CN1095844C - Triptolide derivative, preparation method and application thereof - Google Patents
Triptolide derivative, preparation method and application thereof Download PDFInfo
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- CN1095844C CN1095844C CN99111660A CN99111660A CN1095844C CN 1095844 C CN1095844 C CN 1095844C CN 99111660 A CN99111660 A CN 99111660A CN 99111660 A CN99111660 A CN 99111660A CN 1095844 C CN1095844 C CN 1095844C
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- cancer
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- triptolide
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- DFBIRQPKNDILPW-CIVMWXNOSA-N Triptolide Chemical class O=C1OCC([C@@H]2C3)=C1CC[C@]2(C)[C@]12O[C@H]1[C@@H]1O[C@]1(C(C)C)[C@@H](O)[C@]21[C@H]3O1 DFBIRQPKNDILPW-CIVMWXNOSA-N 0.000 title claims abstract description 28
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- 125000004432 carbon atom Chemical group C* 0.000 claims abstract 3
- YKUJZZHGTWVWHA-UHFFFAOYSA-N triptolide Natural products COC12CC3OC3(C(C)C)C(O)C14OC4CC5C6=C(CCC25C)C(=O)OC6 YKUJZZHGTWVWHA-UHFFFAOYSA-N 0.000 claims description 35
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Landscapes
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Abstract
1. A triptolide derivative has a structural formula: wherein R is1Is H, an alkyl radical having from 1 to 4 carbon atoms, -Ac, -C (═ O) (CH)2)nCO2H or an amino acid group, wherein n is an integer from 1 to 4, R2is-SCN, or-NCS. The compounds of the present invention have selective inhibitory effects on certain tumors.
Description
The present invention relates to a kind of triptolide alcohol derivative, its production method, the application in the medicine of production for treating cancer, and the drug regimen that contains this compound.
Trypterygine has antitumous effect and is confirmed by Chinese scholars.1972 Kuphchan (1) just from trypterygine, separate to obtain to contain the diterpene compound of epoxy, promptly triptolide (triptolide) is used for antitumor research.Triptolide can prolong L 0.2 and during 0.25mg/kg ip
615The survival time of leukemia mouse, and survival mice is through attacking for several times unlikely leukemia.Can suppress human nasopharyngeal carcinoma KB cells in vitro propagation when 1ng/ml dosage (opens a Tan and washes one's hair, Chen Zhengyu, Lin Chen, triptolide antitumor action and to effect of immunologic function, Acta Pharmacologica Sinica, 1981; 2 (2): 128), when suppressing the formation of mammary cancer and stomach cancer cell colony, its intensity is close with the inhibition human leukemia HL-60 cell, IC
50Be 0.504-1.22 μ g/L.The experiment of human cervical carcinoma Hela cell's kinetic effect is found, triptolide to the time synchronised cell lethal effect is all arranged, and to S phase cell sensitivity.Experiment is proof also, triptolide can suppress RNA and proteinic synthetic in antineoplastic while, and optionally makes the sulfydryl inactivation on the phosphofructokinase, it is synthetic to suppress liver glycogen, make the RNA polymerase inactivation, disturb (Xu Jianhua, the Li Changchun of duplicating of DNA, Huang is improved oneself, triptolide is to the cytokinetics influence of Hela cell, Acta Pharmacologica Sinica, 1989; 10 (6): 550).Because trypterygine epoxy diterpenic lactone has stronger biological activity and application prospect, cause investigators' extensive interest, to constant so far (the Berchtold GA et al of their chemosynthesis, structural modification and bioactivity research, J Am Chem Soc, 1980,102:1200; Tamelen EEet al, J Am Chem Soc, 1982,104:1785).As far back as the initial stage eighties, just successfully chemosynthesis triptolide, but because reaction scheme is long, step is many and reaction is harsh, the target product of final acquisition only has theoretical researching value.(Yu Dequan, Zhang Dongming, Wang Huaibin, Liang Xiaotian, the structural modification of triptolide, Acta Pharmaceutica Sinica, 1992,27 (11): 830) triptolide has been made a large amount of chemical structures and modified, once obtained many valuable products such as Yu Dequan.Triptolide is one of main effective constituent in the trypterygine, although consumption is little, its biological activity effective dose is near its LD
50Clinically still show suitable toxicity, so hindered further exploitation and clinical application.In addition from present bibliographical information, domestic and international bioactivity research to trypterygine epoxy diterpene-kind compound is still major part and concentrates in the immunosuppression, and not a lot of to their Antitumor Effects, its reason is to compare with other natural product with immunosuppressive action, and trypterygine epoxy diterpene-kind compound has more characteristic.But recent studies show that, the autoimmunization systemic disease is the same with growth of tumor as rheumatic arthritis, diabetic retinopathy, lupus erythematosus etc., the new vessel that all relates to pathology generates, and these diseases all can be referred to as " angiogenic disease ".Therefore, the compound that generates disease as treatment rheumatic arthritis and other new vessel also may show restraining effect to tumour (Folkman J, Nature Med, 1995,1:27).
Molecular weight tumor learn progress that institute obtains for the treatment tumour provides many as oncogene, cancer suppressor gene and other can be for the target spot of the cancer therapy drug effect of utilization, if can find the especially medicine of antagonism oncogene and unconventionality expression product thereof of target spot that medicine attacks cancer cells, will have significant values the research and development of antitumor drug.Apoptosis and the oncogene relevant with apoptosis are the important target spots of screening anti-tumor medicine.But (Immunopharmacology 1998 for Yang YL, et al for triptolide induction of lymphocyte Study of apoptosis; 40:139) show at apoptosis-induced early stage triptolide activation apoptosis-related protein enzyme.T12 has very strong lethal effect to kinds of tumor cells, and toxicity is also well below triptolide; Also find from the mechanism of action of molecular level research T12, this compound inducing apoptosis of tumour cell, and the activation of initiation apoptosis-related protein enzyme, the gene product that changes apoptosis program or the apoptosis program Bcl-2 cancer protein of promptly degrading, this activity that T12 causes obviously is better than triptolide, makes it promise to be a kind of antitumor drug of novel, low toxicity.
It is low to an object of the present invention is to provide a kind of toxicity, the triptolide alcohol derivative that clinical value is high.
Another purpose of the present invention provides a kind of purposes of triptolide alcohol derivative of the present invention.
Another object of the present invention provides a kind of method of producing triptolide alcohol derivative of the present invention.
A further object of the present invention provides a kind of pharmaceutical composition that contains triptolide alcohol derivative of the present invention.
The invention provides a kind of triptolide alcohol derivative, its structural formula is:
Wherein, R
1Be H, contain the alkyl of 1-4 carbon atom ,-Ac-C (=O) (CH
2)
nCO
2H, or amino acid based, wherein, n is the integer of 1-4; R
2Be-SCN-NCS.Because intramolecular rearrangement ,-SCN and-may change between the NCS.
In compound of the present invention, preferred R
2Be-SCN; R
1Be H.
The different reagent of usefulness such as Yu Dequan are opened 12 of triptolide, 13-epoxy and obtain comprising 12-position halogen (Cl, Br) a series of triptolide alcohol derivative .12-position is after the triptolide alcohol derivative of halogen enters in the body, under enzymatic action, C12,13-may close ring again, replys to be the precursor compound triptolide, thereby shows the toxicity of precursor compound triptolide once again.The present invention's triptolide is a raw material, under mild reaction conditions, carries out C-12 with ammonium thiocyanate, 13 ring-opening reaction, obtain novel high anti-tumor activity, the 12-β-thiocyano of low toxicity-13-Alpha-hydroxy triptolide (hereinafter to be referred as T12), its structural formula is as follows:
Wherein, R
1Be H, contain the alkyl of 1-4 carbon atom ,-Ac-C (=O) (CH
2)
nCO
2H, or amino acid based, wherein, n is the integer of 1-4.R
1Group can add by known method.
The present invention also provides a kind of method of producing above-claimed cpd, comprise the structure that will be shown below under heating condition with the thiocyanic acid reaction,
Wherein, R
1Be H, contain the alkyl of 1-4 carbon atom ,-Ac-C (=O) (CH
2)
nCO
2H, or amino acid based, wherein, n is the integer of 1-4.
In the aforesaid method of the present invention, described reaction is preferably carried out under 65-87 ℃ condition in organic solvent.Described organic solvent is the trimethyl carbinol preferably.
The present invention also provides the application of a kind of above-claimed cpd in the medicine of preparation treatment cancer.
In above-mentioned application, described cancer can be mammary cancer, lung cancer, kidney, malignant melanoma, colorectal carcinoma and ovarian cancer.And to kidney, ovarian cancer, mammary cancer, colorectal carcinoma and malignant melanoma have better effect.
The present invention also provides a kind of pharmaceutical composition, and it contains the compound of the present invention and the pharmaceutically acceptable carrier of anticancer growth significant quantity.
In above-mentioned pharmaceutical composition, described cancer cells can be mammary cancer, lung cancer, kidney, malignant melanoma, colorectal carcinoma and ovarian cancer.Preferably kidney, ovarian cancer, mammary cancer, colorectal carcinoma and malignant melanoma cell.
The present invention also provides another pharmaceutical composition, and it contains the compound of the present invention and the bcl-2 antisense nucleotide of anticancer growth cooperative effective quantity, and pharmaceutically acceptable carrier.
Brief Description Of Drawings: Fig. 1 represents the dna degradation state of T12 cell death inducing.Fig. 2 represents that T12 induces HL-60 cell and Bre-01 cell Bcl-2 to degrade.Fig. 3 represent T12 induce bcl-2 take place degraded intensity and with the comparison of other three kinds of compounds.Fig. 4 represents the result of compound of the present invention and antisense nucleic acid synergy Col-06 cell.
The preparation method of embodiment 112-β-thiocyano-13-Alpha-hydroxy triptolide;
Get 3.6g (0.01mol) triptolide and be dissolved in the 400ml trimethyl carbinol,, after treating to dissolve fully, add 11.4g (0.15mol) ammonium thiocyanate in 50 ℃ of following stirring heating dissolvings.Be warming up to 80 ℃, reaction 24hrs, reaction is finished.Reaction solution adds the 400ml ethyl acetate, and organic layer is washed 3 times with saturated sodium-chloride water solution, and the MgSO4 drying is filtered, and steams down in 60 ℃ to desolventize, and residuum adds acetone solution.TLC detects, chloroform: methyl alcohol (95: 5) launches, and T12 is main point, contains a small amount of T and other by product.Acetone solution adds 10g 100-200 order silica gel mixed sample, eliminates acetone, and 200-300 order silica gel column chromatography separates, and eliminates T with the chloroform wash-out earlier, uses chloroform again instead: methyl alcohol (95: 5) wash-out, TLC follows the tracks of detection, collects the T12 cut, merges T12.Add the small amount of acetone dissolving after removing solvent under reduced pressure, place, separate out T12 granular crystal 3.77g, yield 90%Mp:265-266 ℃ .R
fValue is: 0.15 (CHCl
3/ MeOH, 95:5), developer is a Kedd ' s reagent, is red-purple.Ultimate analysis C
21H
25NSO
6, calculated value %:C, 60.14.H, 6.01.N, 3.34. measured value %:C, 59.85.H, 5.93.N, 3.26.[α]
D 25-114.06 ℃ of (acetone, 4.17mg/ml) .IR (KBr) cm
-1: 3450,3000,2152,1739,1674,1441,1032,980.MS?m/z(％)：419(M+，13)，404(-CH
3，14.5)，361(-
，13)，343(-H
2O，13)，271(14)，241(23)，151(48)，137(89)，72(91)，61(100)。
1HNMR，δppm：0.8(3H，d，J＝6.77Hz，16-CH
3)，0.95(3H，s，20-CH
3)，0.99(3H，d，J＝6.88Hz，17-CH
3)，1.28(1H，m，1-αH)，1.45(1H，m，1-βH)，1.85(1H，t，J＝14.17Hz，6-βH)，2.0(1H，m，2-H)，2.18(1H，m，2-H)，2.20(1H，m，6-αH)，2.25(1H，m，15-H)，2.67(1H，m，5-H)，3.0(1H，d，J＝4.4Hz，14-H)，3.35(1H，m，7-H)，3.75(1H，d，J＝5.61Hz，11-H)，3.90(1H，d，J＝5.87Hz，12-H)，4.85(2H，m，19-H)，5.0(1H，s，13-OH)，5.45(1H，d，J＝4.2Hz，14-OH)；
13CNMR，δppm：14(16-C)，15.5(20-C)，16(17-C)，17(2-C)，22.5(6-C)，29(15-C)，30(1-C)，35(10-C)，39(5-C)，51(11-C)，57.5(12-C)，60.5(8-C)，61.5(7-C)，67(9-C)，70.5(19-C)，74(14-C)，76.5(13-C)，115(SCN)，123(3-C)，162(4-C)，173(18-C)。
Embodiment 2SRB method is measured the restraining effect of T12 to 20 kinds of different tumour cells
The important means that cultured tumor cells in vitro has become the screening cancer therapy drug and studied.Past, means commonly used were to utilize animal such as mouse leukemia/lymphoma cell line, but the cell of people and mouse has tangible difference to the reaction of medicine, so had used the human tumour cell line in recent years mostly instead and carried out antitumor activity in vitro.Six kinds of clones have been used in antitumor test of the present invention, are respectively lung cancer (Lu-06), mammary cancer (Bre-01), ovarian cancer (Ov-01), kidney (Re-01), colorectal carcinoma (Col-06), malignant melanoma (Mel-08).
SRB method for screening anticancer medicine (the Skehan P that adopts national cancer institute to set up, Storeng R, Scudiero D et al, New colorimentric cytotoxicity assay foranticancer-drug screening.J Nalt Cancer Inst 1990; 82:1107) the present invention is carried out the extracorporeal anti-tumor test, and with its lead compound triptolide (T) and another triptolide alcohol derivative 8-OH triptolide (high-activity immune inhibitor, hereinafter to be referred as T8, see Jung, 1/1999 PCT/US98/08562, WO98/52933) and anticancer plants synthetic drug taxol compare.Testing method is as follows:
Tumor cell culture in containing the RPMI1640 substratum of 5% calf serum, is included an amount of penicillin and streptomycin and glutamine, at 5%CO
2, cultivate under 37 ℃ of conditions.Respectively subject cell is inoculated in 96 orifice plates by certain density, the administration group changes the new nutrient solution that contains tested medicine into (medicine is earlier after the DMSO dissolving after 24 hours, add PBS and be diluted to 1mg/ml concentration, during use by the desired concn redilution and add in the cell) continue to cultivate 48 hours.Every hole adds 50% trichoroacetic acid(TCA) (TCA, ultimate density is 10%), the 50 μ l of precooling, leave standstill move after 5 minutes place 4 ℃ 1 hour, washing is 5 times on automatic washer, dry air.Add 100 μ l SRB liquid (being mixed with 0.4% solution) dyeing 10 minutes, put again and wash on the plate machine with 1% acetum washing 4 times, to remove not combination dye, dry air with 1% acetic acid.With the non-buffering of 150 μ l 10mmol/L Tris solution dissolving bonded SRB, fully behind the mixing, use the microwell plate readout instrument at last, carry out automatic rapid colorimetric determination, simultaneously data are handled at wavelength 490nm place.
The GI50 average of table 1 T12 and six tumor cell lines of taxol effect (μ g/ml) relatively
Clone T T8 T12 Taxol
Lu-06 (lung cancer) 0.09 0.5 0.6 0.075
Bre-01 (mammary cancer) 0.04 0.3 0.055 0.06
OV-01 (ovarian cancer) 0.006 0.06 0.02 0.05
Mel-08 (melanoma) 0.02 0.25 0.09 0.07
Re-01 (kidney) 0.03 0.25 0.06 1
Col-6 (colorectal carcinoma) 0.02 0.1 0.03 0.007
The present invention to the exercising result of six tumor cell lines and with the comparison such as the table 1 of T, T8 and taxol.The present invention all has the obvious suppression effect to six tumour cells, and it acts on a little less than T, but apparently higher than T8, anti-kidney effect is significantly higher than taxol; To colorectal carcinoma, ovarian cancer, mammary cancer and melanoma cell also show very strong restraining effect.
Embodiment 3 adherent growth cell colony forming methods are measured the restraining effect of T12 to the tumour clonogenic cell
Clonogenic cell is meant the cell with lasting splitting ability.Growth of tumor, transfer and recurrence are all based on the propagation of clonogenic cell.When individual cells continuous 6 generations of division or when above, can form discontiguous each other colony colony, but, be a kind of than the sensitive detection method by counting colony quantitative analysis clonogenic cell.It is as described below that colony forms testing method:
Getting adherent Re-01 cell exponential phase of growth, make single cell dispersion suspension after Trypsin-EDTA digestion, carry out viable count, is 500/ml with RPMI1640 substratum diluting cells density.Get the 35ml culture dish, every triad add the cell suspension after diluting, and the present invention is diluted to five kinds of different concns (0.001～10 μ g/ml) with 10 times and adds, and establishes the blank group of medicine simultaneously.Cell and medicine mixing postposition contain 5%CO
237 ℃ of incubators in cultivated 14 days.Discard nutrient solution, conventional Giemsa staining cell and counting contain the above colony of 50 cells.Colony inhibiting rate (%)=(1-administration group colony number/control group colony number) * 100
With kidney Re-01 cell is material, has measured the restraining effect that T12 forms its colony, and the result is: when the T12 activity is 0.1ug/ml, can suppress the growth of Re-01 clonogenic cell fully; Activity is 0.01ug/m and 10.001ug/ml, and inhibiting rate is respectively 85% and 28%.
The experimental therapy of embodiment 4 people's cancer bare mouse different species grafts
The antitumour drug of finding by multiple mouse model was mainly effective to malignant lymphoma and leukemia in the past, and mouse tumor cell is higher than human body tumour cell to the susceptibility of antitumor drug.Nude mice has the thymus dependent function and lacks, and does not have rejection during xenotransplantation.Human tumor after the xenotransplantation still keeps its original tissue morphology and amynologic characteristic and karyotype and to original susceptibility of antitumor drug in nude mouse.In human malignant melanoma cell Mel-08 inoculation nude mouse, the xenotransplantation method is as described below:
Medicine preparation: get the respective amount sample, add an amount of tween 80 suspending, be mixed with the suspension (each dosage 0.5ml/ mouse) of desired concn with 0.5%CMC-Na solution.
The immunodeficiency nude mice, body weight 17-19 gram, is female at 6-8 age in week.Test group and positive controls are 6, and negative control group is 12.The positive control medicine is dacarbazine (DTIC), prepares with physiological saline; Negative control is given corresponding solvent.
The key step of test: get eugonic tumor animal, be equipped with the human malignant melanoma with the homogenate legal system under the aseptic condition and become cell suspension, with the every mouse armpit of corresponding host subcutaneous vaccination 0.2ml/ only, next day is by experimental design scheme intraperitoneal administration, continuous fortnight, 4mg/kg/ day.Experiment finishes the back and dissects each treated animal, cuts open to get tumour and weigh, and calculates tumor control rate by following formula: tumor control rate=[(the average knurl of the average knurl weight-administration of control group group is heavy)/average knurl of control group weighs] * 100.
Compare with the medicine untreated fish group, tumor growth is obviously suppressed, and tumour inhibiting rate is 50.17%, and is approaching with the result's (tumour inhibiting rate is 54.92%) who is used for the uncommon miaow amine (20mg/kg) of clinical positive control medicine nitrogen.
The detected through gel electrophoresis of embodiment 5T12 cell death inducing
Apoptosis plays an important role with upgrading, keep the relative constant aspect of cell number to cell decline in the ontogenetic process.Apoptosis changes or unusually, will cause the generation of human diseases.Tumour is considered to propagation, prosoplasia disease in the past, along with the research to apoptosis of tumor cells, thinks that now tumour also is the unusual disease of apoptosis.Apoptosis has been broken the balance adjustment of cell proliferation and apoptosis in the healthy tissues unusually, causes the continuous increase of cell number, shows growth vigor, finally forms tumour.Therefore, but the medicine of inducing apoptosis of tumour cell be the treatment tumour an effective way.
The apoptotic later stage is because the activation of nuclease makes the nuclear chromatin dna degradation, and the oligomerization nucleosome fragment of generation different lengths (180-200bp), can be observed this feature by agarose electrophoresis.This feature is an important symbol of judging that at present apoptosis takes place.Using the agarose electrophoresis method has detected with the dna degradation situation behind the T12 effect human promyelocytic leukemia HL-60 cell of 1 μ g-/ml concentration.Operation steps is as described below.
HL-60 cell about 3 * 10 after the present invention handles
6Wash 2 times with the PBS that contains 2mmol/L EDTA, add 0.3mlTBE damping fluid (Tris.HCl 50mmol/L, pH 8.0, contain 10mmol/LEDTA), 0.25%NP-40 and Rnase A (final concentration is 1%), behind the mixing, 37 ℃ of incubations 30 minutes; Add proteinase K (final concentration is 1mg/ml) again, 37 ℃ were continued incubation 30 minutes.Get supernatant liquor 15 μ l and add 5 μ l sample-loading buffers, electrophoresis on 1.5% sepharose, observations and photograph under the UV-light.
Fig. 1 has shown that T12 handles and the dna degradation state of untreated HL-60 cell.T12 handled cell respectively after 4,12,24 hours, dna ladder shape band (dna degradation sign clearly appears on 1.5% agarose gel, hole 3-5), do not produce dna ladder shape band (hole 1,2) without T12 processing and treated 2 hours cell, but show the T12 inducing apoptosis of tumour cell.
Embodiment 6 flow cytometers detect apoptosis and cell cycle distribution
Apoptotic some feature also can be come out by the detection reaction with flow cytometer, judges the generation of apoptosis with this, simultaneously also is to carry out the reliable tools that cell cycle distribution detects behind the drug effect.The flow cytometry analysis method is as described below.
Handle respectively and untreated Re-01 and HL-60 cell 2 * 10 through the present invention
6, respectively at the different time collecting cell, 1000rpm/ divided centrifugal 5 minutes, removed supernatant, and precipitation is used the PBS washed twice, adds 70% ethanol fixed cell with 1: 9 ratio again, and-20 ℃ are spent the night behind the mixing.The recentrifuge collecting cell, the PBS washed twice adds 1ml 20 μ g/ml PI staining fluids and (uses 40mM Na
2HPO
4/ citrate buffer solution, pH 7.8; 0.25mg/ml Rnase A), room temperature dyeing is 30 minutes.(COULTER company US), is a light source with 488nm laser, detects the red fluorescence signal, and interpretation of result software is Elite 4.0 and DNA Multicycle on Epics Elite flow cytometer.
Table 2 T12 handles dna content analysis (%) treatment times (h) 0248 24Sub-G1 9.1 28.9 74.8 79.8 79.8 of HL-60 cell different time
Table 3 T12 handles dna content analysis (%) treatment times (h) 0 12 24 48 72Sub-G1 5.1 10.3 27.4 54.0 90.0 of Re-01 cell different time shown in table 2,3, handled 2 hours with T12 (1 μ g/ml) difference HL-60 cell, the Re-01 cell can the inferior G1 apoptotic peak of dna content less than 2 times of bodies occur by inducing cell in 12 hours, and increased with the inferior G1 apoptotic peak of the prolongation content of action time.
Table 4 T12 changed the cell cycle of HL-60 cytosis different time
Action time (h) G0/G1 S G2/M
control 32.0 53.7 14.3
2 29.5 59.2 11.3
4 71.8 25.3 2.9
Table 5 T12 changed the cell cycle of Re-01 cytosis different time
Treatment time (h) G0/G1 S G2/M
control 28.4 60.6 11.0
24 38.1 36.9 25.0
48 40.7 43.3 16.0
From table 4,5 as can be seen, HL-60 cell and two kinds of cell major parts of Re-01 without drug treating all are in the S phase, after T12 handles different time respectively, visible cell G0/G1 phase per-cent increases, show under 1 μ g/ml mass action, T12 can obviously reduce the cell colony of S phase, and increases the cell colony of G0/G1 phase, and its effect is time-dependent manner.
Embodiment 7 the present invention make the apoptosis-related protein enzyme activation and the Bcl-2 albumen of degrading
Apoptosis is the programmed death process that is subjected to multifactor regulation and control, and many oncogenes, cancer suppressor gene have participated in this process, as bcl-2.In the growth inhibiting analysis of T12 to six tumour cells, we find that the cancer cells of unconventionality expression Bcl-2 shows special susceptibility of the present invention, and GI50 significantly is lower than the cancer cells (as shown in table 6) that Bcl-2 expresses feminine gender or low expression level.
The GI50 of overexpression of table 6 part and low expression level Bcl-2 cell relatively
Bre-01
*?Bre-07 Col-06
* Col-05 Mel-08
* Mel-05
0.055 0.3 0.03 0.2 0.07 0.25
"
*" clone (Western blot detection) of expression high expression level Bcl-2
Therefore infer that the effect of T12 cell death inducing may be relevant with bcl-2 oncogene or its product.In order to confirm this supposition, use anti-Bcl-2 monoclonal antibody and be material with the HL-60 cell of unconventionality expression Bcl-2, carry out the protein immunoblot reaction, detect Bcl-2 protein expression state after the effect of the present invention.Method is described as follows.
HL-60 cell 1 * 10 after drug treating
6, directly be suspended from sample preparation liquid (20mmol/L Tris/HCl pH 8.0,4%SDS after centrifugal, 30% glycerine, 10% beta-mercaptoethanol and an amount of tetrabromophenol sulfonphthalein), aspirate repeatedly with microsyringe, make lysis, 100 ℃ were boiled 3 minutes, directly went up sample and separated through 12%SDS-PAGE, gel is transferred on the pvdf membrane, the sealing of 5% skim-milk is spent the night, and adds anti-Bcl-2 monoclonal antibody, acts on 2 hours, wash behind the film and to cross with horseradish that the sheep anti mouse two of itching thing enzyme labelling is anti-hatches the chemoluminescence method color development treatment
Figure 2 shows that T12 effect HL-60 cell 24,48 hours (hole 4,5), with untreated cell (hole 6) relatively, the below in Western blot master tape position (p26) increases a degraded band (relative molecular weight is approximately 20kDa) that can react with special monoclonal antibody.The T12 treatments B re-01 breast cancer cell 48 hours (hole 7) of same concentration also can obtain identical trace reaction result.But the present invention does not induce lymphoma Ly-01 cell Bcl-2 degrade (hole 1,2).The Bcl-2 proteolytic degradation can make the Bcl-2 inactivation and quicken apoptosis of tumor cells (Yamamoto AM et al, Leukemia 1998; 12:1467).The degraded prompting cell death inducing process of the present invention of Bcl-2 has caused the activation of the apoptotic proteins enzyme of degradable Bcl-2.Further compare the present invention and its lead compound triptolide, T8 and Tripterysium Glucosides powder and caused the active difference that raises of HL-60 apoptosis protein, four kinds of compounds have been detected under 1 μ g/ml concentration of treatment, the intensity of mediated apoptosis proteasome degradation Bcl-2, result such as Fig. 3: the intensity of the present invention (hole 5) mediation proteasome degradation Bcl-2 is apparently higher than other three kinds of compounds (hole 1: contrast, hole 2-4 is respectively Tripterysium Glucosides powder, T and T8).
The synergy of embodiment 8 the present invention and antisense nucleotide
Antisense nucleotide is a kind of new bio technology medicine that is born the nineties, this medicine specifically, is optionally attacked and is caused the gene of disease and specific base sequence thereof, thereby block its active and proteinic synthesizing, finally reach the purpose of treatment tumour.The high expression level of oncogene bcl-2 and the generation of multiple human tumor and develop relevantly, its gene product suppresses the apoptosis pathway of tumour cell.Antisense nucleotide effect target gene also makes it inactivation, has very high sequence-specific, but feels simply helpless for the cancer protein that has generated.Therefore utilize synthetic the bcl-2 antisense nucleotide (Chinese patent application number: the 98 1 11872.0) cancer cells of effect unconventionality expression Bcl-2, seal the generation that gene product Bcl-2 is promptly sealed in the expression of its oncogene; Utilize the present invention can the effect of apoptosis-induced associated protein enzyme activated again, make the Bcl-2 proteolytic degradation that has generated, the mode of the combination treatment tumour cell of this novelty will provide new thinking for the clinical treatment tumour disease.With colon cancer cell Col-06 is material, successively handles with antisense nucleotide and the present invention.And with independent T12 and separately antisense in contrast, method is as described below:
The antisense nucleotide extracorporeal treatment adopts antisense nucleotide, and the mixed liquid of liposome is pressed the desired concn dilution and handled cell with serum-free 1640, cultivates 6 hours for 37 ℃, discard old nutrient solution, after serum-free 1640 washings once, add the substratum that contains 10% calf serum, 37 ℃ of 5%CO
2Continue to be cultured to 72 hours under the condition; Of the present invention group of processing directly is diluted to desired concn with containing blood serum medium, function cells 48 hours; Coupling antisense nucleotide group of the present invention is then handled through antisense earlier, adds the present invention, continuation effect 48 hours after 24 hours.The cell contrast was three multiple holes when all test concentrations comprised the blank cell contrast of medicine and dosing zero.Detect to adopt and carry out with embodiment 3 identical methods.
The result as shown in Figure 4.Separately with effect Col-06 cell of the present invention, its GI
50Be approximately 0.04 μ g/ml, handle cell GI with the antisense coupling
50Less than 0.001 μ g/ml, reduced more than 50 times than the independent effect of T12.
Embodiment 9 acute toxicity tests of the present invention
Toxicity test is the important step of PTS research, and mouse is an animal the most frequently used in the toxicity research.Toxicity test of the present invention adopts Kunming mouse abdominal injection single-dose, the toxic reaction that animal produces after the observation administration, and measure its medium lethal dose.Kunming mouse, body weight: 20 ± 1 grams, 6 ages in week.24 ± 2 ℃ of room temperatures, relative humidity 65 ± 10% conditions are fed, and raise with standard full nutrition granulated feed, freely ingest and drink water.5 dosage groups are established in test, and the dosage spacing is 0.8.The animal random packet, 20 every group, male and female half and half.Observe immediate reaction after the administration, the death distribution of record mouse in two weeks, dead mouse and two all backs survival mice are dissected gross necropsy, when finding pathological tissues is arranged, this tissue are carried out microscopy.Test-results is calculated with the Bliss method.
With the LD of abdominal injection single-dose to the Kunming mouse acute toxicity test
50Value is: 74.42mg/kg, there was no significant difference between the sex, P value＞0.05.The LD of lead compound triptolide
50Value is: and the existing report of 0.85mg/kg body weight (Folkman J Nature Med 1995,1:27).Compare therewith, toxicity of the present invention is starkly lower than lead compound T.
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| US7626044B2 (en) | 2002-12-27 | 2009-12-01 | Shanghai Institute Of Materia Medica Chinese Academy Of Sciences | Triptolide derivatives and their uses |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100431547C (en) * | 2006-06-12 | 2008-11-12 | 中国人民解放军第三军医大学第一附属医院 | A triptolide composite preparation for treating osteoarthritis by joint intracavity injection |
| CN102286065B (en) * | 2010-06-18 | 2012-11-14 | 中国科学院上海药物研究所 | Abietane-type diterpene derivatives, preparation methods and applications thereof |
| CN102443040A (en) * | 2010-10-14 | 2012-05-09 | 中国科学院上海药物研究所 | N-substituted fluorinated phenyl-14 beta-(aminomethyl) triptodiolide derivative and preparation method and application thereof |
| CN102443044B (en) * | 2010-10-14 | 2013-11-13 | 中国科学院上海药物研究所 | N-substituted ester-containing phenyl-14 Beta-aminomethyl epitriptolide derivatives, preparation method and use thereof |
| CN102443045A (en) * | 2010-10-14 | 2012-05-09 | 中国科学院上海药物研究所 | N-substituted phenyl-14 beta -(aminomethyl) triptodiolide derivative and preparation method and application thereof |
| CN102443042B (en) * | 2010-10-14 | 2013-11-13 | 中国科学院上海药物研究所 | N-substituted methoxy-containing phenyl-14 Beta-aminomethyl epitriptolide derivatives, preparation method and use thereof |
| CN102443039B (en) * | 2010-10-14 | 2013-11-13 | 中国科学院上海药物研究所 | N-substituted amino phenyl-14 beta-(aminomethyl) triptodiolide derivative and preparation method and application thereof |
| CN102443043B (en) * | 2010-10-14 | 2013-11-13 | 中国科学院上海药物研究所 | N-substituted-14 beta -(aminomethyl) triptodiolide derivative and preparation method and application thereof |
| CN110407850A (en) * | 2019-08-16 | 2019-11-05 | 福建省医学科学研究院 | (5R) -5- hydroxy triptolide derivative and its preparation method and application |
| WO2023143330A1 (en) * | 2022-01-25 | 2023-08-03 | 杭州卫本医药科技有限公司 | Tripterygium wilfordii diterpene epoxide having function of killing myc positive cell, preparation method therefor, and application thereof |
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| CN1052860A (en) * | 1989-12-22 | 1991-07-10 | 中国医学科学院皮肤病研究所 | The preparation method of tripchlorolide and antifertility purposes |
| CN1052861A (en) * | 1989-12-22 | 1991-07-10 | 中国医学科学院皮肤病研究所 | The preparation method of wilfordii alcoholic lactone and antifertility purposes |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1052860A (en) * | 1989-12-22 | 1991-07-10 | 中国医学科学院皮肤病研究所 | The preparation method of tripchlorolide and antifertility purposes |
| CN1052861A (en) * | 1989-12-22 | 1991-07-10 | 中国医学科学院皮肤病研究所 | The preparation method of wilfordii alcoholic lactone and antifertility purposes |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7626044B2 (en) | 2002-12-27 | 2009-12-01 | Shanghai Institute Of Materia Medica Chinese Academy Of Sciences | Triptolide derivatives and their uses |
| WO2007112648A1 (en) * | 2006-04-04 | 2007-10-11 | Shanghai Institute Of Materia Medica, Chinese Academy Of Sciences | The medicinal compositions comprising triptolide derivatives and its dosage form and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1261602A (en) | 2000-08-02 |
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