 4.17 (1H, d, J=12.8Hz), 4.06 (1H, d, J=11.2Hz), 3.99 (1H, d, J=11.2Hz), 2.85 (1H, brs), 2.66 (1H, s), 2.63 (1H, s), 2.28 (1H, m), 2.07-2.13 (1H, m), 2.05 (3H, s), 2.02 (3H, s), 1.57-1.93 (10H, m), 1.03-1.09 (2H, m), 0.93 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 207.6,196.1,170.6,170.1,146.0,119.3,64.9,63.6,57.8,51.5,48.0,38.5,38.1,36.6,36.0,35.4,33.3,26.8,26.1,25.7,23.2,20.6 (2CH 3), 17.8.HR-EI-MS m/z416.2220 [M] +(C 24h 32o 6, calcd416.2199).
Compound (32): 1h-NMR (400MHz, CDCl 3) δ: 5.99 (1H, d, J=1.2Hz), 5.35 (1H, d, J=1.2Hz), 4.19 (1H, d, J=13.2Hz), 3.98 (1H, d, J=13.2Hz), 3.93 (1H, d, J=11.6Hz), 3.87 (1H, d, J=11.6Hz), 3.83 (1H, d, J=4.0Hz), 3.47 (1H, m), 2.81 (2H, m), 2.69 (1H, m), 2.12-2.26 (2H, m), 1.98 (3H, s), 1.94 (3H, s), 1.75-1.86 (1H, m), 1.69-1.73 (2H, m), 1.43-1.63 (7H, m), 0.99-1.10 (1H, m), 0.93 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 208.9,199.1,170.8,170.4,145.8,120.7,65.8,65.6,63.3,57.4,54.0,53.6,44.9,38.5,37.8,36.6,35.6,32.9,27.1,26.6,26.4,22.5,20.8,20.7,17.8.HR-EI-MS m/z446.2305 [M] +(C 25h 34o 7, calcd446.2305).
Compound (33): 1h-NMR (400MHz, CDCl 3) δ: 5.98 (1H, s), 5.33 (1H, s), 4.31 (1H, dd, J=3.2,11.2Hz), 4.29 (1H, d, J=13.6Hz), 4.11 (1H, d, J=8.0Hz), 4.08 (1H, d, J=13.6Hz), 4.06 (1H, d, J=8.0Hz), 3.99 (2H, d, J=3.6Hz), 3.10 (1H, m), 2.84 (1H, brs), 2.29-2.30 (2H, m), 2.05 (3H, s), 2.04 (3H, s), 2.00 (3H, s), 1.48-1.96 (9H, m), 1.09-1.25 (2H, m), 1.04 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 206.7,195.9,170.9,170.8,170.5,146.6,119.0,66.3,65.8,63.4,57.6,53.9,49.5,44.7,38.5,38.9,36.8,35.7,33.3,27.1,26.7,26.5,23.3,20.8 (3CH 3), 17.9.HR-EI-MS m/z488.2397 [M] +(C 27h 36o 8, calcd488.2410).
Compound (34): 1h-NMR (400MHz, CDCl 3) δ: 5.94 (1H, s), 5.31 (1H, s), 4.43 (1H, dd, J=6.0, 10.0Hz), 4.27 (1H, dd, J=2.4, 10.0Hz), 4.23 (1H, d, J=13.2Hz), 4.06 (1H, d, J=13.2Hz), 3.99 (1H, d, J=11.6Hz), 3.92 (1H, d, J=11.6Hz), 3.07 (3H, s), 3.01 (1H, m), 2.80 (1H, brs), 2.13-2.27 (2H, m), 2.02 (3H, s), 1.95 (3H, s), 1.70-1.91 (2H, m), 1.58-1.68 (6H, m), 1.46 (1H, t, J=12.0Hz), 1.07-1.21 (2H, m), 1.01 (3H, s), 0.82 (1H, m). 13c-NMR (100MHz, CDCl 3) δ: 206.7,196.0,170.8,170.3,146.3,119.5,71.9,65.8,63.6,57.9,52.3,49.7,44.2,38.3,38.1,36.8 (3CH 3), 35.6,33.0,27.1,26.6,26.5,23.6,20.8,20.7,17.8.HR-EI-MS m/z524.2072 [M] +(C 26h 36o 9s, calcd524.2080).
Compound (35): 1h-NMR (400MHz, CDCl 3) δ: 5.92 (1H, d, J=1.2Hz), 5.26 (1H, d, J=1.2Hz), 4.22 (1H, d, J=13.2Hz), 3.97 (1H, d, J=11.2Hz), 3.92 (1H, d, J=13.2Hz), 3.87 (1H, d, J=11.2Hz), 3.64-3.71 (4H, m), 2.77-2.85 (1H, m), 2.57 (1H, brs), 2.05-2.53 (9H, m), 1.99 (3H, s), 1.96 (3H, s), 1.45-1.83 (9H, m), 1.39 (1H, d, J=7.6Hz), 1.00-1.19 (1H, m), 0.98 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 205.1,197.3,170.8,170.5,146.5,118.9,66.8 (2CH 2-morpholine), 65.9,63.1,62.5,57.6,57.1,53.0 (2CH 2-morpholine), 47.7,45.5,38.7,38.1,36.9,35.7,33.2,26.9,26.7,26.6,22.9,20.8,20.7,17.8.HR-EI-MS m/z515.2879 [M] +(C 29h 41nO 7, calcd515.2833).
Compound (36): 1h-NMR (400MHz, CDCl 3) δ: 6.51 (1H, t, J=3.6Hz), 6.05 (1H, s), 5.82 (1H, t, J=3.6Hz), 5.34 (1H, s), 4.38 (1H, d, J=11.2Hz), 4.30 (1H, d, J=12.8Hz), 4.16 (1H, d, J=11.2Hz), 4.14 (1H, d, J=12.8Hz), 2.84 (1H, s), 2.52 (1H, s), 2.22 (1H, m), 2.13 (1H, m), 2.06 (3H, s), 2.01 (3H, s), 1.51-1.96 (8H, m), 1.21 (3H, s), 1.11 (2H, m), 0.87 (1H, m). 13c-NMR (100MHz, CDCl 3) δ: 197.2,195.8,171.0,170.3,146.1,142.5,125.1,119.6,63.6,63.5,57.4,57.0,47.8,39.6,38.5,37.6,35.7,33.3,28.1,27.2,24.7,20.8,20.7,17.7.HR-EI-MS m/z428.2202 [M] +(C 25h 32o 6, calcd428.2199)
Compound (37): 1h-NMR (400MHz, CDCl 3) δ: 5.98 (1H, d, J=1.2Hz), 5.30 (1H, d, J=1.2Hz), 4.56 (1H, d, J=11.2Hz), 4.24 (1H, d, J=11.2Hz), 4.22 (1H, d, J=13.2Hz), 4.20 (1H, d, J=13.2Hz), 4.07 (1H, d, J=12.0Hz), 3.13 (3H, s), 3.04 (3H, s), 2.8 (1H, s), 2.60 (1H, s), 2.08 (1H, d, J=13.6Hz), 2.00 (1H, dd, J=4.8, 13.6Hz), 1.65-1.84 (6H, m), 1.58-1.65 (1H, m), 1.38-1.49 (3H, m), 1.12-1.23 (1H, m), 1.09 (3H, s), 0.84-0.92 (1H, m). 13c-NMR (100MHz, CDCl 3) δ: 202.9,146.2,118.6,72.3,70.6,67.8,52.3,50.1,46.7,41.5,37.4,36.6,36.9,35.7,35.3,34.7,27.9,27.0,25.6,24.8,19.2,17.9.HR-EI-MSm/z490.1703 [M] +(C 22h 34o 8s 2, calcd490.1695).
Compound (38): 1h-NMR (400MHz, CDCl 3) δ: 6.05 (1H, s), 5.39 (1H, s), 4.80 (1H, d, J=11.2Hz), 4.33 (1H, d, J=11.2Hz), 4.30 (1H, d, J=10.0Hz), 4.15 (1H, d, J=10.0Hz), 3.06 (3H, s), 3.04 (3H, s), 2.89 (1H, brs), 2.66 (1H, dd, J=9.6, 9.6Hz), 2.51 (1H, dd, J=11.6, 11.6Hz), 2.31 (1H, t, J=12.4, 14.0Hz), 2.17 (1H, t, J=8.8, 10.4Hz), 1.86-2.06 (5H, m), 1.76-1.80 (1H, d, J=13.6Hz), 1.60-1.71 (4H, m), 1.08-1.20 (2H, m), 1.03 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 207.3,195.9,145.8,119.9,71.6,65.8,57.9,51.1,47.9,38.8,38.5,37.9,37.1,36.6,35.3,35.2,32.8,26.7,26.1,25.5,23.3,17.5.HR-EI-MS m/z488.1512 [M] +(C 22h 32o 8, calcd488.1539).
Compound (39): 1h-NMR (400MHz, CDCl 3) δ: 6.48 (1H, d, J=2.8Hz), 6.06 (1H, d, J=1.2Hz), 5.79 (1H, d, J=2.8Hz), 5.38 (1H, d, J=2.8Hz), 4.43 (1H, d, J=10.4Hz), 4.41 (1H, d, J=10.8Hz), 4.33 (1H, d, J=10.4Hz), 4.27 (1H, d, J=10.8Hz), 3.05 (3H, s), 2.98 (3H, s), 2.88 (1H, s), 2.66 (1H, t, J=2.8Hz), 2.33 (1H, m), 2.12-2.20 (1H, m), 2.02-2.08 (2H, m), 1.89-1.94 (3H, m), 1.63-1.74 (5H, m), 1.35 (3H, s), 1.13-1.25 (1H, m). 13c-NMR (100MHz, CDCl 3) δ: 197.2,195.5,145.8,142.5,125.2,120.1,70.0,65.9,56.9,47.4,39.8,38.0,37.8,37.5,37.0,35.5,32.8,28.3,26.9,24.5,17.7.HR-EI-MSm/z500.1541 [M] +(C 23h 32o 8s 2, calcd500.1539).
Compound (40): 1h-NMR (400MHz, CDCl 3) δ: 6.06 (1H, d, J=1.2Hz), 5.36 (1H, d, J=1.2Hz), 4.52 (1H, d, J=12.8Hz), 4.20-4.26 (3H, m), 3.97-4.10 (5H, m), 2.86 (1H, s), 2.57-2.70 (2H, m), 2.10-2.31 (2H, m), 1.61-1.98 (8H, m), 1.08-1.25 (3H, m), 1.07 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 207.6,196.1,167.3,167.0,145.9,119.9,66.9,65.4,58.0,51.6,48.3,40.7,40.5,38.6,38.5,37.0,36.0,35.5,33.3,26.9,26.3,25.9,23.6,17.9.HR-EI-MS m/z484.1423 [M] +(C 24h 30o 6cl 2, calcd484.1419).
Compound (41): 1h-NMR (400MHz, CDCl 3) δ: 6.53 (1H, d, J=2.4Hz), 6.06 (1H, s), 5.82 (1H, d, J=2.4Hz), 5.36 (1H, s), 4.48 (1H, m), 4.33 (1H, d, J=11.2Hz), 4.21 (1H, d, J=13.2Hz), 4.10 (2H, s), 4.04 (2H, s), 2.86 (1H, s), 2.57 (1H, s), 2.12-2.30 (2H, m), 1.55-1.93 (10H, m), 1.25 (3H, s), 1.12-1.22 (2H, m). 13c-NMR (100MHz, CDCl 3) δ: 197.2,195.6167.4,166.8,145.9,142.1,125.8,119.8,65.4,65.1,57.3,57.0,47.7,40.7,40.5,39.9,38.2,37.7,35.6,32.9,28.0,27.1,27.0,24.8,17.7.HR-EI-MS m/z496.1422 [M] +(C 25h 30o 6cl 2, calcd496.1419).
Compound (42): 1h-NMR (400MHz, CDCl 3) δ: 6.05 (1H, s), 5.36 (1H, s), 4.39 (1H, d, J=12.8Hz), 4.32 (1H, d, J=12.8Hz), 4.20 (1H, d, J=9.6Hz), 4.06 (1H, d, J=9.6Hz), 3.68 (4H, brs), 3.50 (1H, brs), 3.46 (1H, brs), 3.30 (2H, brs), 3.01 (3H, s), 2.85 (1H, brs), 2.70 (1H, dd, J=8.8, 20.0Hz), 2.46 (1H, dd, J=12.0, 20.0Hz), 2.22 (1H, t, J=12.0Hz), 2.10 (1H, t, J=10.0Hz), 1.59-1.99 (10H, m), 1.05-1.17 (2H, m), 1.01 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 207.6,195.9,154.6,145.8,119.8,70.6,66.4,64.8,57.9,51.1,48.0,38.5,38.3,36.8,35.5,35.4,33.6,26.9,26.3,25.5,23.5,17.8.HR-EI-MS m/z523.2231 [M] +(C 26h 37nO 8s, calcd523.2240).
Compound (43): 1h-NMR (400MHz, CDCl 3) δ: 6.03 (1H, s), 5.34 (1H, s), 4.49 (1H, d, J=11.2Hz), 4.29 (1H, d, J=11.2Hz), 4.19 (1H, d, J=10.8Hz), 4.05 (1H, d, J=10.8Hz), 3.64 (4H, brs), 3.43 (4H, brs), 3.07 (3H, s), 2.83 (1H, brs), 2.65 (1H, dd, J=8.8, 20.0Hz), 2.48 (1H, dd, J=12.4, 19.6Hz), 2.26 (1H, t, J=13.2Hz), 2.07-2.15 (2H, m), 1.81-1.94 (6H, m), 1.59-1.70 (4H, m), 1.04-1.11 (2H, m), 0.94 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 207.4,196.1,155.1,145.9,119.7,66.8,66.1,58.0,51.4,48.3,39.2,38.2,37.6,37.0,35.8,35.5,33.1,26.8,26.4,26.2,23.6,17.7.HR-EI-MS m/z523.2245 [M] +(C 26h 37nO 8s, calcd523.2240).
Compound (44): 1h-NMR (400MHz, CDCl 3) δ: 7.80 (1H, dd, J=1.2, 3.6Hz), 7.59 (1H, dd, J=1.2, 4.8Hz), 7.12 (1H, dd, J=3.6, 4.8Hz), 6.05 (1H, d, J=1.2Hz), 5.35 (1H, d, J=1.2Hz), 4.39 (1H, d, J=11.6Hz), 4.34 (1H, d, J=16.0Hz), 4.18 (1H, d, J=16.0Hz), 4.15 (1H, d, J=11.6Hz), 4.06 (1H, d, J=10.8Hz), 3.69 (1H, d, J=10.8Hz), 2.86 (1H, brs), 2.65 (2H, m), 2.27 (1H, m), 2.03-2.11 (2H, m), 1.84-1.92 (4H, m), 1.57-1.73 (5H, m), 1.08-1.19 (1H, m), 1.00-1.08 (1H, m), 1.06 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 207.8,196.4,162.2,146.1,133.3,133.2,132.6,127.8,119.5,114.7,71.5,66.2,57.9,56.2,51.5,48.1,39.4,38.6,36.9,36.1,35.6,33.7,26.9,26.5,26.0,24.0,18.1.HR-EI-MS m/z481.1932 [M] +(C 27h 31nO 5s, calcd481.1923).
Compound (45): 1h-NMR (400MHz, CDCl 3) δ: 7.80 (1H, d, J=3.2Hz), (7.57 1H, d, J=4.4Hz), 7.12 (1H, m), 6.06 (1H, s), 5.36 (1H, s), 4.20-4.38 (4H, m), 2.85 (1H, s), 2.66-2.81 (2H, m), 2.29 (1H, m), 2.12 (1H, m), 1.99 (3H, s), 1.57-1.97 (10H, m), 1.06-1.25 (2H, m), 1.04 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 207.6,196.3,170.4,162.0,146.1,133.3 (C dand C s), 132.5,127.8,119.6,70.8,65.9,63.8,58.0,51.7,48.3,38.8,38.3,37.2,36.4,35.6,33.6,27.0,26.3,25.9,23.4,20.8,17.9.HR-EI-MS m/z484.1923 [M] +(C 27h 32o 6s, calcd484.1920).
Compound (46): 1h-NMR (400MHz, CDCl 3) δ: 7.81 (1H, dd, J=1.2, 3.6Hz), 7.57 (1H, dd, J=1.2, 5.2Hz), 7.21 (1H, m), 6.54 (1H, d, J=2.4Hz), 6.06 (1H, d, J=1.2Hz), 5.90 (1H, d, J=2.4Hz), 5.34 (1H, d, J=1.2Hz), 4.59 (1H, dd, J=1.2, 11.6Hz), 4.90 (1H, d, J=1.2Hz), 4.32 (1H, d, J=12.8Hz), 4.21 (1H, d, J=12.8Hz), 2.85 (1H, d, J=3.2Hz), 2.58 (1H, m), 2.23-2.31 (1H, m), 2.14-2.19 (1H, m), 2.02 (3H, s), 1.70-2.03 (8H, m), 1.54-1.59 (1H, m), 1.31 (3H, s), 1.10-1.30 (2H, m). 13c-NMR (100MHz, CDCl 3) δ: 197.1,195.8,170.3,162.2,146.1,142.6,133.5 (C dand C s), 132.5,127.8,125.2,119.6,64.2,63.4,57.4,56.9,47.8,39.6,38.5,38.1,35.6,33.4,28.1,27.2,27.1,24.7,20.7,17.8.HR-EI-MSm/z496.1918 [M] +(C 28h 32o 6s, calcd496.1920).
Compound (47): 1h-NMR (500MHz, CDCl 3) δ: 7.87 (1H, d, J=3.8Hz), 7.79 (1H, d, J=3.8Hz), 7.56 (2H, m), 7.11 (2H, m), 6.00 (1H, s), 5.29 (1H, s), 4.98 (1H, d, J=12.9Hz), 4.51 (1H, d, J=11.4Hz), 4.49 (1H, d, J=13.2Hz), 4.38 (1H, d, J=11.4Hz), 4.17 (2H, m), 2.81 (1H, brs), 2.46 (1H, brs), 2.10-2.15 (2H, m), 1.48-1.99 (11H, m), 1.15-1.25 (1H, m), 1.58 (3H, s), 0.88-0.91 (1H, m). 13c-NMR (125MHz, CDCl 3) δ: 203.7,162.1,146.7,133.8,133.7,133.4,133.3,132.7,132.3,128.0,127.8,71.4,67.7,64.0,53.1,50.6,46.8,42.2,36.9,35.7,35.3,28.1,27.8,26.1,25.0,19.6,18.2.HR-EI-MS m/z554.1970 [M] +(C 30h 34o 6s 2, calcd554.1797).
Compound (48): 1h-NMR (400MHz, CDCl 3) δ: 7.67-7.70 (2H, m), 7.50-7.53 (2H, m), 7.02-7.06 (2H, m), 6.06 (1H, d, J=1.6Hz), 5.35 (1H, d, J=1.6Hz), 4.61 (2H, d, J=3.2Hz), 4.37 (1H, d, J=11.2Hz), 4.25 (1H, d, J=11.2Hz), 2.78-2.94 (3H, m), 2.12-2.28 (2H, m), 1.61-2.03 (11H, m), 1.10-1.28 (1H, m), 1.06 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 207.5,195.8,161.6,161.5,145.9,133.7,133.0, (C dand C s), 132.9,132.1,132.0,127.8,127.4,119.3,66.3,64.2,57.8,51.2,48.1,38.5,38.4,36.8,36.6,35.4,33.7,26.7,26.3,26.0,23.8,17.8.HR-EI-MS m/z552.1655 [M] +(C 30h 32o 6s 2, calcd552.1640).
Compound (49): 1h-NMR (400MHz, CDCl 3) δ: 7.75-7.80 (2H, m), 7.55-7.60 (2H, m), 7.09-7.13 (2H, m), 6.70 (1H, d, J=2.8Hz), 6.07 (1H, d, J=1.6Hz), 6.00 (1H, d, J=2.8Hz), 5.35 (1H, d, J=1.6Hz), 4.74 (1H, d, J=11.6Hz), 4.60 (1H, d, J=12.8Hz), 4.49 (1H, d, J=11.6Hz), 4.40 (1H, d, J=12.8Hz), 2.86 (1H, m), 2.60 (1H, t, J=2.8Hz), 1.56-2.28 (12H, m), 1.30 (3H, s), 1.14-1.25 (1H, m). 13c-NMR (100MHz, CDCl 3) δ: 196.8,195.8,162.0,161.7,146.2,141.3,134.2,133.5,133.4,133.1,132.6,132.5,128.1,127.8,126.4,119.6,64.2,63.8,57.4,57.1,47.9,40.4,38.8,38.0,35.7,33.4,27.7,27.2,27.0,25.2,17.6.HR-EI-MS m/z564.1643 [M] +(C 31h 32o 6s 2, calcd564.1640).
Compound (50): 1h-NMR (400MHz, CDCl 3) δ: 6.00 (1H, d, J=1.6Hz), 5.30 (1H, d, J=1.6Hz), 4.14 (1H, dd, J=4.4, J=12.4Hz), 3.65 (2H, t, J=4.8Hz), 2.80 (1H, s), (2.64 1H, d, J=11.2Hz), 2.37-256 (4H, m), 2.22 (1H, d, J=11.2Hz), 2.13 (2H, m), 1.90-1.95 (2H, m), 1.54-1.80 (9H, m), 1.39-1.44 (2H, m), 1.25 (1H, s), 1.14-1.17 (1H, s), 1.03 (1H, m), 0.84 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 203.8,146.4,118.5,70.6,60.7,59.9,57.8,53.5,50.2,47.8,43.0,40.8,39.5,39.2,35.8,33.5,27.5,26.3,25.3,24.6,23.0,18.8.HR-EI-MS m/z359.2452 [M] +(C 22h 33nO 3, calcd359.2460).
Compound (51): 1h-NMR (400MHz, CDCl 3) δ: δ: 5.90 (1H, s), 5.45 (1H, s), 5.08 (2H, s), 3.92 (1H, s), (3.61 2H, t, J=5.6Hz), (2.86 1H, d, J=11.6Hz), 2.67 (1H, s), 2.32-2.53 (7H, m), 2.11-2.14 (2H, dd, J=2.8, J=11.2Hz), 1.90-1.98 (2H, m), 1.50-1.73 (7H, m), 1.33-1.43 (2H, m), 1.24-1.26 (1H, m), 0.96 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 209.4,150.2,145.1,126.4,113.0,74.6,60.9,60.3,57.8,56.5,53.7,48.8,45.6,41.6,37.4,35.8,35.7,35.5,27.3,26.5,25.7,22.4,20.5.HR-EI-MS m/z371.2460 [M] +(C 23h 33nO 3, calcd371.2460).
Compound (52): 1h-NMR (400MHz, CDCl 3) δ: 6.03 (1H, d, J=2.0Hz), (5.32 1H, d, J=2.0Hz), 3.63 (2H, m), 2.82 (2H, m), 2.38-2.53 (5H, m), 2.30 (1H, dd, J=2.4,10.8Hz), 2.11-2.18 (2H, m), 1.89-2.03 (3H, m), 1.51-1.82 (7H, m), 1.37-1.46 (1H, m), 1.15-1.25 (2H, m), 0.82-0.90 (1H, m), 0.73 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 209.6,196.1,146.3,119.8,60.7,59.1,58.3,57.6,53.6,46.6,46.0,40.9,38.4,37.6,37.4,36.0,34.1,27.3,26.4,25.4,25.3,23.0.HR-EI-MS m/z357.2301 [M] +(C 22h 31nO 3, calcd357.2304).
Compound (53): 1h-NMR (400MHz, CDCl 3) δ: 5.98 (1H, d, J=1.6Hz), 5.93 (1H, t, J=2.0Hz), 5.31 (1H, t, J=2.0Hz), 5.30 (1H, d, J=1.6Hz), (3.67 2H, t, J=6.0Hz), (2.96 1H, d, J=11.2Hz), 2.81-2.87 (2H, m), 2.40 (1H, d, J=11.2Hz), 2.29 (1H, m), 2.27 (2H, t, J=6.0Hz), 1.22-2.10 (12H, m), 0.92 (2H, m), 0.90 (12H 4Me, s), 0.05 (6H 2Si-Me, s). 13c-NMR (100MHz, CDCl 3) δ: 203.1,196.1,146.4,146.2,125.5,118.9,62.0,61.5,60.8,57.2,56.7,53.9,44.7,41.7,38.0,36.1,35.9 (2C, 1d, 1s), 27.3,26.9,26.1,25.8 (3CH 3), 23.8,21.7,18.2 ,-5.3 (2Si-CH 3) .HR-EI-MS m/z483.3155 [M] +(C 29h 45o 3si, calcd483.3169)
Compound (54): 1h-NMR (400MHz, CDCl 3) δ: 6.00 (1H, s), 5.96 (1H, s), 5.35 (1H, s), 5.32 (1H, s), 3.60 (2H, m), 2.87 (2H, m), 1.25-2.29 (20H, m), 0.93 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 203.5,196.3,146.4,146.3,126.3,119.7,61.1,60.3,57.9,57.2,56.2,53.8,44.7,41.2,38.1,35.9 (2C), 35.7,27.4,26.9,26.1,23.9,22.5.HR-EI-MS m/z369.2301 [M] +(C 23h 31nO 3, calcd369.2304)
Compound (55): 1h-NMR (400MHz, CDCl 3) δ: 5.91 (1H, s), 5.21 (1H, s), 4.67 (1H, s), 4.20 (1H, s), 3.95 (1H, d, J=5.2Hz), 3.92 (1H, s), 3.68-3.98 (2H, m), 3.46-3.50 (1H, m), 3.22-3.27 (1H, m), 3.04-3.07 (1H, m), 2.79-2.81 (1H, m), 2.26-2.49 (2H, m), 1.96-2.13 (1H, m), 1.01-1.90 (11H, m), 1.06 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 200.8,148.2/148.1,117.3/117.1,95.1/91.1,85.7/83.5,67.8/67.4,64.9/63.1,51.0,49.2/49.1,47.4,45.5/44.3,43.1/43.0,36.8/36.7,35.7/35.4,35.3/35.1,29.9,29.8/29.6,27.4,24.5/24.0,23.5/23.3,22.8/22.7,20.6/20.3.HR-EI-MS m/z355.2137 [M] +(C 22h 29nO 3, calcd355.2147).
Compound (56): 1h-NMR (400MHz, CDCl 3) δ: 6.03 (1H, d, J=1.6Hz), 5.34 (1H, d, J=1.6Hz), 5.09 (1H, s), 4.15-4.21 (2H, m), 3.86-3.98 (2H, m), 3.24-3.32 (1H, m), 2.81 (1H, s), (2.48 1H, d, J=3.6Hz), 2.33-2.37 (1H, m), 2.19-2.25 (1H, m), 2.17 (1H, s), 1.99-2.13 (1H, m), 1.77-1.87 (3H, m), 1.63-1.77 (2H, m), 1.25-1.54 (6H, m), 1.22 (3H, s), 0.82-0.89 (1H, m). 13c-NMR (100MHz, CDCl 3) δ: 202.9,173.1,146.2,119.1,88.9,70.1,64.8,50.9,49.7,42.6,42.2,41.4,40.8,40.2,35.7,33.8,28.4,26.4,25.3,22.3,20.7,18.9.HR-EI-MS m/z371.2107 [M] +(C 22h 29nO 4, calcd371.2097).
Compound (57): 1h-NMR (400MHz, CDCl 3) δ: 5.95 (1H, d, J=1.6Hz), 5.28 (1H, d, J=1.6Hz), 4.92 (1H, d, J=2.4Hz), 3.99 (1H, d, J=3.6Hz), 3.91-3.91 (1H, m), 3.81-3.87 (1H, m), 3.76-3.80 (1H, m), 3.65-3.72 (1H, m), 3.21-3.28 (1H, m), 2.82 (1H, m), 2.27-2.36 (1H, m), 2.11-2.15 (1H, m), 1.97-2.03 (1H, m), 1.83-1.92 (2H, m), 1.74-1.79 (2H, m), 1.28-1.67 (9H, m), 1.16 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 199.9,175.5,147.6,117.9,86.6,67.6,61.8,51.8,48.5,46.5,44.5,44.4,39.4,36.5,33.9,29.5,27.1,26.4,24.7,23.3,21.0,19.7.HR-EI-MS m/z371.2094 [M] +(C 22h 29nO 4, calcd371.2097).
Compound (58): 1h-NMR (400MHz, CDCl 3) δ: 6.47 (1H, s), 5.95 (1H, d, J=1.6Hz), 5.26 (1H, d, J=1.6Hz), 4.88 (1H, m), 3.93 (1H, m), 2.83 (1H, m), 2.27-2.36 (1H, m), 2.11-2.17 (1H, m), 1.97-2.04 (1H, m), 1.17-1.91 (13H, m), 1.14 (3H, s). 13c-NMR (100MHz, CDCl 3) δ: 200.3,177.2,147.8,117.8,80.5,67.2,48.7,46.9,44.6,44.2,39.6,36.6,33.7,29.5,27.3,26.5,24.8,23.4,20.7,20.0.HR-EI-MS m/z327.1841 [M] +(C 22h 29nO 4, calcd327.1834)+.
Embodiment 2:
The compounds of this invention is to the restraining effect of various tumor cell line:
(1) experimental technique
1. inoculating cell: be made into individual cells suspension with the nutrient solution (DMEM or RMPI1640) containing 10% foetal calf serum, be inoculated into 96 orifice plates with every hole 5000-10000 cell, every pore volume 100 μ l, attached cell shifts to an earlier date 12 hours inoculation culture.
2. add testing compound solution (fixed concentration 40 μMs of primary dcreening operations suppress the compound near 50% to establish 5 concentration to enter gradient in this concentration to growth of tumour cell and sieve again), every hole final volume 200 μ l, 3 multiple holes are all established in often kind of process.
3. develop the color: cultivate after 48 hours for 37 degrees Celsius, every hole adds MTT solution 20 μ l.Continue to hatch 4 hours, stop cultivating, inhale and abandon culture supernatant in hole, every hole adds the SDS solution (10%) of 200 μ l, and night incubation (temperature 37 DEG C), makes crystallisate fully melt.
4. colorimetric: select 595nm wavelength, enzyme-linked immunosorbent assay instrument (Bio-Rad680) reads each hole absorbance value, record result, take concentration as X-coordinate, cell survival rate is that ordinate zou draws cell growth curve, the IC of application two-point method (Reed and Muench method) computerized compound 50value [8].
(2) the selection result:
Tumor cytotoxic activity (the IC of table 1 15-oxospiramilactone (S-3) derivative 50, μM)
Compound HL-60 SMMC-7721 A-549 MCF-7 SW-480
1 4.56 4.72 3.16 4.61 3.74
2 2.02 0.72 0.59 0.96 0.66
3 4.43 3.53 2.80 3.55 7.77
4 3.41 0.71 0.83 1.18 1.03
5 3.85 2.48 2.44 3.94 2.80
6 4.69 2.41 3.14 4.83 3.71
7 3.86 0.83 0.73 1.51 1.21
8 3.82 1.96 3.03 2.63 2.38
9 0.80 0.80 0.91 1.44 1.21
10 5.56 12.02 4.77 3.09 11.44
11 4.20 1.87 3.17 5.04 4.19
12 1.46 0.51 0.67 1.57 2.20
13 0.51 0.15 0.20 0.24 0.39
14 4.87 4.92 3.25 4.00 6.33
15 4.90 4.62 2.97 2.85 2.37
16 1.94 1.90 0.77 1.44 1.53
17 3.45 3.12 2.22 2.02 1.58
18 3.60 4.44 6.11 8.86 5.43
19 >40 >40 >40 >40 >40
20 1.31 1.79 0.76 1.71 0.62
21 16.97 10.88 13.69 18.53 24.59
22 28.77 34.76 17.46 21.53 20.73
23 1.58 0.90 2.18 2.24 2.09
24 0.68 0.16 0.58 0.78 0.59
25 3.78 5.97 3.64 4.12 8.01
26 5.28 13.14 4.04 5.96 7.69
27 >40 >40 >40 >40 >40
28 4.28 4.48 4.28 8.07 4.42
29 - - - - -
30 4.74 7.83 3.49 4.82 9.89
31 3.38 3.91 2.95 3.38 3.74
32 5.03 6.13 3.04 2.93 3.94
33 6.33 15.44 11.10 15.13 12.88
34 6.50 13.99 10.97 12.81 15.51
35 6.30 10.83 4.69 4.23 6.57
36 1.21 0.75 0.64 0.65 0.78
37 1.37 0.93 1.86 1.55 1.85
38 3.97 0.77 2.89 2.36 2.57
39 0.51 0.26 0.56 0.53 0.15
40 0.79 0.24 0.55 0.56 0.22
41 0.54 0.19 0.41 0.32 0.14
42 4.18 3.95 4.21 3.31 2.94
43 2.70 2.18 2.32 2.05 3.37
44 0.62 1.06 1.17 0.65 0.80
45 0.71 0.57 0.80 0.89 0.76
46 0.24 0.13 0.14 0.15 0.10
47 - - - - -
48 0.61 0.57 0.70 0.67 0.59
49 0.29 0.22 0.35 0.33 0.16
50 7.95 11.16 9.92 16.57 14.41
51 4.82 1.48 3.04 6.98 9.84
52 14.32 15.19 9.24 15.70 11.82
53 1.39 2.63 1.55 2.90 2.78
54 6.61 11.52 6.50 3.57 2.81
55 1.95 1.18 2.93 5.22 2.38
Embodiment 3:
The compounds of this invention is to the restraining effect of Wnt signal path reporter gene Top-flash:
(1) experimental technique:
1) cell cultures and conditioned medium preparation:
The DMEM(Invitrogen of HEK293T cell containing 10% foetal calf serum) cultivate, 37 DEG C, CO 2concentration 5%.Go down to posterity every other day.When keeping going down to posterity, density is 70 ~ 80%.The L cell strain (CRL-2647 purchased from American ATCC cell bank) of stably excreting mouse Wnt3a albumen and contrast strain (CRL-2648 purchased from American ATCC cell bank), maintain the DMEM containing 50ug/ml G418 and 10% foetal calf serum, 37 DEG C, CO 2concentration is under the condition of 5%.Change liquid (DMEM containing 10% foetal calf serum) to during about 70% density to it at Growth of Cells, cultured continuously is after four days, and collection nutrient solution is also centrifugal, stays supernatant, obtains Wnt conditioned medium.After titration, liquid nitrogen flash freezer, preserves for a long time, can keep titer plateaus for-80 DEG C.
2) cell transfecting:
By cell by often coiling 2.0 ~ 2.5 × 10 6the density kind of individual cell enters 48 porose discs, transfection after 20 hours.Transfection plasmid transfection used reagent calculates with the consumption in the 48 every holes of porose disc: plasmid total amount is 125ng/ hole; First plasmid adds mixing in the training liquid (25 μ L/ hole) of serum-free, then adds PLUS reagent (Invitrogen) mixing according to 0.5 μ L/ hole, leaves standstill 15 minutes; According to the amount in 0.5 μ L/ hole by Lipofectamine(Invitrogen) liposome adds serum-free DMEM and trains mixing in liquid (25 μ L/ hole), then mix with the mixing solutions of above-mentioned plasmid and PLUS, standing 15 minutes; Cell is changed to serum-free DMEM and trains liquid (100 μ L/ hole), in cell, be added dropwise to final plasmid, the packing mixt of PLUS and Lipofectamine, hatch after 3 hours with the substratum termination transfection reaction of 200 μ L/ holes containing 10% foetal calf serum.
3) Wnt reporter gene is surveyed and is lived and IC 50calculate:
To the HEK293T cell of examining report gene activity be used for by 2) shown in method carry out transfection.Wnt reporter gene activity detect: each plasmid amount of transfection is: 5ng/ hole TOP-flash and 5ng/ hole as interior target GFP plasmid, with lacZ polishing 125ng/ hole.Within 18 hours, add after transfection containing DMSO contrast nutrient solution and containing different concns (DMSO, 2.5 μMs, 5 μMs, 10 μMs, 15 μMs, 20 μMs, 30 μMs; The small molecules that 30 μMs be greater than to IC50 then use instead DMSO, 5 μMs, 15 μMs, 30 μMs, 40 μMs, 50 μMs, 60 μMs) micromolecular Wnt3a conditioned medium process 6 hours, with Boehringer Mannheim Luci-ferase Assay Kit lysing cell (200 μ L/ hole).Respectively get 50 μ L and enter 96 orifice plates, with photofluorometer FL600(BIO-TEK Inc.Winooski, VT) intensity of GFP albumen in cell pyrolysis liquid is surveyed, mark as in cell transfecting efficiency, then the substrate of 10 μ L luciferases is added to every hole, with Micro Lumate Plus(Perkin Elmer Inc.Wellesley, MA) luminometer measures uciferase activity.Be finally the activity value of interior mark correction luciferase by GFP intensity, be Wnt reporter gene activity.
IC 50calculate (to calculate the IC50 to Wnt signal path): by not containing or containing the reporter gene activity deduction background (the conditioned medium treatment group gained only containing DMSO is active) measured by different concns micromolecular Wnt3a conditioned medium treatment group, the numerical value of gained is defined as Wnt and induces or reporter gene activity remaining after adding small molecules.The all residual activities of reporter gene activity homogenization induced with Wnt, obtain relative residual activity.Make point and line chart in conjunction with small molecules concentration again, with the method matching amount effect curve based on four parameter logistic, when relative residual activity is 0.5, corresponding small molecules concentration is the IC of this small molecules to Wnt signal path 50.
(2) the selection result:
Table 2 15-oxospiramilactone (S-3) derivative suppresses the activity (IC of Wnt signal reporter gene Top-flash 50, μM)
Compound Inhibitory Compound Inhibitory
S-3 18.35±1.82 7 16.46±1.16
1 12.05±1.88 8 20.06±1.32
6 19.75±1.34
Table 3 15-oxospiramilactone (S-3) derivative suppresses the activity (IC of Wnt signal reporter gene Top-flash 50, μM)
Compound Inhibitory Compound Inhibitory
S-3 18.73±0.58 27 >60(0)
11 27.35±0.98 30 9.98±1.00
12 17.56±1.90 31 16.89±1.89
14 21.71±0.63 37 12.59±1.04
15 9.12±0.21 38 42.99±5.35
21 18.05±2.67 40 10.36±0.57
22 >60(15%) 45 8.39±0.63
23 >60(20%) 48 7.83±0.70
Table 4 15-oxospiramilactone (S-3) derivative suppresses the activity (IC of Wnt signal reporter gene Top-flash 50, μM)
Compound Inhibitory Compound Inhibitory
S-3 17.97±0.28 25 >60(33%)
2 4.09±0.07 26 >60(21%)
3 8.56±1.15 32 46.93±1.26
4 9.54±1.34 33 >60(10%)
5 6.89±0.27 34 58.22±1.18
9 11.05±0.63 35 56.04±0.92
10 8.27±1.24 36 7.30±0.22
13 6.72±1.11 39 11.18±1.54
19 >60(24%) 41 7.2±0.71
20 7.10±0.70 46 4.92±0.69
24 12.85±0.13 49 7.68±0.19
Table 5 15-oxospiramilactone (S-3) derivative suppresses the activity (IC of Wnt signal reporter gene Top-flash 50, μM)
Compound Inhibitory Compound Inhibitory
S-3 22.49±0.56 54 17.04±1.28
50 >60(0) 55 >60(35%)
51 8.34±0.90 56 28.67±0.97
52 49.35±1.42 57 >60(26%)
53 31.93±0.21 58 60.63±3.09
Table 6 15-oxospiramilactone (S-3) derivative suppresses the activity (IC of Wnt signal reporter gene Top-flash 50, μM)
Compound Inhibitory Compound Inhibitory
S-3 13.9100±2.0100 29 44.8350±3.3450
16 15.4150±1.5050 42 43.7800±3.0500
17 19.8000±1.0400 43 32.3000±3.0100
18 12.0600±1.2500 44 12.1400±1.2500
28 27.7400±2.8000
The present invention's research in earlier stage shows: the 15-oxospiramilactone (S-3) coming from pink blossom Ramulus et Folium Spiraeae Salicifolia has brand-new mechanism of action, the apoptosis that S-3 induces non-Bax/Bak to rely on, further investigation finds that S-3 induces the high expression level of pro apoptotic protein Bim specifically, itself and Bcl-2 have an effect at mitochondrial level, Bcl-2 conformational change is caused to be converted to short apoptosis function by anti-apoptotic, thus the release of activating cells pigment C causes the dependent apoptosis of the non-Bax/Bak of cell, this is the new mechanism of cell death inducing; Find that S-3 is tumour cell Wnt signal path optionally inhibitor, suppressed the expression of Wnt downstream target gene by the interaction weakening β-catenin and TCF4; Expressing by suppressing downstream target gene causes cell cycle arrest in the G2/M phase; SW480 transplants experiment of nude mouse and shows, the S-3 of low dosage gets final product the growth of Tumor suppression.The present invention on this basis, to the large intestine of S-3 and colon tumor strain, Normocellular cytotoxic activity, and the early stage Fast Evaluation of ADME/T has done analysis, finds that S-3 has " first pass effect ", it eliminates the transformation period is 0.43 hour, the hydrolysate anti-tumor activity completely dissolve of lactonic ring.For the defect of its druggability, structure of modification and composition optimizes are carried out to it, using the cytotoxic activity of the inhibit activities of Wnt signal path, different tumor line etc. as foundation, find patent medicine more reasonably antineoplastic compound be very important.
Embodiment 4:
Tablet: any one compound of embodiment 1 gained 10mg, lactose 180mg, starch 55mg, Magnesium Stearate 5mg
Preparation method: by compound, the mixing of newborn sugar and starch, evenly moistening with water, sieves moistening mixture and drying, after sieve, adds Magnesium Stearate, and then by mixture compressing tablet, the heavy 250mg of every sheet, compounds content is 10mg.
Embodiment 5:
Ampulla: any one compound of embodiment 1 gained 2mg, sodium-chlor 10mg;
Preparation method: compound and sodium-chlor are dissolved in appropriate water for injection, filters gained filtrate, aseptically loads in ampoule.
Embodiment 6:
Capsule: any one compound of embodiment 1 gained 10mg, lactose 187mg, Magnesium Stearate 3mg
Preparation method: mixed with auxiliary agent by compound, sieves, Homogeneous phase mixing, and the mixture obtained is loaded in hard gelatin capsule, and the heavy 200mg of each capsule, activeconstituents is 10mg.

Claims (5)

1. 15 shown in following structural formula ?oxo Ramulus et Folium Spiraeae Salicifolia lactone (S ?3) derivative,
2. there is a pharmaceutical composition for anti-tumor activity, it is characterized in that, comprise 15 ?oxo Ramulus et Folium Spiraeae Salicifolia lactone derivatives and pharmaceutically acceptable auxiliary materials involved in one or more claims 1 for the treatment of significant quantity.
3. the preparation method of compound shown in claim 1, comprises the steps:
(1) compound 1 ?5,7,9 preparation method:
(2) preparation method of compound 10:
(3) preparation method of compound 12,13:
(4) preparation method of compound 15,18,20:
(5) preparation method of compound 21:
(6) preparation method of compound 24,30,31,36,39:
(8) preparation method of compound 40,41:
(9) syntheti c route of compound 44:
(10) preparation method of compound 45,46,48,49:
(11) preparation method of compound 51,54,56:
4. compound shown in claim 1 is preparing the application in antitumor drug.
5. the application of compound shown in claim 1 in preparation Wnt signal path inhibitor medicine.
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