CN102731526B - New ansamitocin compounds and preparation method thereof - Google Patents
New ansamitocin compounds and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to ansamitocin compounds and a preparation method thereof. A structural formula of the ansamitocin compound is shown as follow, wherein R is CH2CH3 or CH2CH(CH3)2. The preparation method for the ansamitocin compound comprises: adopting macroporous resin adsorption column chromatography, silica gel column chromatography and other methods to carry out separation and purification on fermentation products of Actinosynnema sp FIM06-0063 to obtain two ansamitocin compounds, wherein structures of the compounds are characterized by mass spectrometry, nuclear magnetic resonance spectroscopy, ultraviolet spectrum and other technologies. The prepared ansamitocin compounds of the present invention have strong anti-tumor activities. According to the present invention, the microbial fermentation method is adopted to prepare the ansamitocin compound, such that advantages of simple process and low production cost are provided.
Description
[technical field]
The present invention relates to a kind of compound, relate in particular to a kind of ansamitocin compounds, the invention still further relates to the preparation method of this ansamitocin compounds.
[background technology]
Kupchan in 1972 etc. isolate first natural antitumor activeconstituents from the tingia Caulis Mayteni (M.serrata) of original Tropical Africa---and macrolides compound maytenin (Maytansine), researchist is separated to Caulis Mayteni compounds in succession from higher plant Mao Wei section, Rhamnaceae, euphorbia plant, bryophyte thereafter.The most attractive is from microorganism, to have found this compounds, 1977, Higashide and colleague thereof are in the process of screening anticancer active constituent, from separation, from the orange synnema actinomycetes (Actinosynnema pretiosum) of Cyperaceae carex leaf, obtain six maytenin derivatives, called after ansamitocin P0, P1, P2, P3, P4, P4 '.From another orange synnema actinomycetes subspecies (A pretiosum ssp.Auranticum), separation obtains 15 new ansamitocins except above-mentioned six compounds again subsequently, its parent nucleus is the Macrocyclic lactams ring of 19-C, C-3 connects the carbochain of different lengths, and main active ingredient is P-3.
Above-mentioned all these ansamitocins all can be reduced and resolve into C-3 Ansamitocin Po (P-0), and this alcohol is the precursor for the synthesis of sulfur-bearing alcohol maytansinoid.Ansamitocin is become the maytansinoid (maytansinoids) containing mercaptan by chemical transformation, and the medical use that this alkaloid reveals with the form body of Cell binding agent-maytansinoid conjugate is in the news.
Along with deepening continuously that CHROMATOGRAPHIC FRACTIONATION AND MASS antibody conjugates is studied, market progressively expands the demand of ansamitocin compounds.Because Caulis Mayteni class plant is very low containing the content of maytenin and homologue thereof, cutting down in a large number Caulis Mayteni class plant can do great damage to ecology, and from wild plant, finds raw material and be difficult to meet industrial needs; Adopt chemosynthesis, again because of shortcomings such as ansamitocin complex structure exist complete synthesis and semi-synthesizing technology complicated, process is loaded down with trivial details, productive rate is very low, reaction specificity is poor.
[summary of the invention]
One of technical problem to be solved by this invention is to provide a kind of ansamitocin compounds, and described ansamitocin compounds has anti-tumor activity.
The present invention is what one of to solve the problems of the technologies described above by the following technical programs: the ansamitocin compounds of formula (Ι) representative,
Wherein, R is CH
2cH
3or CH
2cH (CH
3)
2.
Two of technical problem to be solved by this invention is to provide a kind of preparation method of ansamitocin compounds, prepares ansamitocin compounds have advantages of that technique is simple, production cost is low by microbial fermentation processes.
The present invention be solve the problems of the technologies described above by the following technical programs two: a kind of preparation method of described ansamitocin compounds, its operation steps is as follows:
Step 1: prepare synnema actinomycetes bacterial strain FIM06-0063 (Actinosynnema sp) fermented liquid; Described synnema actinomycetes bacterial strain FIM06-0063, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 30th, 2011, and address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and deposit number is CGMCC NO.5677.
Step 2: the synnema actinomycetes bacterial strain FIM06-0063 fermented liquid that step 1 is obtained carries out macroporous resin adsorption column chromatography, obtains crude extract;
Step 3: the crude extract that step 2 is obtained carries out purification on normal-phase silica gel column chromatography, and adopt chloroform-methanol gradient elution crude extract, then each elutriant of Fractional Collections;
Step 4: each elutriant that step 3 is obtained carries out respectively thin-layer chromatography, and adopt high performance liquid chromatography to detect respectively analysis to each elutriant, the elutriant then aobvious orange red and high performance liquid chromatography detected result of thin-layer chromatography result to identical peak merges, and the elutriant after this merging of concentrating under reduced pressure obtains the first enriched material;
Step 5: the first enriched material that step 4 is obtained carries out middle pressure reversed-phase silica gel column chromatography to obtain crude product, while carrying out middle pressure reversed-phase silica gel column chromatography, first adopting volume fraction is that 10% methanol aqueous solution is that moving phase balance is after 10 minutes, then moving phase adopts the methanol-water solvent systems gradient elution 90min of volume ratio 10%～100%:1, and the flow velocity of moving phase is 5ml/min;
Step 6: the crude product that step 5 is obtained carries out Sephadex LH-20 gel filtration chromatography, described Sephadex LH-20 gel filtration chromatography adopts the chloroform-methanol solvent systems wash-out of volume ratio 1:1, and according to flow point and each flow point of bismuth potassium iodide color reaction Fractional Collections, then the flow point identical with bismuth potassium iodide reaction solution merged, concentrating under reduced pressure is to obtain the second enriched material;
Step 7: the second enriched material that step 6 is obtained adopts preparative high performance liquid chromatography to carry out separation, obtains 3 components; Finally, 3 components are passed through respectively to the separation of preparative silica-gel plate to obtain ansamitocin compounds; The separation condition of described preparative high performance liquid chromatography is: the methanol aqueous solution that moving phase is 70%, and the flow velocity of moving phase is 1.5ml/min, detection wavelength is 254nm.
Further, the separation method of described synnema actinomycetes bacterial strain FIM06-0063 is as follows:
Step 100: get Lao Ye and the petiole of Yunnan Caulis Mayteni, rinse after 30min with tap water, then clean and be placed in sterile petri dish with distilled water; Then by described Lao Ye and petiole successively with 75% alcohol scouring, after aseptic water washing, proceed to massfraction and be in 0.1% mercuric chloride solution and soak 3min, then use aseptic water washing 5 times, remove thimerosal; Then described Lao Ye and petiole are cut into small pieces or segment, put into aseptic mortar and smash into mashed prod to pieces;
Step 200: pick with aseptic glass spreading rod the mashed prod obtaining in a small amount of step 100 and directly coat starch asparagine agar plate, then flat board is placed at 28 ℃ and cultivates 7～20 days, obtain the single bacterium colony of actinomycetes; The component of described starch asparagine agar: Zulkovsky starch 2%, L-asparagine 0.05%, KNO
30.1%, K
2hPO
43H
2o0.05%, NaCl0.05%, MgSO
47H
2o0.05%, CaCO
30.05%, agar 1.5%, distilled water preparation, pH is 7.5, and the percentage ratio in this starch asparagine agar component is massfraction;
Step 300: transfer in inorganic salt Starch Agar inclined-plane process the single bacterium colony of the actinomycetes that obtain through step 200, obtain the slant culture of synnema actinomycetes bacterial strain FIM06-0063; The component on described inorganic salt Starch Agar inclined-plane is: Zulkovsky starch 10g; K
2hPO
43H
2o1g; MgSO
47H
2o1g; NaCl1g; (NH
4)
2sO
42g; CaCO
32g; FeSO
47H
2o0.001g; MnCl
24H
2o0.001g; ZnSO
47H
2o0.001g; Agar 20g; Distilled water 1000mL, pH is 7.0～7.4.
Further, described step 1 is specially:
(1) prepare seed liquor: will after synnema actinomycetes bacterial strain FIM06-0063 slant culture maturation, be inoculated in seed culture medium, and be placed on shaking culture 40～48h in constant-temperature table, 28 ℃ of temperature, rotating speed 220r/min;
(2) prepare fermented liquid: the seed liquor that the culture transferring amount by 10% obtains step (1) is transferred and cultivated 96～120h, 28 ℃ of temperature, rotating speed 220r/min into the fermention medium of sterilization.
Further, the component of described seed culture medium: Zulkovsky starch 1.5%, glucose 0.5%, analysis for soybean powder 1%, sodium-chlor 0.1%, peptone 0.5%, yeast extract 0.5%, calcium carbonate 0.5%, pH is 7.0, and the percentage ratio in this seed culture medium component is massfraction.
Further, the component of described fermention medium: dextrin 1%, glycerine 3%, analysis for soybean powder 2%, Dried Corn Steep Liquor Powder 1%, potassium primary phosphate 0.05%, ammonia chloride 0.05%, calcium carbonate 0.5%, pH is 7.0, and the percentage ratio in this fermention medium component is massfraction.
Further, described step 2 is specially: by synnema actinomycetes bacterial strain FIM06-0063 fermented liquid, at rotating speed, be centrifugal 15min under 3000r/min, and get supernatant liquor; Then supernatant liquor is splined on to macroporous adsorbent resin, then obtain mixed solution by methanol-eluted fractions; Mixed solution is removed methyl alcohol to obtain concentrated solution through concentrating under reduced pressure; Concentrated solution is used with the ethyl acetate of this concentrated solution equal volume and extracted three times, then combined ethyl acetate extraction liquid; Acetic acid ethyl acetate extract arrives to obtain crude extract after concentrating under reduced pressure.
Further, the chloroform-methanol gradient elution crude extract in described step 3 refers to uses volume ratio 100:0,98:2, and 95:5,90:10,85:15,80:20,70:30,60:40, the chloroform-methanol solvent systems of 50:50 carries out gradient elution to crude extract.
Further, the developing agent of the thin-layer chromatography in described step 4 is trichloromethane-methanol solvate of volume ratio 15:1, and developer is bismuth potassium iodide developer.
Further, the high performance liquid chromatography testing conditions in described step 4 is: moving phase is the water-methanol containing 0.1% trifluoroacetic acid of volume ratio 35:65, wash-out 20min, and flow velocity lmL/min, detects wavelength 254nm, 40 ℃ of column temperatures.
Further, described synnema actinomycetes bacterial strain FIM06-0063 (Actinosynnema sp), is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 30th, 2011, and deposit number is CGMCC NO.5677.
Beneficial effect of the present invention is: the ansamitocin compounds that the present invention makes has anti-tumor activity; The present invention prepares ansamitocin compounds by microbial fermentation processes, has advantages of that technique is simple, production cost is low.
[accompanying drawing explanation]
The invention will be further described in conjunction with the embodiments with reference to the accompanying drawings.
Fig. 1 is the uv absorption spectra of compound 1 in the present invention.
Fig. 2 is the uv absorption spectra of compound 2 in the present invention.
Fig. 3 is compound 1 in the present invention
1h-
1h COSY spectrogram.
Fig. 4 is compound 2 in the present invention
1h-
1h COSY spectrogram.
Fig. 5 is the hsqc spectrum figure of compound 1 in the present invention.
Fig. 6 is the hsqc spectrum figure of compound 2 in the present invention.
Fig. 7 is the HMBC spectrogram of compound 1 in the present invention.
Fig. 8 is the HMBC spectrogram of compound 2 in the present invention.
Fig. 9 is the structure elucidation procedure chart of compound 1 in the present invention.
Figure 10 is the structure elucidation procedure chart of compound 2 in the present invention.
Figure 11 is the dose response curve figure of 1 pair of HL60 cyto-inhibition of compound in the present invention.
Figure 12 be in the present invention 2 pairs of HL60 cyto-inhibitions of compound dose response curve figure.
Bacterial strain preservation
The synnema actinomycetes bacterial strain FIM06-0063 (Actinosynnema sp) that the present invention is used, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 30th, 2011, and deposit number is CGMCC NO.5677.
[embodiment]
The separation method of synnema actinomycetes bacterial strain FIM06-0063 in the present invention is as follows:
Step 100: get Lao Ye and the petiole of Yunnan Caulis Mayteni, rinse after 30min with tap water, then clean and be placed in sterile petri dish with distilled water; Then described Lao Ye and petiole are cleaned with 75% alcohol successively, after aseptic water washing, the mercury chloride that proceeds to massfraction 0.1% (is HgCl
2) soak 3min in solution, then use aseptic water washing 5 times, remove thimerosal; Then described Lao Ye and petiole are cut into small pieces or segment, put into aseptic mortar and smash into mashed prod to pieces;
Step 200: pick with aseptic glass spreading rod the mashed prod obtaining in a small amount of step 100 and directly coat starch asparagine agar plate, then flat board is placed at 28 ℃ and cultivates 7～20 days, obtain the single bacterium colony of actinomycetes; In embodiment 1, flat board is placed at 28 ℃ and is cultivated 7 days; In embodiment 2, flat board is placed at 28 ℃ and is cultivated 13 days; In embodiment 3, flat board is placed at 28 ℃ and is cultivated 20 days.
The component of described starch asparagine agar: Zulkovsky starch 2%, L-asparagine 0.05%, KNO
30.1%, K
2hPO
43H
2o0.05%, NaCl0.05%, MgSO
47H
2o0.05%, CaCO
30.05%, agar 1.5%, distilled water preparation, pH is 7.5, and the percentage ratio in this starch asparagine agar component is massfraction;
Step 300: transfer in inorganic salt Starch Agar inclined-plane process the single bacterium colony of the actinomycetes that obtain through step 200, obtain the slant culture of synnema actinomycetes bacterial strain FIM06-0063; The component on described inorganic salt Starch Agar inclined-plane is: Zulkovsky starch 10g; K
2hPO
43H
2o1g; MgSO
47H
2o1g; NaCl1g; (NH
4)
2sO
42g; CaCO
32g; FeSO
47H
2o0.001g; MnCl
24H
2o0.001g; ZnSO
47H
2o0.001g; Agar 20g; Distilled water 1000mL, pH is 7.0～7.4.
The evaluation of this bacterial strain FIM06-0063:
The slant culture of synnema actinomycetes bacterial strain FIM06-0063 is lined to inorganic salt Starch Agar dull and stereotyped, then oblique cutting cover glass is cultivated 5～20 days at 28 ℃.In embodiment 1, adopt and at 28 ℃, cultivate 5 days; In embodiment 2, adopt and at 28 ℃, cultivate 13 days; In embodiment 3, adopt and at 28 ℃, cultivate 20 days; Observe gas silk, Ji Si and pigment, take out opticmicroscope and electron microscope observation for cover glass, Main Morphology and the biological characteristics of bacterial strain FIM06-0063 are as follows:
Bacterial strain FIM06-0063 bacterium colony is orange-yellow, forms rhizomorph on solid medium, on the surface of rhizomorph top or dome-shaped body, produces gas silk; Gas silk is separated and is produced spore chain; Ripe hyphal diameter 0.5-1.2 is micron, and appearance presents bamboo shape, forms the shaft-like or oval spore that can move about; Milk peptonizes, energy hydrolyzed starch, and tyrosine, can utilize glucose, sucrose, wood sugar, seminose is carbon source for growth; Cell walls III type: only having meso-diaminopimelic acid is characteristic component; The sugary type of synnema actinomycetes bacterial strain FIM06-0063 cell walls is: sugared MODEL C, atypism sugar.
The strains A ctinosynnema pretiosum subsp.Pretiosum ATCC31281 of the upper known array of the 16SrDNA sequencing result of this bacterial strain FIM06-0063 and GeneBank
t(GenBank:AF114800) homology is the highest, reaches 99.82%.According to the measurement result of the proximate analysis of the physiology and morphology biochemical character of bacterial strain FIM06-0063, cell walls and 16S rRNA sequence, identify that this bacterial strain is synnema actinomycetes (Actinosynnema sp).
A preparation method for ansamitocin compounds, its operation steps is as follows:
Step 1: prepare synnema actinomycetes bacterial strain FIM06-0063 (Actinosynnema sp) fermented liquid, particularly:
(1) prepare seed liquor: will after synnema actinomycetes bacterial strain FIM06-0063 slant culture maturation, be inoculated in seed culture medium, and be placed on shaking culture 40～48h in constant-temperature table, 28 ℃ of temperature, rotating speed 220r/min.In embodiment 1, adopt and be placed in constant-temperature table shaking culture 40h; In embodiment 2, adopt and be placed in constant-temperature table shaking culture 44h; In embodiment 3, adopt and be placed in constant-temperature table shaking culture 48h.Wherein, the substratum of described slant culture is inorganic salt Starch Agar (being ISP-4) slant medium; The component of described seed culture medium: Zulkovsky starch 1.5%, glucose 0.5%, analysis for soybean powder 1%, sodium-chlor 0.1%, peptone 0.5%, yeast extract 0.5%, calcium carbonate 0.5%, pH is 7.0, and the percentage ratio in this seed culture medium component is massfraction.
(2) prepare fermented liquid: the seed liquor that the culture transferring amount by 10% obtains step (1) is transferred and cultivated 96～120h, 28 ℃ of temperature, rotating speed 220r/min into the fermention medium of sterilization.In embodiment 1, adopt seed liquor is transferred and cultivated 96h into the fermention medium of sterilization; In embodiment 2, adopt seed liquor is transferred and cultivated 109h into the fermention medium of sterilization; In embodiment 3, adopt seed liquor is transferred and cultivated 120h into the fermention medium of sterilization.The component of described fermention medium: dextrin 1%, glycerine 3%, analysis for soybean powder 2%, Dried Corn Steep Liquor Powder 1%, potassium primary phosphate 0.05%, ammonia chloride 0.05%, calcium carbonate 0.5%, pH is 7.0, and the percentage ratio in this fermention medium component is massfraction.
Step 2: the synnema actinomycetes bacterial strain FIM06-0063 fermented liquid that step 1 is obtained carries out macroporous resin adsorption column chromatography, obtain crude extract, particularly: by synnema actinomycetes bacterial strain FIM06-0063 fermented liquid, at rotating speed, be centrifugal 15min under 3000r/min, and get supernatant liquor; Then supernatant liquor is splined on to macroporous adsorbent resin, then obtain mixed solution by methanol-eluted fractions; Mixed solution is removed methyl alcohol to obtain concentrated solution through concentrating under reduced pressure; Concentrated solution is used with the ethyl acetate of this concentrated solution equal volume and extracted three times, then combined ethyl acetate extraction liquid; Acetic acid ethyl acetate extract arrives to obtain crude extract after concentrating under reduced pressure.
Step 3: the crude extract that step 2 is obtained carries out purification on normal-phase silica gel column chromatography (silica gel G 200-300 order), and adopt chloroform-methanol gradient elution crude extract, then each elutriant of Fractional Collections; Described chloroform-methanol gradient elution crude extract refers to uses volume ratio 100:0,98:2, and 95:5,90:10,85:15,80:20,70:30,60:40, the chloroform-methanol solvent systems of 50:50 carries out gradient elution to crude extract.
Step 4: each elutriant that step 3 is obtained carries out respectively thin-layer chromatography, and adopt high performance liquid chromatography to detect respectively analysis to each elutriant, then thin-layer chromatography result is aobvious orange red, and the elutriant that high performance liquid chromatography detected result has identical peak merges, and the elutriant after this merging of concentrating under reduced pressure obtains the first enriched material.Wherein, the developing agent of described thin-layer chromatography is trichloromethane-methanol solvate of volume ratio 15:1, and developer is bismuth potassium iodide developer; Described high performance liquid chromatography testing conditions is: moving phase is the water-methanol containing 0.1% trifluoroacetic acid of volume ratio 35:65, wash-out 20min, and flow velocity l mL/min, detects wavelength 254nm, 40 ℃ of column temperatures; The water of described 0.1% trifluoroacetic acid refers to volume ratio trifluoroacetic acid: water=0.1:99.9.
Step 5: the first enriched material that step 4 is obtained carries out middle pressure reversed-phase silica gel column chromatography to obtain crude product, while carrying out middle pressure reversed-phase silica gel column chromatography, first adopting volume fraction is that 10% methanol aqueous solution is that moving phase balance is after 10 minutes, then moving phase adopts the methanol-water solvent systems gradient elution 90min of volume ratio 10%～100%:1, and the flow velocity of moving phase is 5ml/min;
Step 6: the crude product that step 5 is obtained carries out Sephadex LH-20 gel filtration chromatography, described Sephadex LH-20 gel filtration chromatography adopts the chloroform-methanol solvent systems wash-out of volume ratio 1:1, and according to flow point and each flow point of bismuth potassium iodide color reaction Fractional Collections, then the flow point identical with bismuth potassium iodide reaction solution merged, concentrating under reduced pressure is to obtain the second enriched material;
Step 7: the second enriched material that step 6 is obtained adopts preparative high performance liquid chromatography to carry out separation, obtains 3 components; Finally, by 3 components, through preparative silica-gel plate, separation is compound 1 and compound 2 to obtain ansamitocin compounds respectively.The separation condition of described preparative high performance liquid chromatography is: the methanol aqueous solution that moving phase is 70%, and the flow velocity of moving phase is 1.5ml/min, detection wavelength is 254nm.
By Structural Identification, prove that the formula for structure (Ι) of compound 1 that above-mentioned preparation method obtains and compound 2 represents, wherein, the R of compound 1 is CH
2cH
3, the structure of compound 1 is formula II; The R of compound 2 is CH
2cH (CH
3)
2, the structure of compound 2 is formula III
Described compound 1 is 9-methoxyl group-ansamitocin P-2, and its molecular formula is C
32h
43clN
2o
9, molecular weight is 634.Compound 1 is faint yellow unsetting powder,
be soluble in the organic solvents such as methyl alcohol, acetone, ethyl acetate, trichloromethane, it is aobvious orange-yellow that it meets bismuth potassium iodide, meet iodine vapor colour developing, and (254nm) fluorescence is blackening point under ultraviolet lamp.The ultraviolet absorption peak of compound 1 is respectively 280.60nm (seeing Fig. 1), 201.60nm (seeing Fig. 1).The ESI-MS spectrum of compound 1 shows molecular ion peak [M] at m/z635.3 place
+.The isotropic substance m/z635.3 of characteristic ion bunch and the abundance ratio of m/z637.3 are 3:1, tentatively infer that this compound 1 contains a chlorine element, and its molecular weight is 634.The quasi-molecular ions of m/z635.3 is carried out to second order ms scanning, obtain m/z and be 561.1 feature fragment ion peak.High resolution mass spectrum (HR-ESI-MS): [M+Na]
+measured value is 657.2552; Theoretical value is 657.2554, and supposition molecular formula is C
32h
43n
2o
9cl, degree of unsaturation is 12.
Compound 1
1h NMR (CDCl
3, 600MHz) data are as follows: 0.77 (3H, s, H-4-CH
3), 1.14 (3H, t, J=7.4Hz, H-3 '), 1.18 (1H, brd, H-8b), 1.20 (3H, d, J=5.7Hz, H-6-CH
3), 1.37 (1H, m, H-6), 1.57 (1H, brd, H-8a), 1.60 (3H, brs, H-14-CH
3), 2.12 (1H, dd, J=2.7,13.7Hz, H-2b), 2.35 (1H, q, J=7.4Hz, H-2 ' is a), 2.42 (1H, q, J=7.4Hz, H-2 ' b), 2.45 (1H, dd, J=12.0,13.7Hz, H-2a), 2.81 (1H, d, J=9.8Hz, H-5), 3.11 (3H, s, H-18-NCH
3), 3.15 (1H, d, J=12.8Hz, H-15b), 3.22 (3H, s, H-10-OCH
3), 3.32 (3H, s, H-9-OCH
3), 3.37 (1H, d, J=9.1Hz, H-10), 3.45 (1H, d, J=12.8Hz, H-15a), 3.93 (3H, s, H-20-OCH
3), 4.08 (1H, m, H-7), 4.83 (1H, dd, J=2.7,12.0Hz, H-3), 5.47 (1H, dd, J=9.1,15.3Hz, H-11), 6.10 (1H, d, J=11.0Hz, H-13), 6.30 (1H, brs, H-9-NHCO), 6.33 (1H, dd, J=11.0,15.3Hz, H-12), 6.71 (1H, s, H-17), 6.77 (1H, s, H-21).Make a concrete analysis of as follows: compound 1
1h-NMR (CDCl
3, 600MH
z) in spectrum, at high field region, show 4 groups of methyl hydrogen signals, wherein there is 1 group of bimodal methyl hydrogen signal [δ
h1.20 (3H, d, J=5.7Hz)] show that it is directly connected with tertiary carbon; 2 unimodal methyl hydrogen signal [δ
h0.77 (3H, s), δ
h1.60 (3H, brs)] show that it is connected with quaternary carbon; 1 group of triplet signal [δ
h1.14 (3H, t, J=7.4Hz)] show that it is connected with secondary carbon.3 methoxyl group hydrogen signal [δ
h3.22 (3H, s), δ
h3.32 (3H, s), δ
h3.93 (3H, s)] and 1 n-formyl sarcolysine base hydrogen signal [δ
h3.11 (3H, s)].There are 5 and sp in low place
2the connected hydrogen signal of the carbon atom of hydridization wherein 3 be 2 couples of hydrogen signal [δ on trans connected two keys
h5.47 (1H, dd, J=9.1,15.3Hz, H-11), δ
h6.33 (1H, dd, J=11.0,15.3Hz, H-12), δ
h6.10 (1H, d, J=11.0Hz, H-13)]; 2 phenyl hydrogen signal [δ
h6.77 (1H, s), δ
h6.71 (1H, s)].
Compound 1
13c NMR (CDCl
3, 150MHz) (DETP) spectrum data are as follows: 173.0 (C-1 '), 169.0 (C-1), 156.2 (C-20), 153.0 (C-9-NHCO), 142.7 (C-18), 140.2 (C-16), 140.1 (C-14), 132.7 (C-12), 128.0 (C-11), 124.5 (C-13), 122.3 (C-17), 119.5 (C-19), 113.2 (C-21), 91.1 (C-10), 83.3 (C-9), 77.4 (C-3), 75.3 (C-7), 66.6 (C-5), 60.5 (C-4), 57.3 (C-10-OCH
3), 56.8 (C-20-OCH
3), 52.2 (C-9-OCH
3), 47.4 (C-15), 38.6 (C-6), 36.9 (C-8), 35.9 (C-18-NCH
3), 33.0 (C-2), 27.3 (C-2 '), 16.0 (C-14-CH
3), 14.7 (C-6-CH
3), 12.3 (C-4-CH
3), 8.9 (C-3 ').Make a concrete analysis of as follows:
13c-NMR (CDCl
3, 150MH
z) (DETP) collection of illustrative plates show altogether 32 spectral lines, comprising: (10 quaternary carbon signals are specially 10 quaternary carbon signals: 1 amide group carbon signal (δ
c169.0), 1 amide oxygen base carbon signal (δ
c153.0), 1 ester group carbon signal (δ
c173.0), 4 phenyl ring carbon signal (δ
c119.5, δ
c140.2, δ
c142.7, δ
c156.2), 1 olefinic carbon signal (δ
c140.1), 2 carbon signal (δ that are connected with Sauerstoffatom
c60.5, δ
c83.3)], (10 methine carbon signals are specially 10 methine carbon signals: 3 olefinic carbon signal (δ
c124.5, δ
c128.0, δ
c132.7), 2 phenyl ring carbon signal (δ
c113.2, δ
c122.3), 4 carbon signal (δ that are connected with Sauerstoffatom
c66.6, δ
c91.1, δ
c77.4, δ
c75.3) and 1 sp
3carbon signal (the δ of hydridization
c38.6)], 4 is mesomethylene carbon signal (δ
c27.3, δ
c33.0, δ
c36.9, δ
c47.4), 3 methoxyl group carbon signal (δ
c52.2, δ
c57.3, δ
c56.8), 1 n-formyl sarcolysine base carbon signal (δ
c35.9), 4 methyl carbon signal (δ
c8.9, δ
c12.3, δ
c14.7, δ
c16.0).
Refer to Fig. 3 and Fig. 5, Fig. 3 is compound 1
1h-
1h COSY spectrogram,
1h-
1h chemical shift correlated spectroscopy figure; Fig. 5 is the HSQC collection of illustrative plates of compound 1, i.e. heteronuclear list quantum coherent spectrogram.At compound 1
1h-
1in H COSY spectrum, visible δ 2.45 (H-2a), δ 2.12 (H-2b) are all relevant to δ 4.83 (H-3); δ 2.34 (H-2 ' a), δ 2.44 (H-2 ' b) is all relevant to δ 1.14 (H-3 '), relevant in turn between δ 2.81 (H-5)-δ 1.37 (H-6)-δ 4.08 (H-7)-δ 1.18 (H-8b), relevant in turn between δ 3.37 (H-10)-δ 5.47 (H-11)-δ 6.33 (H-12)-δ 6.10 (H-13), in addition, can also see δ 1.37 (H-6)-δ 1.20 (H-6-CH
3) between relevant, the HSQC collection of illustrative plates (seeing Fig. 5) of binding compounds 1, can contain following carbon skeleton fragment: C in pushing-out structure
2-C
3, C
5-C
6-C
7-C
8, C
10-C
11-C
12-C
13, C
2'-C
3'.
Fig. 7 is the HMBC spectrogram of compound 1,
1the heteronuclear multikey Correlated Spectroscopy that H detects.In the HMBC of compound 1 collection of illustrative plates, visible δ 2.45 (H-2a) and C-1 (δ 169.0), C-3 (δ 77.4), C-4 (δ 60.5); H-3 (δ 4.83) and C-2 (δ 33.0), C-4 (δ 60.5), C-1 ' (δ 173.0), C-4-CH
3(δ 12.3); 2.35 (H-2 ' a), δ 2.42 (H-2 ' b) equal and C-1 ' (δ 173.0), C-3 ' (δ 8.9); Distant relation between H-3 ' (δ 1.14) and C-1 ' (δ 173.0), C-2 ' (δ 27.3), so just can add up in the connection of bar structure Segment A, as shown in Segment A:
Referring again to Fig. 7, in the HMBC of compound 1 collection of illustrative plates, visible H-5 (δ 2.81) and C-3 (δ 77.4), C-6 (δ 38.6), C-7 (δ 75.3), C-6-CH
3(δ 14.7); H-6 (δ 1.37) and C-5 (δ 66.6), C-7 (δ 75.3); H-6-CH
3(δ 1.20) and C-5 (δ 66.6), C-6 (δ 38.6); H-8a (δ 1.57) and C-9 (δ 83.3); H-8b (δ 1.18) and C-6 (δ 38.6), C-7 (δ 75.3), C-10 (δ 91.1); H-9-OCH
3(δ 3.32) and C-9 (δ 83.3); Distant relation between H-9-NHCO (δ 6.30) and C-8 (δ 36.9), C-10 (δ 91.1), C-9-NHCO (δ 153.0), so just can add up in bar structure fragment B connection, and fragment B is:
Referring again to Fig. 7, in the HMBC of compound 1 collection of illustrative plates, visible H-10 (δ 3.37) and C-9 (δ 83.3), C-12 (δ 132.7); H-11 (δ 5.47) and C-13 (δ 124.5); H-12 (δ 6.33) and C-10 (δ 91.1), C-13 (δ 124.5), C-14 (δ 140.1); H-13 (δ 6.10) and C-11 (δ 128.0), C-12 (δ 132.7), C-15 (δ 47.4), C-14-CH
3distant relation between (δ 16.0), so just can add up in bar structure fragment C connection, and fragment C is:
Referring again to Fig. 7, in the HMBC of compound 1 collection of illustrative plates, visible H-15a (δ 3.45), H-15b (δ 3.15) and C-13 (δ 124.5), C-14 (δ 140.1), C-16 (δ 140.2), C-17 (δ 122.3), C-21 (δ 113.2), C-14-CH
3(δ 16.0); H-17 (δ 6.71) and C-15 (δ 47.4), C-18 (δ 142.7), C-19 (δ 119.5), C-21 (δ 113.2); H-21 (δ 6.77) and C-15 (δ 47.4), C-16 (δ 140.2), C-17 (δ 122.3), C-19 (δ 119.5), C-20 (δ 156.2); H-20-CH
3(δ 3.93) and C-20 (δ 156.2); H-18-NCH
3(δ 3.11) and C-1 (δ 169.0), distant relation between C-18 (δ 142.7), so just can add up in bar structure fragment D connection, and fragment D is:
By Segment A, fragment B, the shared carbon atom of fragment C and fragment D just can couple together the skeleton of compound 1, so just infers the two dimensional structure of compound, and details are referring to Fig. 9.
By comparative compound 1 and document (Kupchan S M, Sneden A T, Branfman A T, et al.Structural requirements for antileukemic activity among the naturally occurring and semisynthetic maytansinoid.[J] J Med Chem, 1978,21:31-37) the ansamitocin P-2 of middle report is ansamitocin P-2's
1h-NMR data, find that compound 1 is closely similar with the nuclear magnetic data of ansamitocin P-2, but compound more than 1 one-OCH
3(δ H3.32, s; δ C52.2) signal, and the J of compound 1
h-5 ,-6(9.8), J
h-10 ,-11(9.1) substantially identical with the data of ansamitocin P-2, illustrate that this compound 1 has identical relative configuration with AMSA compound, therefore, determine that the configuration of this compound 1 is as shown in the structure of the compound 1 in Fig. 9.
Described compound 2 is 9-methoxyl group-ansamitocin P-4, and its molecular formula is C
34h
47clN
2o
9, molecular weight is 662.Compound 2 is faint yellow unsetting powder,
be soluble in the organic solvents such as methyl alcohol, acetone, ethyl acetate, trichloromethane, meet bismuth potassium iodide and show orange-yellow, meet iodine vapor and show brown, under ultraviolet lamp, (254nm) fluorescence is blackening point.The ultraviolet absorption peak of compound 2 is respectively 281.00 (seeing Fig. 2), 250.40 (seeing Fig. 2), 230.80 (seeing Fig. 2), 203.60 (seeing Fig. 2).The ESI-MS spectrum of compound 2 shows molecular ion peak [M] at m/z663.3 place
+, the isotropic substance m/z663.3 of characteristic ion bunch and the abundance ratio of m/z665.3 are 3:1, tentatively infer that this compound 2 contains a chlorine element, its molecular weight is 662.The quasi-molecular ions of m/z663.3 is carried out to second order ms scanning, obtain m/z and be 561.1 feature fragment ion peak.High resolution mass spectrum (HR-ESI-MS): [M+Na]
+measured value is 685.2862; Theoretical value is 685.2867, and supposition molecular formula is C
34h
47n
2o
9cl, degree of unsaturation is 12.
Compound 2
1h NMR (CDCl
3, 600MHz) data are as follows: 0.78 (3H, s, H-4-CH
3), 0.95 (3H, d, J=6.1Hz, H-3 ' b-CH
3), 0.98 (3H, d, J=6.4Hz, H-3 ' a-CH
3) 1.17 (1H, brd, H-8b), 1.19 (1H, d, J=6.9Hz, H-6-CH
3), 1.37 (1H, m, H-6), 1.58 (1H, brd, H-8a), 1.61 (3H, brs, H-14-CH
3), 2.12 (1H, dd, J=4.0,13.4Hz, H-2b), 2.14 (1H, m, H-3 '), 2.15 (1H, d, J=8.6Hz, H-2 ' b), 2.36 (1H, d, J=8.6Hz, H-2 ' a), 2.45 (1H, dd, J=12.0,13.4Hz, H-2a), 2.79 (1H, d, J=9.8Hz, H-5), 3.08 (3H, s, H-18-NCH
3), 3.17 (1H, d, J=12.8Hz, H-15b), 3.23 (3H, s, H-10-OCH
3), 3.32 (3H, s, H-9-OCH
3), 3.37 (1H, d, J=9.1Hz, H-10), 3.45 (1H, d, J=12.8Hz, H-15a), 3.93 (3H, s, H-20-OCH
3), 4.07 (1H, m, H-7), 4.88 (1H, dd, J=4.0,12.0Hz, H-3), 5.47 (1H, dd, J=9.1,15.3Hz, H-11), 6.09 (1H, d, J=10.6Hz, H-13), 6.31 (1H, brs, H-9-NHCO), 6.33 (1H, dd, J=10.6,15.3Hz, H-12), 6.75 (1H, s, H-17), 6.77 (1H, s, H-21).Make a concrete analysis of as follows: compound 2
1h-NMR (CDCl
3, 600MH
z) in spectrum, at high field region, show 5 groups of methyl hydrogen signals, wherein there are 3 groups of bimodal methyl hydrogen signal (δ
h1.19 (3H, d, J=6.9Hz), δ
h0.98 (3H, d, J=6.4Hz), δ
h0.95 (3H, d, J=6.1Hz)) show that it is directly connected with tertiary carbon; 2 unimodal methyl hydrogen signal (δ
h0.78 (3H, brs), δ
h1.61 (3H, brs)) show that it is connected with quaternary carbon; 3 methoxyl group hydrogen signal (δ
h3.23 (3H, s), δ
h3.32 (3H, s), δ
h3.93 (3H, s)) and 1 n-formyl sarcolysine base hydrogen signal (δ
h3.08 (3H, s)).There are 5 and sp in low place
2the connected hydrogen signal of carbon atom of hydridization, wherein 3 is 2 couples of trans hydrogen signal (δ that are connected on two keys
h5.47 (1H, dd, J=9.1,15.3Hz, H-11), δ
h6.33 (1H, dd, J=10.6,15.3Hz, H-12), δ
h6.09 (1H, d, J=10.6Hz, H-13)); 2 phenyl hydrogen signal (δ
h6.77 (1H, s), δ
h6.75 (1H, s)).
Compound 2
13c NMR (CDCl
3, 150MHz) data are as follows: 171.7 (C-1 '), 168.8 (C-1), 156.2 (C-20), 152.9 (C-9-NHCO), 142.7 (C-18), 140.2 (C-16), 140.0 (C-14), 132.5 (C-12), 128.1 (C-11), 124.6 (C-13), 122.2 (C-17), 119.6 (C-19), 113.2 (C-21), 91.0 (C-10), 83.4 (C-9), 77.0 (C-3), 75.2 (C-7), 66.5 (C-5), 60.6 (C-4), 57.4 (C-10-OCH
3), 56.8 (C-20-OCH
3), 52.0 (C-9-OCH
3), 47.5 (C-15), 43.7 (C-2 '), 38.5 (C-6), 36.8 (C-8), 35.8 (C-18-NCH
3), 32.9 (C-2), 26.1 (C-3 '), 22.9 (C-3 ' a-CH
3), 22.7 (C-3 ' b-CH
3), 16.0 (C-14-CH
3), 14.6 (C-6-CH
3), 12.4 (C-4-CH
3).Make a concrete analysis of as follows: compound 2
13c-NMR (CDCl
3, 150MH
z) (DETP) in collection of illustrative plates, show altogether 34 spectral lines, comprising 10 quaternary carbon signals, (10 quaternary carbon signals are specially: 1 amide group carbon signal (δ
c168.8), 1 amide oxygen base carbon signal (δ
c152.9), 1 ester group carbon signal (δ
c171.7), 4 phenyl ring carbon signal (δ
c119.6, δ
c140.2, δ
c142.7, δ
c156.2), 1 olefinic carbon signal (δ
c140.0), 2 carbon signal (δ that are connected with Sauerstoffatom
c60.6, δ
c83.4)), (11 methine carbon signals are specially 11 methine carbon signals: 3 olefinic carbon signal (δ
c124.6, δ
c128.1, δ
c132.5), 2 phenyl ring carbon signal (δ
c113.2, δ
c122.2), 4 carbon signal (δ that are connected with Sauerstoffatom
c66.5, δ
c91.0, δ
c77.0, δ
c75.2) and 2 sp
3carbon signal (the δ of hydridization
c38.5, δ
c26.1)), 4 is mesomethylene carbon signal (δ
c43.7, δ
c32.9, δ
c36.8, δ
c47.5), 3 is methoxyl group carbon signal (δ
c52.0, δ
c57.4, δ
c56.8), 1 is n-formyl sarcolysine base carbon signal (δ
c35.8), 5 methyl carbon signal (δ
c12.4, δ
c14.6, δ
c16.0, δ
c22.9, δ
c22.7).
Refer to Fig. 4, Fig. 4 is compound 2
1h-
1in H COSY spectrum, visible δ 2.12 (H-2b), δ 2.45 (H-2a) are all relevant to δ 4.88 (H-3); δ 2.36 (H-2 '
a)-δ 2.14 (H-3 ')-δ 0.98 (H-3 '
a-CH
3), relevant in turn between δ 2.79 (H-5)-δ 1.37 (H-6)-δ 4.07 (H-7)-δ 1.17 (H-8b), relevant in turn between δ 3.37 (H-10)-δ 5.47 (H-11)-δ 6.33 (H-12)-δ 6.09 (H-13), in addition, can also see δ 1.37 (H-6)-δ 1.19 (H-6-CH
3) between relevant, the HSQC collection of illustrative plates (seeing Fig. 6) of binding compounds 2, can contain following carbon skeleton fragment: C in pushing-out structure
2-C
3, C
2 '-C
3 '-C
3 ' a-CH3, C
5-C
6-C
7-C
8, C
10-C
11-C
12-C
13.
Referring again to Fig. 8, Fig. 8 is in the HMBC collection of illustrative plates of compound 2, visible H-2a (δ 2.45) and C-1 (δ 168.8), C-3 (δ 77.0), C-4 (δ 60.6); H-2b (δ 2.12) and C-1 (δ 168.8); H-3 (δ 4.88) and C-2 (δ 32.9), C-4 (δ 60.6), C-1 ' (δ 171.7), C-4-CH
3(δ 12.4); H-2 '
a(δ 2.36) and H-2 '
b(δ 2.15) all and C-1 ' (δ 171.7), C-3 ' (δ 26.1), C-3 ' a-CH
3(δ 22.9), C-3 ' b-CH
3(δ 22.7); H-3 ' (δ 2.14) and C-2 ' (δ 43.7), C-3a '-CH
3(δ 22.9), C-3 ' b-CH
3distant relation between (δ 22.7), so just can add up in the connection of bar structure Segment A, and Segment A is:
Referring again to Fig. 8, in the HMBC collection of illustrative plates of compound 2, visible H-5 (δ 2.79) and C-3 (δ 77.0), C-6 (δ 38.5), C-7 (δ 75.2), C-6-CH
3(δ 14.6); H-6 (δ 1.37) and C-5 (δ 66.5), C-7 (δ 75.2); H-6-CH
3(δ 1.19) and C-5 (δ 66.5), C-6 (δ 38.5); H-8a (δ 1.58) and C-9 (δ 83.4); H-8b (δ 1.17) and C-6 (δ 38.5), C-7 (δ 75.2), C-10 (δ 91.0); H-9-OCH
3(δ 3.32) and C-9 (δ 83.4); Distant relation between H-9-NHCO (δ 6.31) and C-8 (δ 36.8), C-9-NHCO (δ 152.9), so just can add up in bar structure fragment B connection, and fragment B is:
Referring again to Fig. 8, in the HMBC collection of illustrative plates of compound 2, visible H-10 (δ 3.37) and C-9 (δ 83.4), C-12 (δ 132.5); H-11 (δ 5.47) and C-13 (δ 124.6); H-12 (δ 6.33) and C-10 (δ 91.0), C-13 (δ 124.6), C-14 (δ 140.0); H-13 (δ 6.09) and C-11 (δ 128.1), C-12 (δ 132.5), C-15 (δ 47.5), C-14-CH
3distant relation between (δ 16.0), so just can add up in bar structure fragment C connection, and fragment C is:
Referring again to Fig. 8, in the HMBC collection of illustrative plates of compound 2, visible H-17 (δ 6.75) and C-15 (δ 47.5), C-18 (δ 142.7), C-19 (δ 119.6), C-21 (δ 113.2); H-21 (δ 6.77) and C-15 (δ 47.5), C-16 (δ 140.2), C-17 (δ 122.2), C-19 (δ 119.6), C-20 (δ 156.2); H-20-CH
3(δ 3.93) and C-20 (δ 156.2); H-18-NCH
3(δ 3.08) and C-18 (δ 142.7), C-1 (δ 168.8); H-15
a(δ 3.45), H-15
b(δ 3.17) and C-13 (δ 124.6), C-14 (δ 140.0), C-16 (δ 140.2), C-17 (δ 122.2), C-21 (δ 113.2), C-14-CH
3distant relation between (δ 16.0), so just can add up in bar structure fragment D connection, and fragment D is:
By Segment A, fragment B, the shared carbon atom of fragment C and fragment D just can couple together the skeleton of compound 2, so just infers the two dimensional structure of compound, and details are referring to Figure 10.
By comparative compound 2 and document (reference: [1] Suwanborirux K, Chang CJ, Spjut RW, et al.Ansamitocin P-3, a maytansinoid, from Claopodium crispifolium and Anomodon attenuatus or associated actinomycetes[J] .Experientia, 1990,46:117-120; [2] Higashide E, Asai M, Ootsu K, et al.Ansamitocin, a group of novel maytansinoid antibiotics with antitumour properties from Nocardia[J] .Nature, 1977,270:721-722; [3] Kupchan S M, Sneden A T, Branfman A T, et al.Structural requirements for antileukemic activity among the naturally occurring and semisynthetic maytansinoid.[J] J Med Chem, 1978,21:31-37; [4] Izawa M, Tanida S, Asai M.Ansamitocin analogs from a mutant strain of Nocardia II .isolation and structure[J] .J Antibiot, 1981,34 (5): the ansamitocin compounds of reporting 496-506.)
1h-NMR data, find that compound 2 is closely similar with the nuclear magnetic data of ansamitocin compounds.But more than 2 one-OCH of compound
3(δ
h3.32, s; δ
c52.0) signal, and J
h-5 ,-6(9.8), J
h-10 ,-11(9.1) substantially identical with the data of ansamitocin compounds, illustrate that this compound 2 has identical relative configuration with ansamitocin compounds, therefore, determine that the configuration of this compound 2 is as shown in the structure of the compound 2 in Figure 10.
Compound (compound 1 or compound 2) is carried out to inhibiting rate determination experiment to HL60 cell, investigate the half-inhibition concentration of chemical combination 1 and compound 2, its experimental procedure is as follows:
(1) preparation of cell culture fluid: add 50ml foetal calf serum, the dual anti-solution of the blue or green chain of 5ml in the RPMI1640 of 450ml solution.Wherein, the manufacturer of described RPMI1640 solution, foetal calf serum, the dual anti-solution of blue or green chain is Sai Mo and flies generation that biological chemistry goods (Beijing) company limited.In the dual anti-solution of described blue or green chain, the concentration of penicillin is 10000IU/ml, and the concentration of Streptomycin sulphate is 10000 μ g/ml.
(2) cultivation of HL60 cell: the cell culture fluid that HL60 cell (being people's acute myeloid leukemia cells in children) is obtained by step (1) at 37 ℃, 5%CO
2in incubator, cultivate; Then get HL60 logarithmic phase cell on 96 well culture plates, wherein, the concentration of HL60 logarithmic phase cell is 2 * 10
5individual/ml, every hole solution is 180 μ l.
(3) compound (being compound 1 or compound 2) is dissolved in dimethyl sulfoxide (DMSO) (being DMSO), being made into concentration is 1 * 10
-3the solution of mol/L is storage liquid (solute of storage liquid is compound 1 or compound 2).
(4) cell culture fluid of step for storage liquid (1) gained step (3) being obtained is diluted to the solution of 5 kinds of concentration, the i.e. compound of 5 kinds of concentration (compound 1 or compound 2) solution, the concentration of compound (compound 1 or compound 2) is respectively 500 * 10
-6mol/L, 1000 * 10
-6mol/L, 2000 * 10
-6mol/L, 3000 * 10
-6mol/L, 4000 * 10
-6mol/L, 5000 * 10
-6mol/L.
(5) establish 6 experimental group and a control group, 6 experimental group are:
Experimental group 1: add 500 * 10 of 20 μ l in the culture plate that HL60 logarithmic phase cell is housed of step (2) gained
-6the compound solution of mol/L, and experimental group 1 is established three parallel holes;
Experimental group 2: add 1000 * 10 of 20 μ l in the culture plate that HL60 logarithmic phase cell is housed of step (2) gained
-6the compound solution of mol/L, and experimental group 2 is established three parallel holes;
Experimental group 3: add 2000 * 10 of 20 μ l in the culture plate that HL60 logarithmic phase cell is housed of step (2) gained
-6the compound solution of mol/L, and experimental group 3 is established three parallel holes;
Experimental group 4: add 3000 * 10 of 20 μ l in the culture plate that HL60 logarithmic phase cell is housed of step (2) gained
-6the compound solution of mol/L, and experimental group 4 is established three parallel holes;
Experimental group 5: add 4000 * 10 of 20 μ l in the culture plate that HL60 logarithmic phase cell is housed of step (2) gained
-6the compound solution of mol/L, and experimental group 5 is established three parallel holes;
Experimental group 6: add 5000 * 10 of 20 μ l in the culture plate that HL60 logarithmic phase cell is housed of step (2) gained
-6the compound solution of mol/L, and experimental group 6 is established three parallel holes.
The final concentration of above-mentioned experimental group compound (compound 1 or compound 2) is respectively 50 * 10
-6mol/L, 100 * 10
-6mol/L, 200 * 10
-6mol/L, 300 * 10
-6mol/L, 400 * 10
-6mol/L, 500 * 10
-6mol/L.
Control group is: toward the cell culture fluid that adds 20 μ l step (1) gained in the culture plate that HL60 logarithmic phase cell is housed of step (2) gained, and control group is established three parallel holes.
(6) by the culture plate after step (5) is processed at 37 ℃, 5%CO
2in incubator, cultivate 48h.
(7) preparation of working fluid: be 3-(4,5-dimethylthiazole-2)-2 by 50mg MTT[, 5-phenylbenzene tetrazole bromine salt] (manufacturer of MTT is SIGMA company) dissolve in the phosphate buffered saline buffer of 10ml, is made into the working fluid of 5mg/ml.
(8) toward the culture plate of processing through step (6), in every hole, add the above-mentioned working fluid of 20 μ l, then put culture plate in 37 ℃, 5%CO
2in incubator, cultivate 4h.Then, with the centrifugal culture plate of dull and stereotyped whizzer, carefully with vacuum pump, inhale and abandon supernatant liquor, then in every hole, add the methyl-sulphoxide of 150 μ l, finally by microplate reader, at 570nm wavelength place, detect the light absorption value in every hole.
Inhibitory rate of cell growth=(control group light absorption value-experimental group light absorption value)/control group light absorption value * 100%
Compound (compound 1 or compound 2) to the concrete outcome of HL60 cell inhibitory rate determination experiment in Table 1 and table 2.
1 pair of HL60 cell inhibitory rate determination experiment result of table 1 compound
2 pairs of HL60 cell inhibitory rate determination experiment results of table 2 compound
Different concns cell growth inhibiting rate mapping with same compound (compound 1 or compound 2), can obtain the dose response curve of compound (compound 1 or compound 2) to HL60 cyto-inhibition, as shown in Figure 11-12.According to IC
50counter is obtained the half-inhibition concentration IC of this compound (compound 1 or compound 2)
50, drug level when cell survival rate reduces 50%.Wherein, the IC of compound 1
50be 72.68 * 10
-6mol/L, the IC of compound 2
50be 119.33 * 10
-6mol/L, hence one can see that, and compound 1 and compound 2 have the very strong anti-tumor activity that has.Document (reference: Guo-Zhu Wei; Lin-Quan Bai; Tao Yang; Juan Ma; Ying Zeng; Yue-Mao Shen; Pei-Ji Zhao.A new antitumour ansamitocin from Actinosynnema pretiosum[J] .Natural Product Research Vol.24, No.12,20July2010,1146 – 1150) IC of the compound N-demethyl-desepoxy-9-methoxy-maytansinol of report to HL-60
50data (IC
50be 0.12 μ M) close with compound 2, both are the same to the influential key position of activity.
The present invention screens a strain synnema actinomycetes bacterial strain FIM06-0063 from Caulis Mayteni callus, utilize its growing environment unique, by microorganism, ferment and multiple separation purification method, from this bacterial strain FIM06-0063 fermented liquid, it is compound 1 and compound 2 that separation obtains two ansamitocin compounds, for research and development antitumor drug provides Natural Medicine Chemistry Research foundation and lead compound, and compound 1 and compound 2 likely become for targeted molecular coupling formation target mixtures such as the bullet molecule of target therapeutic agent and antibody or parts.Ansamitocin compounds can be reduced into C-3 Ansamitocin Po, and this alcohol is the precursor for the synthesis of sulfur-bearing alcohol maytansinoid.In addition, the present invention prepares ansamitocin compounds by microbial fermentation processes, has advantages of that technique is simple, production cost is low.
Claims (5)
1. the ansamitocin compounds that formula (Ι) represents,
Wherein, R is CH
2cH
3or CH
2cH (CH
3)
2.
2. a preparation method for ansamitocin compounds as claimed in claim 1, is characterized in that: its operation steps is as follows:
Step 1: the separation method of synnema actinomycetes bacterial strain FIM06-0063 (Actinosynnema sp):
A: get Lao Ye and the petiole of Yunnan Caulis Mayteni, rinse after 30min with tap water, then clean and be placed in sterile petri dish with distilled water; Then by described Lao Ye and petiole successively with 75% alcohol scouring, after aseptic water washing, proceed to massfraction and be in 0.1% mercuric chloride solution and soak 3min, then use aseptic water washing 5 times, remove thimerosal; Then described Lao Ye and petiole are cut into small pieces or segment, put into aseptic mortar and smash into mashed prod to pieces;
B: pick with aseptic glass spreading rod the mashed prod obtaining in a small amount of steps A and directly coat starch asparagine agar plate, then flat board is placed at 28 ℃ and cultivates 7～20 days, obtain the single bacterium colony of actinomycetes; The component of described starch asparagine agar: Zulkovsky starch 2%, L-asparagine 0.05%, KNO
30.1%, K
2hPO
43H
2o0.05%, NaCl0.05%, MgSO
47H
2o0.05%, CaCO
30.05%, agar 1.5%, distilled water preparation, pH is 7.5, and the percentage ratio in this starch asparagine agar component is massfraction;
C: transfer in inorganic salt Starch Agar inclined-plane process the single bacterium colony of the actinomycetes that obtain through step B, obtain the slant culture of synnema actinomycetes bacterial strain FIM06-0063; The component on described inorganic salt Starch Agar inclined-plane is: Zulkovsky starch 10g; K
2hPO
43H
2o1g; MgSO
47H
2o1g; NaCl1g; (NH
4)
2sO
42g; CaCO
32g; FeSO
47H
2o0.001g; MnCl
24H
2o0.001g; ZnSO
47H
2o0.001g; Agar 20g; Distilled water 1000mL, pH is 7.0～7.4;
Step 2: prepare synnema actinomycetes bacterial strain FIM06-0063 (Actinosynnema sp) fermented liquid, be specially:
(1) prepare seed liquor: will after synnema actinomycetes bacterial strain FIM06-0063 slant culture maturation, be inoculated in seed culture medium, and be placed on shaking culture 40～48h in constant-temperature table, 28 ℃ of temperature, rotating speed 220r/min;
(2) prepare fermented liquid: the seed liquor that the culture transferring amount by 10% obtains step (1) is transferred and cultivated 96～120h, 28 ℃ of temperature, rotating speed 220r/min into the fermention medium of sterilization;
Step 3: the synnema actinomycetes bacterial strain FIM06-0063 fermented liquid that step 2 is obtained carries out macroporous resin adsorption column chromatography, obtains crude extract, is specially:
By synnema actinomycetes bacterial strain FIM06-0063 fermented liquid, at rotating speed, be centrifugal 15min under 3000r/min, and get supernatant liquor; Then supernatant liquor is splined on to macroporous adsorbent resin, then obtain mixed solution by methanol-eluted fractions; Mixed solution is removed methyl alcohol to obtain concentrated solution through concentrating under reduced pressure; Concentrated solution is used with the ethyl acetate of this concentrated solution equal volume and extracted three times, then combined ethyl acetate extraction liquid; Acetic acid ethyl acetate extract arrives to obtain crude extract after concentrating under reduced pressure;
Step 4: the crude extract that step 3 is obtained carries out purification on normal-phase silica gel column chromatography, and adopt volume ratio 100:0,98:2,95:5,90:10,85:15,80:20,70:30,60:40, the chloroform-methanol solvent systems of 50:50 carries out gradient elution to crude extract, then each elutriant of Fractional Collections;
Step 5: each elutriant that step 4 is obtained carries out respectively thin-layer chromatography, and adopt high performance liquid chromatography to detect respectively analysis to each elutriant, the elutriant then aobvious orange red and high performance liquid chromatography detected result of thin-layer chromatography result to identical peak merges, and the elutriant after this merging of concentrating under reduced pressure obtains the first enriched material;
Wherein, the developing agent of thin-layer chromatography is trichloromethane-methanol solvate of volume ratio 15:1, and developer is bismuth potassium iodide developer; High performance liquid chromatography testing conditions is: moving phase is the water-methanol containing 0.1% trifluoroacetic acid of volume ratio 35:65, wash-out 20min, and flow velocity lmL/min, detects wavelength 254nm, 40 ℃ of column temperatures;
Step 6: the first enriched material that step 5 is obtained carries out middle pressure reversed-phase silica gel column chromatography to obtain crude product, while carrying out middle pressure reversed-phase silica gel column chromatography, first adopting volume fraction is that 10% methanol aqueous solution is that moving phase balance is after 10 minutes, then moving phase adopts the methanol-water solvent systems gradient elution 90min of volume ratio 10%～100%:1, and the flow velocity of moving phase is 5ml/min;
Step 7: the crude product that step 6 is obtained carries out Sephadex LH-20 gel filtration chromatography, described Sephadex LH-20 gel filtration chromatography adopts the chloroform-methanol solvent systems wash-out of volume ratio 1:1, and according to flow point and each flow point of bismuth potassium iodide color reaction Fractional Collections, then the flow point identical with bismuth potassium iodide reaction solution merged, concentrating under reduced pressure is to obtain the second enriched material;
Step 8: the second enriched material that step 7 is obtained adopts preparative high performance liquid chromatography to carry out separation, obtains 3 components; Finally, 3 components are passed through respectively to the separation of preparative silica-gel plate to obtain ansamitocin compounds; The separation condition of described preparative high performance liquid chromatography is: the methanol aqueous solution that moving phase is 70%, and the flow velocity of moving phase is 1.5ml/min, detection wavelength is 254nm.
3. the preparation method of ansamitocin compounds as claimed in claim 2, is characterized in that:
The component of described seed culture medium: Zulkovsky starch 1.5%, glucose 0.5%, analysis for soybean powder 1%, sodium-chlor 0.1%, peptone 0.5%, yeast extract 0.5%, calcium carbonate 0.5%, pH is 7.0, and the percentage ratio in this seed culture medium component is massfraction.
4. the preparation method of ansamitocin compounds as claimed in claim 2, is characterized in that:
The component of described fermention medium: dextrin 1%, glycerine 3%, analysis for soybean powder 2%, Dried Corn Steep Liquor Powder 1%, potassium primary phosphate 0.05%, ammonia chloride 0.05%, calcium carbonate 0.5%, pH is 7.0, and the percentage ratio in this fermention medium component is massfraction.
5. the preparation method of ansamitocin compounds as claimed in claim 2, it is characterized in that: described synnema actinomycetes bacterial strain FIM06-0063 (Actinosynnema sp), on December 30th, 2011, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC NO.5677.
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