CN102219822A - Ganoderic acid T amide derivative TLTO-A and synthetic method and application thereof - Google Patents
Ganoderic acid T amide derivative TLTO-A and synthetic method and application thereof Download PDFInfo
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- CN102219822A CN102219822A CN2011100718954A CN201110071895A CN102219822A CN 102219822 A CN102219822 A CN 102219822A CN 2011100718954 A CN2011100718954 A CN 2011100718954A CN 201110071895 A CN201110071895 A CN 201110071895A CN 102219822 A CN102219822 A CN 102219822A
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Abstract
The invention relates to a ganoderic acid T amide derivative TLTO-A in the technical field of biological medicines and a synthetic method and application thereof. In the synthetic method, the amide derivative TLTO-A with higher antitumor activity is obtained by modifying the structure of ganoderic acid T serving as a lead compound. The ganoderic acid T amide derivative TLTO-A has an obvious inhibitory effect on the growth and proliferation of human cervical cancer cell lines HeLa, human highly metastatic lung cancer 95 D and human liver cancer cell lines HepG2 and can induce the apoptosis of tumor cells, so the ganoderic acid T amide derivative TLTO-A is a potential antitumor medicine, and has a good application prospect.
Description
Technical field
What the present invention relates to is the compound and the methods for making and using same thereof in a kind of biological medicine technology field, specifically is a kind of ganoderic acid T amide derivatives TLTO-A and synthetic and application thereof.
Background technology
Cancer is serious harm human life and healthy disease, and its mortality ratio is only second to cardiovascular and cerebrovascular diseases, and its incidence is the trend that rises year by year.Therefore the research of cancer therapy drug is significant at life science, and is rich in challenge.Exploitation is novel, efficient, the new antitumoral medicine of low toxicity, high specificity is the vital task of cancer therapy drug research.
Triterpene is the important secondary metabolite of a class, extensively is present in plant and the higher fungi.A lot of researchs have reported that triterpene compound has good antitumor activity, can suppress growth of tumour cell, and the apoptosis of inducing tumor cell or the like.Glossy ganoderma (Ganoderma lucidum) is Basidiomycetes, polyporaceae, Ganoderma fungi, is exactly the famous and precious medicinal fungi of China since ancient times, and applicating history in several thousand has been arranged.Modern medicine study shows that one of wherein main activeconstituents is exactly a triterpene substance.
Ganoderic acid T is a tetracyclic triterpene, belongs to highly oxidized lanostane derivative.We can produce (Biotechnology Progress, 18,51-54,2002) efficiently by two stage fermentation cultured method in the laboratory.Further pharmacological evaluation proof ganoderic acid T has good antitumor activity.Ganoderic acid T can be induced lung carcinoma cell 95-D apoptosis (Life Science by the plastosome approach, 80,205-211,2006), in addition, the interior experiment of external and body proves that also it can be by suppressing transfer (Pharmacology Report, 62 that the proteic expression of MMPs suppress tumour cell, 157-170,2010).
Summary of the invention
The present invention is directed to the prior art above shortcomings, a kind of ganoderic acid T amide derivatives TLTO-A and synthetic and application thereof are provided, with the ganoderic acid T is lead compound, it is carried out structure of modification, obtain having the amide derivatives TLTO-A of better anti-tumor activity, obtained novel and high-efficiency on the one hand, had better optionally triterpenes anticancer compound, on the other hand the structure and the active relation of Ganodenic acid are carried out desk study, significant to the exploitation of cancer therapy drug.
The present invention is achieved by the following technical solutions:
The present invention relates to a kind of ganoderic acid T amide derivatives TLTO-A, its chemical name (22S, 24E)-3 α, 15 α, 22-triacetoxy-5 α-lanosta-7,9 (11), 24-trien-26-oic acid amide, chemical structural formula is:
The preparation route that the present invention relates to above-mentioned ganoderic acid T amide derivatives is as follows:
Its concrete steps are: ganoderic acid T is dissolved in the methylene dichloride, ice bath cooling and the environment that stirs drip oxalyl chloride down, dropwise the back to room temperature, continue to feed the exsiccant ammonia then, ammonia and excessive oxalyl chloride are reacted completely, add the entry termination reaction at last and adopt ethyl acetate to stir extraction and obtain the ganoderic acid T amide derivatives.
The present invention relates to the application of above-mentioned ganoderic acid T amide derivatives TLTO-A, be used to prepare the medicine that suppresses human cervical carcinoma cell strain HeLa, the high lung cancer cell line 95D of transfer of people and human hepatoma cell strain HepG2.
Through the external activity test shows, this compound is to human cervical carcinoma cell strain HeLa, the high lung cancer cell line 95D that shifts of people, the growth of human hepatoma cell strain HepG2 and propagation have the obvious suppression effect, further experiment is an example with the HeLa cell, proving the apoptosis that it can the inductive tumour cell, is a kind of potential antitumor drug therefore, has a good application prospect.
Description of drawings
Fig. 1 is TLTO-A and the hydroxycamptothecine synoptic diagram that influences to the HeLa cell survival rate.
Fig. 2 is the effect synoptic diagram of TLTO-A to the tumour cell HeLa cell cycle.
Fig. 3 is the apoptotic effect synoptic diagram of TLTO-A inducing tumor cell HeLa.
Fig. 4 reduces the effect synoptic diagram of tumour cell HeLa mitochondrial membrane potential for TLTO-A.
Fig. 5 activates tumour cell HeLa caspase-3 and the active effect synoptic diagram of caspase-9 for TLTO-A.
Embodiment
Below embodiments of the invention are elaborated, present embodiment is being to implement under the prerequisite with the technical solution of the present invention, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Embodiment 1
TLTO-A synthesizes and separates
Get the there-necked flask of volume 100mL, ganoderic acid T 100mg (0.16mmol) is dissolved in the 30mL methylene dichloride, ice bath is cooled to 0-5 ℃, stirs down and drips oxalyl chloride 0.14mL (1.6mmol), finishes and rises to room temperature, continues to stir 3h.Continue to feed the exsiccant ammonia then, 3h is carried out in reaction, and ammonia and excessive oxalyl chloride are reacted completely.Question response back fully adds 10mL water termination reaction, and the ethyl acetate that adds 20mL again stirs extraction.Get organic layer, use 20mL ethyl acetate extraction twice respectively again, merge organic phase, concentrating under reduced pressure.Separate moving phase condition ether: sherwood oil=4: 1 with the preparation silica-gel plate.Purpose product band scraped with ethyl acetate it soak proposed, drying under reduced pressure, white crystalline solid 40.8mg, product yield: 40.8%.Product is composed through EI-MS,
1H NMR composes mensuration.Analytical results is as follows:
EI-MS spectrum: m/z:612.3884[M+H]
+, 634.3727[M+Na]
+, 1245.7642[2M+Na]
+.
1H-NMR composes (CDCl
3, 400MHz): δ: 0.61 (s, 3H), 0.83 (s, 3H), 0.91-0.94 (m, 9H), 0.99 (s, 3H), 1.09 (d, J=6.4Hz, 4H), 1.21 (s, 1H), 1.42-1.78 (m, 7H), 1.80 (s, 3H), 2.00 (s, 3H), 2.01 (s, 3H), 2.03 (s, 3H), 2.31-2.53 (m, 2H), and 2.45-2.53 (m, 1H), 4.62 (s, 1H), 4.96 (t, J=7.0Hz, 1H), 5.02-5.06 (m, 1H), 5.28 (d, J=6.0Hz, 1H), 5.44 (s, 1H), 5.52 (m, 2H), 6.59 (t, J=7.2Hz, 1H).
Embodiment 2
TLTO-A is to the toxic action of tumour cell:
Testing used cell strain is: HeLa (human cervical carcinoma cell), and 95D (the high lung carcinoma cell that shifts of people), HepG2 (human liver cancer cell) is all available from Chinese science research institute Shanghai cell institute; HF (people inoblast of former generation) is by the week swallow teacher of engineering seminar of animal tissues of biotechnology institute of East China University of Science present.HeLa, 95D, HepG2, HF cell all add conventional cultivation of 10% foetal calf serum with the DMEM substratum and go down to posterity.
Experimental technique is international mtt assay: according to cell growth rate, the tumour cell that will be in logarithmic phase is inoculated in 96 well culture plates with 100 μ l/ holes, and inoculum density is 10000～20000/ holes, and adherent growth 5～18h is dosing 1 μ l/ hole again.Each concentration is established 5 multiple holes.And the solvent that respective concentration is set contrasts and acellular zeroing hole.Tumour cell is at 37 ℃, 5%CO
2Cultivated 24 hours under the condition.Add MTT, final concentration is 50 μ g/mL.5%CO
2After 37 ℃ of incubators are hatched 4 hours, measure the absorbance of 570nm and 630nm on the microplate reader.Calculate inhibiting rate.
Cell inhibitory rate=[1-(test group OD mean value-blank group OD mean value)/(control group OD mean value-blank group OD mean value)] * 100%
Experimental result such as table 1 and shown in Figure 1.
Table 1:
Above embodiment explanation, TLTO-A compares with the lead compound ganoderic acid T, has stronger inhibition tumour cell HeLa, 95D, the ability of HepG2 growth, IC
50See Table 1.This compound all has restraining effect to each tumour cell as shown in Table 1, and to normal cell inhibiting less than inhibition to tumour cell, can think that TLTO-A has tangible anti-tumor activity and selectivity.The activity of accompanying drawing 1 usefulness positive drug hydroxycamptothecine and TLTO-A compares, our synthetic compound exhibits the effect of better inhibition tumour cell HeLa growth.The test triplicate is averaged.
TLTO-A tests the effect of HeLa cell cycle:
General culture method is cultivated the HeLa cell, administration, and with the equal-volume solvent in contrast, collecting cell after 24 hours is washed twice back with PBS and is spent the night with 70% alcohol fixation.The centrifugal ethanol that goes of 1000rpm/10min washes twice with the PBS that contains 1% foetal calf serum, and is resuspended with the PBS that contains 1% foetal calf serum again, adjusts cell concn about 10
6Individual/mL, add the RNA enzyme of no DNA enzyme, final concentration is 100U/mL, 37 ℃ of insulation 30min add PI, final concentration is 50 μ g/mL, normal temperature reaction 30min down darkling, place 20min on ice after, carry out flow cytometer and detect, write down 10000 cells at every turn.Experimental result is seen accompanying drawing 2.
Above embodiment explanation, the effect of TLTO-A different concns can make the HeLa cell stop at the G0/G1 phase, and have concentration dependent after 24 hours.The SubG1 phase also obviously increases in addition, and the ability that shows this compound cell death inducing also increases along with concentration and strengthens.
Embodiment 4
TLTO-A induces the detection of human cervical carcinoma cell apoptosis
The HeLa cell is through 5,10, and after the TLTO-A of three kinds of concentration of 20 μ M stimulated 24h, PBS washing twice added trysinization, and serum is ended, and (1500rpm 10min), makes single-cell suspension liquid with Binding liquid to centrifugal collecting cell, and cell concn is 2 * 10
5Individual/mL.Add AnnexinV-FITC liquid, final concentration is 10ng/mL, and room temperature was hatched 15 minutes in the dark, and with PI dyeing, final concentration is 5 μ g/mL then.Use flow cytometry analysis, write down 10000 cells at every turn.Experimental result is seen accompanying drawing 4.
Above embodiment explanation, the effect of TLTO-A different concns was obviously induced the apoptosis of HeLa cell after 24 hours, comprise early apoptosis and late period apoptosis, and have concentration dependent.
TLTO-A reduces the detection of cell mitochondrial membrane potential
Plastosome changes membrane potential and adopts flow cytometry analysis.After the TLTO-A of different concns handled HeLa cell 24h, collecting cell PBS cleaned twice, and cell concn is 2 * 10
5Individual/mL, add 8 μ M Rhodamine123 (Molecular Probes) and under 37 ℃, hatched 15 minutes.The corresponding change in fluorescence flow cytometry analysis of cell.The test triplicate is averaged.The result as shown in Figure 5.
Above embodiment explanation, inducing apoptosis of tumour cell is relevant with mitochondrial functional status, as changing the mitochondrial membrane integrity.Utilize flow cytometer to detect the mitochondrial inner membrane commentaries on classics membrane potential influence of TLTO-A to cancer cells, Intramitochondrial fluorescence intensity reduces along with the increase of concentration of treatment, and after 20 μ M handled back 24h, fluorescence intensity approximately reduced by 60%.Results suggest TLTO-A has influenced the commentaries on classics membrane potential of cancer cells, and making changes membrane potential decline.
Embodiment 6
TLTO-A activates caspase-3 and-9 active detections
Caspase-3 and caspase-9 use narrow spectrum test kit detect (KeyGen Biotech Co., Nanjing, China).After the TLTO-A of different concns handled HeLa cell 24h, collecting cell PBS cleaned twice, adds lysate ice bath cracking 1h then, and 12000rpm/min centrifuging and taking supernatant is adjusted protein concentration.According to working instructions, add the substrate of supernatant liquor and reaction in 96 orifice plates, hatch 2h under 37 ℃.Detect a sample light absorption value down with microplate reader 405nm.The test triplicate is averaged.
Above embodiment explanation, the effect of TLTO-A different concns obviously increased the activity of caspase-3 and caspase-9 after 24 hours, and had concentration dependent.Further proof TLTO-A has the ability of inducing apoptosis of tumour cell.
Claims (4)
1. ganoderic acid T amide derivatives TLTO-A is characterized in that having following chemical structural formula:
2. the application of a ganoderic acid T amide derivatives TLTO-A according to claim 1 is characterized in that, is used to prepare the medicine of anticancer.
3. application according to claim 2 is characterized in that, described cancer cells comprises: human cervical carcinoma cell strain HeLa, high lung cancer cell line 95D and the human hepatoma cell strain HepG2 of shifting of people.
4. one kind according to the preparation method of above-mentioned people with the described ganoderic acid T amide derivatives of claim, it is characterized in that: ganoderic acid T is dissolved in the methylene dichloride, ice bath cooling and the environment that stirs drip oxalyl chloride down, dropwise the back to room temperature, continue to feed the exsiccant ammonia then, ammonia and excessive oxalyl chloride are reacted completely, add the entry termination reaction at last and adopt ethyl acetate to stir extraction and obtain the ganoderic acid T amide derivatives, described preparation method's chemical formula is:
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104000829A (en) * | 2014-05-15 | 2014-08-27 | 上海应用技术学院 | Application of ganoderic acid as p53-MDM2 interaction inhibitor |
| MD4300C1 (en) * | 2012-12-28 | 2015-03-31 | Государственный Университет Молд0 | Inhibitor of HepG2 cell proliferation in liver cancer based on chloro-[2-phenyl(pyridine-2-yl)methanone-4-(3-methoxyphenyl)thiosemicarbazono]nickel |
| CN105294815A (en) * | 2015-11-23 | 2016-02-03 | 江苏康缘药业股份有限公司 | Compound and preparation method and application thereof |
| CN112574272A (en) * | 2020-12-25 | 2021-03-30 | 中国医学科学院药用植物研究所 | Preparation and application of ganoderic acid A derivative |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| MD4300C1 (en) * | 2012-12-28 | 2015-03-31 | Государственный Университет Молд0 | Inhibitor of HepG2 cell proliferation in liver cancer based on chloro-[2-phenyl(pyridine-2-yl)methanone-4-(3-methoxyphenyl)thiosemicarbazono]nickel |
| CN104000829A (en) * | 2014-05-15 | 2014-08-27 | 上海应用技术学院 | Application of ganoderic acid as p53-MDM2 interaction inhibitor |
| CN105294815A (en) * | 2015-11-23 | 2016-02-03 | 江苏康缘药业股份有限公司 | Compound and preparation method and application thereof |
| CN105294815B (en) * | 2015-11-23 | 2017-06-13 | 江苏康缘药业股份有限公司 | A kind of compound and its preparation method and application |
| CN112574272A (en) * | 2020-12-25 | 2021-03-30 | 中国医学科学院药用植物研究所 | Preparation and application of ganoderic acid A derivative |
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