CN102731526A - 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 new ansamitocin compounds and a preparation method thereof. A structural formula of the new ansamitocin compound is 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 new 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 new ansamitocin compounds, the invention still further relates to the preparation method of this ansamitocin compounds.
[background technology]
Kupchan in 1972 etc. isolate the natural antitumor activeconstituents first from the tingia leaf of Hooker's Mayten (M.serrata) that originates in Tropical Africa---and macrolides compound maytenin (Maytansine), the researchist defends from the higher plant lance in succession and is separated to the leaf of Hooker's Mayten compounds section, Rhamnaceae, euphorbia plant, the bryophyte thereafter.The most attractive is from mikrobe, to have found this compounds; 1977; Higashide and colleague thereof are in the process of screening anticancer active constituent; From the orange synnema actinomycetes (Actinosynnema pretiosum) of Cyperaceae carex leaf, obtain six maytenin verivates, called after ansamitocin P0, P1, P2, P3, P4, P4 ' from separating.From another orange synnema actinomycetes subspecies (A pretiosum ssp.Auranticum), separate again subsequently and obtain 15 new ansamitocins except that above-mentioned six compounds; 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 that is used for synthetic sulfur-bearing alcohol maytansinoid.Ansamitocin has been become to contain the maytansinoid (maytansinoids) of mercaptan by chemical transformation, this vegeto-alkali is in the news with the medical use that the cell node mixture-form body of maytansinoid conjugate reveals.
Deepen continuously along with what the CHROMATOGRAPHIC FRACTIONATION AND MASS antibody conjugates was studied, market progressively enlarges the demand of ansamitocin compounds.The content of maytenin and homologue thereof is very low because leaf of Hooker's Mayten class plant contains, and cutting down leaf of Hooker's Mayten class plant in a large number can do great damage to ecology, and the searching raw material is difficult to satisfy industrial needs from wild plant; Adopt chemosynthesis, exist complete synthesis because of the ansamitocin complex structure again and shortcomings such as semi-synthesizing technology is complicated, process is loaded down with trivial details, productive rate is very low, reaction specificity difference.
[summary of the invention]
One of technical problem to be solved by this invention is to provide a kind of new ansamitocin compounds, and said new ansamitocin compounds has anti-tumor activity.
The present invention one of solves the problems of the technologies described above through following technical scheme: the new ansamitocin compounds of formula (I) 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 new ansamitocin compounds, prepares the ansamitocin compounds through microbial fermentation processes and has the advantage that technology is simple, production cost is low.
The present invention through following technical scheme solve the problems of the technologies described above two: a kind of preparation method of described new ansamitocin compounds, its operation steps is following:
Step 1: preparation synnema actinomycetes bacterial strain FIM06-0063 (Actinosynnema sp) fermented liquid; Said synnema actinomycetes bacterial strain FIM06-0063 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on December 30th, 2011, and the 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 obtained is carried out the macroporous resin adsorption column chromatography, obtain crude extract;
Step 3: the crude extract that step 2 obtained is carried out the purification on normal-phase silica gel column chromatography, and adopt chloroform-methanol gradient elution crude extract, each elutriant of Fractional Collections then;
Step 4: each elutriant that step 3 obtained is carried out thin-layer chromatography respectively; And adopt performance liquid chromatography that each elutriant is carried out check and analysis respectively; Then the thin-layer chromatography result is shown the elutriant that orange red and performance liquid chromatography detected result has identical peak and merge, and the elutriant after this merging of concentrating under reduced pressure obtains first enriched material;
Step 5: first enriched material that step 4 obtained is carried out the middle reversed-phase silica gel column chromatography of pressing to obtain crude product; When pressing reversed-phase silica gel column chromatography in carrying out; Adopting volume(tric)fraction earlier is that 10% methanol aqueous solution is that the moving phase balance is after 10 minutes; Moving phase adopts the methanol-water solvent systems gradient elution 90min of volume ratio 10%～100%: 1 then, and the flow velocity of moving phase is 5ml/min;
Step 6: the crude product that step 5 obtained is carried out Sephadex LH-20 gel filtration chromatography; Said Sephadex LH-20 gel filtration chromatography adopts 1: 1 chloroform-methanol solvent systems wash-out of volume ratio; And according to flow point and each flow point of bismuth potassium iodide coupling reaction Fractional Collections; Flow point that then will be identical with the bismuth potassium iodide reaction solution merges, and concentrating under reduced pressure is to obtain second enriched material;
Step 7: adopt preparative high performance liquid chromatography to separate second enriched material that step 6 obtained, obtain 3 components; At last, 3 components are separated to obtain the ansamitocin compounds through preparation type silica-gel plate respectively; The separation condition of said preparative high performance liquid chromatography is: moving phase is 70% methanol aqueous solution, and the flow velocity of moving phase is 1.5ml/min, and the detection wavelength is 254nm.
Further, the separation method of said synnema actinomycetes bacterial strain FIM06-0063 is following:
Step 100: get the Lao Ye and the petiole of Yunnan leaf of Hooker's Mayten, behind tap water flushing 30min, clean with zero(ppm) water again and place sterile petri dish; Then with said Lao Ye and petiole successively with the scouring of 75% alcohol, behind the aseptic water washing, change massfraction over to and be in 0.1% the mercuric chloride solution and soak 3min, use aseptic water washing again 5 times, remove thimerosal; Then said 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 the mashed prod that obtains in the few steps 100 with aseptic glass spreading rod and directly coat starch asparagine agar plate, place 28 ℃ to cultivate 7～20 days down flat board then, obtain the single bacterium colony of actinomycetes; The component of said starch asparagine agar: Zulkovsky starch 2%, L-asparagine 0.05%, KNO
30.1%, K
2HPO
43H
2O 0.05%, and NaCl 0.05%, MgSO
47H
2O 0.05%, CaCO
30.05%, agar 1.5%, the zero(ppm) water preparation, pH is 7.5, and the percentage ratio in this starch asparagine agar component is massfraction;
Step 300: the single bacterium colony of actinomycetes that will pass through step 200 processing acquisition is transferred in inorganic salt Starch Agar inclined-plane, obtains the slant culture of synnema actinomycetes bacterial strain FIM06-0063; The component on said inorganic salt Starch Agar inclined-plane is: Zulkovsky starch 10g; K
2HPO
43H
2O 1g; MgSO
47H
2O 1g; NaCl 1g; (NH
4)
2SO
42g; CaCO
32g; FeSO
47H
2O 0.001g; MnCl
24H
2O 0.001g; ZnSO
47H
2O 0.001g; Agar 20g; Zero(ppm) water 1000mL, pH are 7.0～7.4.
Further, said step 1 is specially:
(1) preparation seed liquor: with being inoculated in the seed culture medium after the synnema actinomycetes bacterial strain FIM06-0063 slant culture maturation, and be placed on shaking culture 40～48h in the constant temperature shaking table, 28 ℃ of temperature, rotating speed 220r/min;
(2) preparation fermented liquid: the seed liquor that the culture transferring amount by 10% is obtained step (1) is transferred into the fermention medium cultivation 96～120h of sterilization, 28 ℃ of temperature, rotating speed 220r/min.
Further, the component of said 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%; Lime carbonate 0.5%, pH are 7.0, and the percentage ratio in this seed culture medium component is massfraction.
Further, the component of said 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%; Lime carbonate 0.5%, pH are 7.0, and the percentage ratio in this fermention medium component is massfraction.
Further, said step 2 is specially: is centrifugal 15min under the 3000r/min with synnema actinomycetes bacterial strain FIM06-0063 fermented liquid at rotating speed, and gets supernatant; Then supernatant is splined on macroporous adsorbent resin, obtains mixed solution with methanol-eluted fractions again; Mixed solution is removed methyl alcohol to obtain liquid concentrator through concentrating under reduced pressure; Liquid concentrator is used and the ethyl acetate extraction of this liquid concentrator equal volume three times, then the combined ethyl acetate extraction liquid; Acetic acid ethyl acetate extract behind concentrating under reduced pressure, arrive crude extract.
Further, the chloroform-methanol gradient elution crude extract in the said step 3 is meant that 98: 2,95: 5,90: 10,85: 15,80: 20,70: 30,60: 40,50: 50 chloroform-methanol solvent systems carried out gradient elution to crude extract with volume ratio 100: 0.
Further, the developing agent of the thin-layer chromatography in the said step 4 is trichloromethane-methanol solvate of 15: 1 of volume ratio, and developer is the bismuth potassium iodide developer.
Further, the performance liquid chromatography testing conditions in the said step 4 is: moving phase is 35: 65 the water-methanol that contains 0.1% trifluoroacetic acid of volume ratio, wash-out 20min, and flow velocity 1mL/min detects wavelength 254nm, 40 ℃ of column temperatures.
Further, said synnema actinomycetes bacterial strain FIM06-0063 (Actinosynnema sp) is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on December 30th, 2011, and deposit number is CGMCC NO.5677.
Beneficial effect of the present invention is: the new ansamitocin compounds that the present invention makes has anti-tumor activity; The present invention prepares the ansamitocin compounds through microbial fermentation processes, has the advantage that technology is simple, production cost is low.
[description of drawings]
Combine embodiment that the present invention is done further description with reference to the accompanying drawings.
Fig. 1 is the uv absorption spectra of compound 1 among the present invention.
Fig. 2 is the uv absorption spectra of compound 2 among the present invention.
Fig. 3 is for compound 1 among the present invention
1H-
1H COSY spectrogram.
Fig. 4 is for compound 2 among the present invention
1H-
1H COSY spectrogram.
Fig. 5 is the hsqc spectrum figure of compound 1 among the present invention.
Fig. 6 is the hsqc spectrum figure of compound 2 among the present invention.
Fig. 7 is the HMBC spectrogram of compound 1 among the present invention.
Fig. 8 is the HMBC spectrogram of compound 2 among the present invention.
Fig. 9 is the structure elucidation procedure chart of compound 1 among the present invention.
Figure 10 is the structure elucidation procedure chart of compound 2 among the present invention.
Figure 11 is the dose response curve figure of 1 pair of HL60 cyto-inhibition of compound among the present invention.
Figure 12 for 2 pairs of HL60 cyto-inhibitions of compound among the present invention dose response curve figure.
The 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 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 among the present invention is following:
Step 100: get the Lao Ye and the petiole of Yunnan leaf of Hooker's Mayten, behind tap water flushing 30min, clean with zero(ppm) water again and place sterile petri dish; Then said Lao Ye and petiole are cleaned with 75% alcohol successively, behind the aseptic water washing, the mercury chloride that changes massfraction 0.1% over to (is HgCl
2) soak 3min in the solution, use aseptic water washing again 5 times, remove thimerosal; Then said 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 the mashed prod that obtains in the few steps 100 with aseptic glass spreading rod and directly coat starch asparagine agar plate, place 28 ℃ to cultivate 7～20 days down flat board then, obtain the single bacterium colony of actinomycetes; In embodiment 1, place 28 ℃ to cultivate 7 days down flat board; In embodiment 2, place 28 ℃ to cultivate 13 days down flat board; In embodiment 3, place 28 ℃ to cultivate 20 days down flat board.
The component of said starch asparagine agar: Zulkovsky starch 2%, L-asparagine 0.05%, KNO
30.1%, K
2HPO
43H
2O 0.05%, and NaCl 0.05%, MgSO
47H
2O 0.05%, CaCO
30.05%, agar 1.5%, the zero(ppm) water preparation, pH is 7.5, and the percentage ratio in this starch asparagine agar component is massfraction;
Step 300: the single bacterium colony of actinomycetes that will pass through step 200 processing acquisition is transferred in inorganic salt Starch Agar inclined-plane, obtains the slant culture of synnema actinomycetes bacterial strain FIM06-0063; The component on said inorganic salt Starch Agar inclined-plane is: Zulkovsky starch 10g; K
2HPO
43H
2O 1g; MgSO
47H
2O 1g; NaCl 1g; (NH
4)
2SO
42g; CaCO
32g; FeSO
47H
2O 0.001g; MnCl
24H
2O 0.001g; ZnSO
47H
2O 0.001g; Agar 20g; Zero(ppm) water 1000mL, pH are 7.0～7.4.
The evaluation of this bacterial strain FIM06-0063:
The slant culture of synnema actinomycetes bacterial strain FIM06-0063 is lined inorganic salt Starch Agar flat board, and then the oblique cutting deckglass was cultivated 5～20 days down at 28 ℃.In embodiment 1, be employed in 28 ℃ and cultivated 5 days down; In embodiment 2, be employed in 28 ℃ and cultivated 13 days down; In embodiment 3, be employed in 28 ℃ and cultivated 20 days down; Observe gas silk, Ji Si and pigment, take out deckglass with opticmicroscope and electron microscope observation, main form and the biological characteristics of bacterial strain FIM06-0063 are following:
Bacterial strain FIM06-0063 bacterium colony is orange-yellow, on solid medium, forms rhizomorph, produces the gas silk on the surface of rhizomorph top or dome-shaped body; The gas silk is separated and the generation spore chain; Ripe hyphal diameter 0.5-1.2 is a micron, and appearance presents the bamboo shape, forms the shaft-like or oval spore that can move about; Milk peptonizes, the ability hydrolyzed starch, and tyrosine, glucose capable of using, sucrose, wood sugar, seminose are carbon source for growth; Cell walls III type: having only meso-diaminopimelic acid is characteristic component; The sacchariferous type of synnema actinomycetes bacterial strain FIM06-0063 cell walls is: sugared MODEL C, atypism sugar.
The 16SrDNA sequencing result of this bacterial strain FIM06-0063 and GeneBank go up the strains A ctinosynnema pretiosum subsp.Pretiosum ATCC31281 of known array
T(GenBank:AF114800) homology is the highest, reaches 99.82%.Form and physiological and biochemical property, the proximate analysis of cell walls and the mensuration result of 16S rRNA sequence according to bacterial strain FIM06-0063 identify that this bacterial strain is synnema actinomycetes (Actinosynnema sp).
A kind of preparation method of new ansamitocin compounds, its operation steps is following:
Step 1: preparation synnema actinomycetes bacterial strain FIM06-0063 (Actinosynnema sp) fermented liquid, particularly:
(1) preparation seed liquor: with being inoculated in the seed culture medium after the synnema actinomycetes bacterial strain FIM06-0063 slant culture maturation, and be placed on shaking culture 40～48h in the constant temperature shaking table, 28 ℃ of temperature, rotating speed 220r/min.In embodiment 1, adopt to place constant temperature shaking table shaking culture 40h; In embodiment 2, adopt to place constant temperature shaking table shaking culture 44h; In embodiment 3, adopt to place constant temperature shaking table shaking culture 48h.Wherein, the substratum of said slant culture is inorganic salt Starch Agar (being an ISP-4) slant medium; The component of said 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%; Lime carbonate 0.5%, pH are 7.0, and the percentage ratio in this seed culture medium component is massfraction.
(2) preparation fermented liquid: the seed liquor that the culture transferring amount by 10% is obtained step (1) is transferred into the fermention medium cultivation 96～120h of sterilization, 28 ℃ of temperature, rotating speed 220r/min.In embodiment 1, adopting transfers seed liquor cultivates 96h into the fermention medium of sterilization; In embodiment 2, adopting transfers seed liquor cultivates 109h into the fermention medium of sterilization; In embodiment 3, adopting transfers seed liquor cultivates 120h into the fermention medium of sterilization.The component of said 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%, lime carbonate 0.5%, pH are 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 obtained is carried out the macroporous resin adsorption column chromatography; Obtain crude extract; Particularly: is centrifugal 15min under the 3000r/min with synnema actinomycetes bacterial strain FIM06-0063 fermented liquid at rotating speed, and gets supernatant; Then supernatant is splined on macroporous adsorbent resin, obtains mixed solution with methanol-eluted fractions again; Mixed solution is removed methyl alcohol to obtain liquid concentrator through concentrating under reduced pressure; Liquid concentrator is used and the ethyl acetate extraction of this liquid concentrator equal volume three times, then the combined ethyl acetate extraction liquid; Acetic acid ethyl acetate extract behind concentrating under reduced pressure, arrive crude extract.
Step 3: the crude extract that step 2 obtained is carried out purification on normal-phase silica gel column chromatography (silica gel G 200-300 order), and adopt chloroform-methanol gradient elution crude extract, each elutriant of Fractional Collections then; Said chloroform-methanol gradient elution crude extract is meant that 98: 2,95: 5,90: 10,85: 15,80: 20,70: 30,60: 40,50: 50 chloroform-methanol solvent systems carried out gradient elution to crude extract with volume ratio 100: 0.
Step 4: each elutriant that step 3 obtained is carried out thin-layer chromatography respectively; And adopt performance liquid chromatography that each elutriant is carried out check and analysis respectively; Then the thin-layer chromatography result is shown orange red; And the elutriant that the performance liquid chromatography detected result has identical peak merges, and the elutriant after this merging of concentrating under reduced pressure obtains first enriched material.Wherein, the developing agent of said thin-layer chromatography is trichloromethane-methanol solvate of 15: 1 of volume ratio, and developer is the bismuth potassium iodide developer; Said performance liquid chromatography testing conditions is: moving phase is 35: 65 the water-methanol that contains 0.1% trifluoroacetic acid of volume ratio, wash-out 20min, and flow velocity 1mL/min detects wavelength 254nm, 40 ℃ of column temperatures; The water of said 0.1% trifluoroacetic acid is meant the volume ratio trifluoroacetic acid: water=0.1: 99.9.
Step 5: first enriched material that step 4 obtained is carried out the middle reversed-phase silica gel column chromatography of pressing to obtain crude product; When pressing reversed-phase silica gel column chromatography in carrying out; Adopting volume(tric)fraction earlier is that 10% methanol aqueous solution is that the moving phase balance is after 10 minutes; Moving phase adopts the methanol-water solvent systems gradient elution 90min of volume ratio 10%～100%: 1 then, and the flow velocity of moving phase is 5ml/min;
Step 6: the crude product that step 5 obtained is carried out Sephadex LH-20 gel filtration chromatography; Said Sephadex LH-20 gel filtration chromatography adopts 1: 1 chloroform-methanol solvent systems wash-out of volume ratio; And according to flow point and each flow point of bismuth potassium iodide coupling reaction Fractional Collections; Flow point that then will be identical with the bismuth potassium iodide reaction solution merges, and concentrating under reduced pressure is to obtain second enriched material;
Step 7: adopt preparative high performance liquid chromatography to separate second enriched material that step 6 obtained, obtain 3 components; At last, separating to obtain the ansamitocin compounds through preparation type silica-gel plate 3 components respectively is compound 1 and compound 2.The separation condition of said preparative high performance liquid chromatography is: moving phase is 70% methanol aqueous solution, and the flow velocity of moving phase is 1.5ml/min, and the detection wavelength is 254nm.
Identify through structure, prove that the compound 1 that above-mentioned preparation method obtains and the structure of compound 2 represent that with formula (I) wherein, the R of compound 1 is CH
2CH
3, promptly the structure of compound 1 is formula (II); The R of compound 2 is CH
2CH (CH
3)
2, promptly the structure of compound 2 is formula (III)
Said 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;
is soluble in organic solvents such as methyl alcohol, acetone, ETHYLE ACETATE, trichloromethane; It is met bismuth potassium iodide and shows orange-yellow; The colour developing of chance iodine vapor, and (254nm) fluorescence is the blackening point under uv lamp.The ultraviolet absorption peak of compound 1 is respectively 280.60nm (see figure 1), 201.60nm (see figure 1).The ESI-MS spectrum of compound 1 shows molecular ion peak [M] at the m/z635.3 place
+The isotropic substance m/z 635.3 of characteristic ion bunch is 3: 1 with the abundance ratio of m/z 637.3, infers that tentatively this compound 1 contains a chlorine element, and its molecular weight is 634.The quasi-molecular ions of m/z 635.3 is carried out second order ms scanning, obtain m/z and be 561.1 characteristic fragment ion peak.High resolution mass spectrum (HR-ESI-MS): [M+Na]
+Measured value is 657.2552; Theoretical value is 657.2554, and the 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 ' 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, 600MHZ) in the spectrum, show 4 groups of methyl hydrogen signals at high field region, 1 group of bimodal methyl hydrogen signal [δ is wherein arranged
H1.20 (3H, d, J=5.7Hz)] show that it directly links to each other with tertiary carbon; 2 unimodal methyl hydrogen signal [δ
H0.77 (3H, s), δ
H1.60 (3H, brs)] show that it links to each other with quaternary carbon; 1 group of triplet signal [δ
H1.14 (3H, t, J=7.4Hz)] show that it links to each other 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 hydrogen signal that the carbon atom of hydridization links to each other wherein 3 be 2 couples of hydrogen signal [δ on trans continuous 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, 150MHZ) (DETP) collection of illustrative plates shows 32 spectral lines altogether, comprising: (10 quaternary carbon signals are specially 10 quaternary carbon signals: 1 carboxamido-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 link to each other 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 link to each other 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 be 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).
See also Fig. 3 and Fig. 5, Fig. 3 is a compound 1
1H-
1H COSY spectrogram, promptly
1H-
1The H chemical shift spectrogram of being correlated with; Fig. 5 is the HSQC collection of illustrative plates of compound 1, i.e. the single quantum coherent spectrogram of heteronuclear.At compound 1
1H-
1In the H COSY spectrum, visible δ 2.45 (H-2a), δ 2.12 (H-2b) are all relevant with δ 4.83 (H-3); δ 2.34 (H-2 ' a), δ 2.44 (H-2 ' b) is all relevant with δ 1.14 (H-3 '); Relevant in order between δ 2.81 (H-5)-δ 1.37 (H-6)-δ 4.08 (H-7)-δ 1.18 (H-8b); Relevant in order 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 (see figure 5) of binding compounds 1 can be released and contain following carbon skeleton fragment: C in the 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, promptly
1The relevant spectrum of the heteronuclear multikey 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) all and C-1 ' (δ 173.0), C-3 ' (δ 8.9); Long-range relevant between H-3 ' (δ 1.14) and C-1 ' (δ 173.0), the C-2 ' (δ 27.3), so just can the connection of bar structure Segment A add up, shown in Segment A:
Please consult Fig. 7 again, 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); Long-range relevant between H-9-NHCO (δ 6.30) and C-8 (δ 36.9), C-10 (δ 91.1), the C-9-NHCO (δ 153.0), so just can bar structure fragment B connection add up, fragment B is:
Please consult Fig. 7 again, 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
3Long-range relevant between (δ 16.0), so just can bar structure fragment C connection add up, fragment C is:
Please consult Fig. 7 again, 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), long-range relevant between the C-18 (δ 142.7), so just can bar structure fragment D connection add up, fragment D is:
Through 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.
Through 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
1The H-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 H 3.32, s; δ C 52.2) signal, and the J of compound 1
H-5 ,-6(9.8), J
H-10 ,-11(9.1) identical basically with the data of ansamitocin P-2, explain that this compound 1 has identical relative configuration with the AMSA compound, therefore, the configuration of confirming this compound 1 is shown in the structure of the compound among Fig. 91.
Said 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;
is soluble in organic solvents such as methyl alcohol, acetone, ETHYLE ACETATE, trichloromethane; Meet bismuth potassium iodide and show orange-yellow; Meet iodine vapor and show brown, (254nm) fluorescence is the blackening point under the uv lamp.The ultraviolet absorption peak of compound 2 is respectively 281.00 (see figure 2)s, 250.40 (see figure 2)s, 230.80 (see figure 2)s, 203.60 (see figure 2)s.The ESI-MS spectrum of compound 2 shows molecular ion peak [M] at m/z 663.3 places
+, the isotropic substance m/z 663.3 of characteristic ion bunch is 3: 1 with the abundance ratio of m/z 665.3, infers that tentatively this compound 2 contains a chlorine element, its molecular weight is 662.The quasi-molecular ions of m/z 663.3 is carried out second order ms scanning, obtain m/z and be 561.1 characteristic fragment ion peak.High resolution mass spectrum (HR-ESI-MS): [M+Na]
+Measured value is 685.2862; Theoretical value is 685.2867, and the 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, 600MHZ) in the spectrum, show 5 groups of methyl hydrogen signals at high field region, 3 groups of bimodal methyl hydrogen signal (δ are wherein arranged
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 directly links to each other with tertiary carbon; 2 unimodal methyl hydrogen signal (δ
H0.78 (3H, brs), δ
H1.61 (3H, brs)) shows that it links to each other with quaternary carbon; 3 methoxyl group hydrogen signal (δ
H3.23 (3H, s), δ
H3.32 (3H, s), δ
H(3.93 3H, s)) and 1 n-formyl sarcolysine base hydrogen signal (δ
H(3.08 3H, s)).There are 5 and sp in low place
2The hydrogen signal that the carbon atom of hydridization links to each other, wherein 3 is 2 couples of hydrogen signal (δ on trans two keys that link to each other
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
H(6.75 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, 150MHZ) show 34 spectral lines altogether in (DETP) collection of illustrative plates, (10 quaternary carbon signals are specially: 1 carboxamido-group carbon signal (δ comprising 10 quaternary carbon signals
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 link to each other 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 link to each other 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 be mesomethylene carbon signal (δ
C43.7, δ
C32.9, δ
C36.8, δ
C47.5), 3 be methoxyl group carbon signal (δ
C52.0, δ
C57.4, δ
C56.8), 1 be 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).
See also Fig. 4, Fig. 4 is a compound 2
1H-
1In the H COSY spectrum, visible δ 2.12 (H-2b), δ 2.45 (H-2a) are all relevant with 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 order between δ 2.79 (H-5)-δ 1.37 (H-6)-δ 4.07 (H-7)-δ 1.17 (H-8b); Relevant in order 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 (see figure 6) of binding compounds 2 can be released and contain following carbon skeleton fragment: C in the 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
Please consult Fig. 8 again, 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
3Long-range relevant between (δ 22.7), so just can the connection of bar structure Segment A add up, Segment A is:
Please consult Fig. 8 again, 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); Long-range relevant between H-9-NHCO (δ 6.31) and C-8 (δ 36.8), the C-9-NHCO (δ 152.9), so just can bar structure fragment B connection add up, fragment B is:
Please consult Fig. 8 again, 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
3Long-range relevant between (δ 16.0), so just can bar structure fragment C connection add up, fragment C is:
Please consult Fig. 8 again, 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
3Long-range relevant between (δ 16.0), so just can bar structure fragment D connection add up, fragment D is:
Through 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.
Through 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.)
1The H-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) identical basically with the data of ansamitocin compounds, explain that this compound 2 has identical relative configuration with the ansamitocin compounds, therefore, the configuration of confirming this compound 2 is shown in the structure of the compound among Figure 10 2.
Compound (compound 1 or compound 2) is carried out the inhibiting rate determination experiment to the HL60 cell, investigate the half-inhibition concentration of chemical combination 1 and compound 2, its experimental procedure is following:
(1) preparation of cell culture fluid: in the RPMI1640 of 450ml solution, add 50ml foetal calf serum, the two anti-solution of the blue or green chain of 5ml.Wherein, the manufacturer of said RPMI1640 solution, foetal calf serum, the two anti-solution of blue or green chain is Sai Mo and flies generation that biological chemistry goods (Beijing) ltd.In the two anti-solution of said blue or green chain, the concentration of penicillium mould 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 leukaemia cell) is obtained with step (1) under 37 ℃, 5%CO
2Cultivate in the incubator; Get HL60 logarithmic phase cell then 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 the DMSO 99.8MIN. (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) storage liquid that step (3) is obtained is diluted to the solution of 5 kinds of concentration with the cell culture fluid of step (1) gained, i.e. the compound of 5 kinds of concentration (compound 1 or compound 2) solution, and 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: in the culture plate that HL60 logarithmic phase cell is housed of step (2) gained, add 500 * 10 of 20 μ l
-6The compound solution of mol/L, and experimental group 1 is established three parallel holes;
Experimental group 2: in the culture plate that HL60 logarithmic phase cell is housed of step (2) gained, add 1000 * 10 of 20 μ l
-6The compound solution of mol/L, and experimental group 2 is established three parallel holes;
Experimental group 3: in the culture plate that HL60 logarithmic phase cell is housed of step (2) gained, add 2000 * 10 of 20 μ l
-6The compound solution of mol/L, and experimental group 3 is established three parallel holes;
Experimental group 4: in the culture plate that HL60 logarithmic phase cell is housed of step (2) gained, add 3000 * 10 of 20 μ l
-6The compound solution of mol/L, and experimental group 4 is established three parallel holes;
Experimental group 5: in the culture plate that HL60 logarithmic phase cell is housed of step (2) gained, add 4000 * 10 of 20 μ l
-6The compound solution of mol/L, and experimental group 5 is established three parallel holes;
Experimental group 6: in the culture plate that HL60 logarithmic phase cell is housed of step (2) gained, add 5000 * 10 of 20 μ l
-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: add the cell culture fluid of 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) will pass through culture plate after step (5) is handled under 37 ℃, 5%CO
2Cultivate 48h in the incubator.
(7) preparation of working fluid: 50mg MTT [being 3-(4,5-dimethylthiazole-2)-2,5-phenylbenzene tetrazole bromine salt] (manufacturer of MTT is a SIGMA company) is dissolved in the phosphate buffered saline buffer of 10ml, be made into the working fluid of 5mg/ml.
(8) add the above-mentioned working fluid of 20 μ l in every hole toward the culture plate of handling through step (6), put culture plate again in 37 ℃, 5%CO
2Cultivate 4h in the incubator.Then,, carefully abandon supernatant, in every hole, add the methyl-sulphoxide of 150 μ l again, detect the light absorption value in every hole at last with ELIASA in the 570nm wavelength with vacuum pump suction with the centrifugal culture plate of dull and stereotyped whizzer.
Inhibitory rate of cell growth=(control group light absorption value one experimental group light absorption value)/control group light absorption value * 100%
Compound (compound 1 or compound 2) is seen table 1 and table 2 to the concrete outcome of HL60 cell inhibitory rate determination experiment.
1 pair of HL60 cell inhibitory rate of table 1 compound determination experiment result
2 pairs of HL60 cell inhibitory rates of table 2 compound determination experiment result
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 the HL60 cyto-inhibition, 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, promptly cell survival rate reduces by 50% o'clock drug level.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 has the very strong anti-tumor activity that has with compound 2.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; 20July 2010, and 1146-1150) compound N-demethyl-desepoxy-9-methoxy-maytansinol of report is to the IC of 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 the leaf of Hooker's Mayten callus; Utilize its growing environment unique; Through microbial fermentation and multiple separation purification method; From this bacterial strain FIM06-0063 fermented liquid; It is compound 1 and compound 2 that separation obtains two new ansamitocin compounds, for researching and developing new antitumor drug Natural Medicine Chemistry research basis and lead compound is provided, and compound 1 might become bullet molecule and the targeted molecular couplings formation target mixtures such as antibody or part that are used for target therapeutic agent with compound 2.The ansamitocin compounds can be reduced into the C-3 Ansamitocin Po, and this alcohol is the precursor that is used for synthetic sulfur-bearing alcohol maytansinoid.In addition, the present invention prepares the ansamitocin compounds through microbial fermentation processes, has the advantage that technology is simple, production cost is low.
Claims (11)
1. the new ansamitocin compounds of formula (I) representative,
Wherein, R is CH
2CH
3Or CH
2CH (CH
3)
2
2. the preparation method of a new ansamitocin compounds as claimed in claim 1, it is characterized in that: its operation steps is following:
Step 1: preparation synnema actinomycetes bacterial strain FIM06-0063 (Actinosynnema sp) fermented liquid;
Step 2: the synnema actinomycetes bacterial strain FIM06-0063 fermented liquid that step 1 obtained is carried out the macroporous resin adsorption column chromatography, obtain crude extract;
Step 3: the crude extract that step 2 obtained is carried out the purification on normal-phase silica gel column chromatography, and adopt chloroform-methanol gradient elution crude extract, each elutriant of Fractional Collections then;
Step 4: each elutriant that step 3 obtained is carried out thin-layer chromatography respectively; And adopt performance liquid chromatography that each elutriant is carried out check and analysis respectively; Then the thin-layer chromatography result is shown the elutriant that orange red and performance liquid chromatography detected result has identical peak and merge, and the elutriant after this merging of concentrating under reduced pressure obtains first enriched material;
Step 5: first enriched material that step 4 obtained is carried out the middle reversed-phase silica gel column chromatography of pressing to obtain crude product; When pressing reversed-phase silica gel column chromatography in carrying out; Adopting volume(tric)fraction earlier is that 10% methanol aqueous solution is that the moving phase balance is after 10 minutes; Moving phase adopts the methanol-water solvent systems gradient elution 90min of volume ratio 10% ~ 100%:1 then, and the flow velocity of moving phase is 5ml/min;
Step 6: the crude product that step 5 obtained is carried out Sephadex LH-20 gel filtration chromatography; Said 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 coupling reaction Fractional Collections; Flow point that then will be identical with the bismuth potassium iodide reaction solution merges, and concentrating under reduced pressure is to obtain second enriched material;
Step 7: adopt preparative high performance liquid chromatography to separate second enriched material that step 6 obtained, obtain 3 components; At last, 3 components are separated to obtain the ansamitocin compounds through preparation type silica-gel plate respectively; The separation condition of said preparative high performance liquid chromatography is: moving phase is 70% methanol aqueous solution, and the flow velocity of moving phase is 1.5ml/min, and the detection wavelength is 254nm.
3. the preparation method of new ansamitocin compounds as claimed in claim 2, it is characterized in that: the separation method of said synnema actinomycetes bacterial strain FIM06-0063 is following:
Step 100: get the Lao Ye and the petiole of Yunnan leaf of Hooker's Mayten, behind tap water flushing 30min, clean with zero(ppm) water again and place sterile petri dish; Then with said Lao Ye and petiole successively with the scouring of 75% alcohol, behind the aseptic water washing, change massfraction over to and be in 0.1% the mercuric chloride solution and soak 3min, use aseptic water washing again 5 times, remove thimerosal; Then said 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 the mashed prod that obtains in the few steps 100 with aseptic glass spreading rod and directly coat starch asparagine agar plate, place 28 ℃ to cultivate 7～20 days down flat board then, obtain the single bacterium colony of actinomycetes; The component of said starch asparagine agar: Zulkovsky starch 2%, L-asparagine 0.05%, KNO
30.1%, K
2HPO
43H
2O 0.05%, and NaCl 0.05%, MgSO
47H
2O 0.05%, CaCO
30.05%, agar 1.5%, the zero(ppm) water preparation, pH is 7.5, and the percentage ratio in this starch asparagine agar component is massfraction;
Step 300: the single bacterium colony of actinomycetes that will pass through step 200 processing acquisition is transferred in inorganic salt Starch Agar inclined-plane, obtains the slant culture of synnema actinomycetes bacterial strain FIM06-0063; The component on said inorganic salt Starch Agar inclined-plane is: Zulkovsky starch 10g; K
2HPO
43H
2O 1g; MgSO
47H
2O 1g; NaCl 1g; (NH
4)
2SO
42g; CaCO
32g; FeSO
47H
2O 0.001g; MnCl
24H
2O 0.001g; ZnSO
47H
2O 0.001g; Agar 20g; Zero(ppm) water 1000mL, pH are 7.0～7.4.
4. the preparation method of new ansamitocin compounds as claimed in claim 2, it is characterized in that: said step 1 is specially:
(1) preparation seed liquor: with being inoculated in the seed culture medium after the synnema actinomycetes bacterial strain FIM06-0063 slant culture maturation, and be placed on shaking culture 40～48h in the constant temperature shaking table, 28 ℃ of temperature, rotating speed 220r/in;
(2) preparation fermented liquid: the seed liquor that the culture transferring amount by 10% is obtained step (1) is transferred into the fermention medium cultivation 96～120h of sterilization, 28 ℃ of temperature, rotating speed 220r/min.
5. the preparation method of new ansamitocin compounds as claimed in claim 4 is characterized in that:
The component of said 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%, lime carbonate 0.5%, pH are 7.0, and the percentage ratio in this seed culture medium component is massfraction.
6. the preparation method of new ansamitocin compounds as claimed in claim 4 is characterized in that:
The component of said 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%, lime carbonate 0.5%, pH are 7.0, and the percentage ratio in this fermention medium component is massfraction.
7. the preparation method of new ansamitocin compounds as claimed in claim 2, it is characterized in that: said step 2 is specially: is centrifugal 15min under the 3000r/min with synnema actinomycetes bacterial strain FIM06-0063 fermented liquid at rotating speed, and gets supernatant; Then supernatant is splined on macroporous adsorbent resin, obtains mixed solution with methanol-eluted fractions again; Mixed solution is removed methyl alcohol to obtain liquid concentrator through concentrating under reduced pressure; Liquid concentrator is used and the ethyl acetate extraction of this liquid concentrator equal volume three times, then the combined ethyl acetate extraction liquid; Acetic acid ethyl acetate extract behind concentrating under reduced pressure, arrive crude extract.
8. the preparation method of new ansamitocin compounds as claimed in claim 2 is characterized in that: the chloroform-methanol gradient elution crude extract in the said step 3 is meant with volume ratio 100: 0 98: 2; 95: 5,90: 10,85: 15; 80: 20; 70: 30,60: 40,50: 50 chloroform-methanol solvent systems carried out gradient elution to crude extract.
9. the preparation method of new ansamitocin compounds as claimed in claim 2, it is characterized in that: the developing agent of the thin-layer chromatography in the said step 4 is trichloromethane-methanol solvate of 15: 1 of volume ratio, and developer is the bismuth potassium iodide developer.
10. the preparation method of new ansamitocin compounds as claimed in claim 2; It is characterized in that: the performance liquid chromatography testing conditions in the said step 4 is: moving phase is 35: 65 the water-methanol that contains 0.1% trifluoroacetic acid of volume ratio; Wash-out 20min; Flow velocity 1mL/min detects wavelength 254nm, 40 ℃ of column temperatures.
11. preparation method like claim 2,3,4 or 6 described new ansamitocin compounds; It is characterized in that: said synnema actinomycetes bacterial strain FIM06-0063 (Actinosynnema sp); Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on December 30th, 2011, deposit number is CGMCC NO.5677.
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|---|---|---|---|---|
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2012
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