CN106083898A - A kind of tumor-targeting gambogic acid compounds and its production and use - Google Patents
A kind of tumor-targeting gambogic acid compounds and its production and use Download PDFInfo
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Abstract
The invention belongs to natural medicine field, disclose a kind of tumor-targeting gambogic acid compounds and its production and use.This compound has the structure as shown in formula (I).Wherein, G is gambogic acid compounds；P is oligomerization ethylene glycol or the derivant of one Amino End Group；FA is folic acid or folacin；FA is with ester bond or amido link and P coupling, and P is with the carboxyl coupling of terminal hydroxy group Yu G.By external MTT test experience, find that it has more preferable inhibition to tumor cell line (HeLa and HepG2 cell), stronger 4 10 times than gambogic acid medicament, but low more than 6 times to the toxicity of normal liver cell LO2.The gambogic acid compounds of this modified with folic acid has the characteristic of folacin receptor mediated targeted tumor cell, the problem that can overcome the low and easy Multidrug resistance of gambogic acid cancer therapy drug selectivity, reaches to be removed the effect of cancerous cell by approach such as apoptosis, induction autophagy.G‑P‑FA(I).
Description
Technical field
The invention belongs to natural medicine field, particularly to a kind of tumor-targeting gambogic acid compounds and preparation side thereof
Method and purposes.
Background technology
Cancer is a big pertinacious disease of serious harm human health, has become the second largest killer being only second to cardiovascular diseases,
Finding cancer therapy drug and study its mechanism of action, especially exploitation has the medicine of target tumor effect, significant.
Total cambogic acid (containing gamlogic acid, neogambogic acid and allogambogic acid) is to extract separation from Resina garciniae secretion dry resin to obtain
Gambogic acid active component, according to existing research report, it has the active anticancer of excellence, but the cancerous cell of this compounds
The deficiencies such as targeting selectivity shortage, poorly water-soluble, toxicity are big, seriously limit its use clinically.
Folacin receptor (folate receptor, FR) is a kind of transmembrane glycoprotein, mainly has FR-α, three kinds of Asias of-β ,-γ
Type, molecular weight, folic acid and the like is had higher affinity (Kd, and there is saturability, specificity=0.1～10nM)
And the feature such as reversibility.Existing research report FR is expressed at kinds of tumor cells excessive surface, if FR-α is at the ovum more than 90%
Nest cancer, breast carcinoma, carcinoma of endometrium, hepatocarcinoma, renal carcinoma, colon cancer and pulmonary carcinoma camber are expressed, and FR-β mainly comes at non-epithelium
High expressed in the malignant tumor in source, granulocyte leukemia and cancer of pancreas and arthritis activity macrophage and inflammation.FR-γ
Then overexpression in some hematopoietic cells are such as hematological malignancies.High conservative in the normal tissue, FR seldom expresses not even
Express, and different from the FR of cancer cell surfaces, in polar contribution, its with in aqueous solution vitamin B family folic acid (folate,
Folic acid, FA) and the like affinity more weak much than the FR adhesion of tumor cell surface, combine hardly.FR can
With by mediated cell endocytosis by FA and the like, or FA conjugate is played a role to intracellular by extracellular transport, and
Little in FA relative molecular mass, to be prone to modify and penetrate cancerous cell, immunogenicity low, the target spot time is short, blood is clear to have arrival
Except advantages such as speed are fast, penetration power is strong, Human immune responses is low.This tumor tissues targeted imaging being FR mediation and drug targeting
Treatment in cancer and rheumatic arthritis (the same overexpression of FR) is had laid a good foundation.
By domestic and foreign literature and patent retrieval, not yet it is found to have folacin coupling gambogic acid medicament and realizes cancer
Or the targeted dosing regimen precedent of rheumatic arthritis.
Summary of the invention
In order to overcome the shortcoming of prior art with not enough, the primary and foremost purpose of the present invention is to provide a kind of tumor-targeting rattan
Yellow acid compounds；This compounds is the gambogic acid compounds by modified with folic acid.
Another object of the present invention is to provide the preparation method of a kind of above-mentioned tumor-targeting gambogic acid compounds.
It is still another object of the present invention to provide the purposes of above-mentioned tumor-targeting gambogic acid compounds.
The purpose of the present invention is achieved through the following technical solutions:
A kind of tumor-targeting gambogic acid compounds, this compound has a structure as shown in formula (I):
G-P-FA
(I)
Wherein, G is gambogic acid compounds；
P is HOCH2(CH2OCH2)nCH2OH or HOCH2(CH2OCH2)nCH2NH2, wherein n is 0 or nature between 1～10
Number；
FA is folic acid or folacin, preferably folic acid；
Described FA is with ester bond or amido link and P coupling, and P is with the carboxyl coupling of terminal hydroxy group Yu G.
Described gambogic acid compounds is gamlogic acid, neogambogic acid, allogambogic acid, TH-2 or their mixture.
The mixture of gamlogic acid, neogambogic acid, allogambogic acid and TH-2 is properly termed as total cambogic acid.
The chemical formula of gamlogic acid (Gambogic acid, GA) is:
The chemical formula of neogambogic acid (Gambogenic acid, NGA) is:
The chemical formula of allogambogic acid (Allogambogic acid, AA) is:
The mean molecule quantity of described P is 61～600.
The preferred molecular weight 61 of described P, 62,105,106,149,150,237,238,281,282,325,326,413,
414,501 or 502, be designated as P61, P62, P105, P106, P149, P150, P237, P238, P281, P282, P325, P326,
P413, P414, P501 or P502.
Described folacin is can be with NH on P2Or the similar biology of folic acid of OH coupling, preferably 5-methyl tetrahydrochysene leaf
Acid, 5-formoxyl tetrahydrofolic acid, methotrexate or 5,10-methylene tetrahydrofolate.
The preparation method of above-mentioned tumor-targeting gambogic acid compounds, including following operating procedure:
(1) take folic acid to be dissolved in a solvent, at dehydrant N, N'-dicyclohexylcarbodiimide or 1-(3-dimethylamino third
Base)-3-ethyl-carbodiimide hydrochloride, and catalyst is under dimethylamino naphthyridine effect, 0 DEG C of stirring reacts 1～6h；Then
With folic acid equimolar than adding oligomerization ethylene glycol, from ice bath, rise high-temperature be stirred overnight to room temperature, lucifuge；Cross and filter by-product
Thing, filtrate concentrates, and ice ether sedimentation recrystallization or preparation liquid phase purification, lyophilizing i.e. obtains folacin coupled oligomerization ethylene glycol；
Or the active ester taking folic acid and the like is dissolved in anhydrous DMSO/TEA, add equimolar with folic acid active ester
The amidized derivant in oligomerization ethylene glycol one end, reacts under anhydrous condition overnight, and vacuum freeze-drying separates or chromatogram purification, to obtain final product
Folacin coupled oligomerization ethylene glycol amination derivant；
Described oligomerization ethylene glycol is HOCH2(CH2OCH2)nCH2OH, described oligomerization ethylene glycol one end is amidized derivative
Thing is HOCH2(CH2OCH2)nCH2NH2), wherein n is 0 or natural number between 1～10；
(2) gambogic acid compounds is dissolved in a solvent, at dehydrant N, N'-dicyclohexylcarbodiimide or 1-(3-bis-
Methylaminopropyl)-3-ethyl-carbodiimide hydrochloride, and catalyst is under dimethylamino naphthyridine effect, 0 DEG C is stirred reaction 1
～6h；Then press gambogic acid compounds mol ratio 1:1-3 amount add step (1) gained folacin coupled oligomerization ethylene glycol or
Folacin coupled oligomerization ethylene glycol amination derivant, rises high-temperature from ice bath and is stirred overnight to room temperature, lucifuge；Filtrate is dense
Contracting, ice ether or recrystallisation from isopropanol, chromatograph or prepare liquid phase purification, lyophilizing can obtain structure tumor as shown in formula (I)
Targeting gambogic acid compounds.
Step (1) described a solvent is CH2Cl2, DMSO or DMF；The time of described stirring reaction is 2.5h；Described room temperature is
25℃；In described anhydrous DMSO/TEA, the volume ratio of DMSO and TEA is 2:1；
Step (2) described a solvent is CH2Cl2, DMSO or DMF；The time of described stirring reaction is 2.5h；Described room temperature is
25℃。
The mol ratio of step (1) described folic acid, dehydrant and catalyst is 1:1:1～1:30:30；Step (2) described rattan
Yellow acid compounds, dehydrant, the mol ratio of catalyst are 1:1:1～1:25:25.
There is the synthesis schematic diagram of tumor-targeting gambogic acid compounds of logical formula I structure as shown in Figure 1.
Above-mentioned tumor-targeting gambogic acid compounds or its pharmaceutically acceptable salt are at preparation treatment Cancerous disease
Purposes in medicine.
Containing the tumor-targeting gambogic acid compounds of therapeutically effective amount or it is pharmaceutically acceptable in described medicine
Salt and pharmaceutically acceptable carrier.
Described pharmaceutically acceptable salt may is that the chemistry of folacin coupled gamlogic acid, neogambogic acid or allogambogic acid
In structure, phenolic hydroxyl group becomes sodium, magnesium, calcium salt or other salt.
The principle of the present invention is:
The present invention is based on folacin receptor mediated tumor cell targeting endocytosis principle, with modified with folic acid gambogic acid compounds solution
The certainly cancer target problem of gambogic acid compounds, the PEG derivant (HOCH of folic acid and linking group low polymerization degree simultaneously2
(CH2OCH2)nCH2OH or HOCH2(CH2OCH2)nCH2NH2, MW be less than 600, wherein n=0,1～10) have the most water-soluble
Property, biocompatibility and without advantages such as immunogenicities, it is possible to obtain there is the folacin coupled gambogic acid medicine of cancer target effect
Thing, improves drug effect, increases tumor cells selectivity, reduces toxic and side effects, is more suitable for anticancer Clinical practice.
The folacin coupled gambogic acid compounds technology preparation technology of the present invention is simple, and the extraction of gambogic acid compounds divides
Refer to pertinent literature from purification and prepare (Chen Baoren, the research I of Resina garciniae anticancer component, the separation of gamlogic acid and the mirror of structure
Fixed.Jiangxi Medical College's journal 1980；37(2):1-7；Lu returns treasured, Yang Xiuxian, Huang Qiaoshu.In Resina garciniae neogambogic acid separation and
Structure.Acta Pharmaceutica Sinica 1984；19(8):636-639.)
The present inventor finds the gambogic acid by water soluble molecules folic acid chemical modification in design and experiment invention
Compound (containing total cambogic acid, gamlogic acid, neogambogic acid, allogambogic acid and TH-2) has more preferable water solublity, dramatically increases
Tumor cells selectivity, improves active anticancer, greatly reduces the toxicity of proto-drug, compare (little with gamlogic acid sodium salt
The LD of Mus lumbar injection50=38.8mg/kg), the LD of part modified with folic acid gambogic acid medicament50Dramatically increase (G-P62-FA,
The mouse peritoneal injection LD of G-P106-FA, G-P150-FA and G-P238-FA50It is respectively 75.12,74.6,81.21 and
92.4mg/kg, converts with Resina garciniae acid number).
2% rabbit erythrocyte hemolytic test shows that the gambogic acid compounds of the modified with folic acid of the present invention non-show in 4h is thin
Cell lysis blood is assembled, gamlogic acid dosage of equal value > 20mg.Rabbit auricular vein irritant experiment shows the modified with folic acid Resina garciniae of the present invention
Acid compounds is dissolved in normal saline iv drip vascular irritation.
Showing through the test of cellulotoxic experiment mtt assay, the compounds of this invention is to having the tumor that folate receptor-positive is expressed
Cell (cervical cancer HeLa and hepatocarcinoma HepG2 etc.) growth in vitro inhibitory action IC50Significantly carry than proto-drug (as a example by gamlogic acid)
High 4-10 times.Normal liver cell LO2 toxicity data is shown that non-cancerous cells is had greatly by the compounds of this invention than proto-drug
In the IC of 6 times50Value, prompting modified with folic acid gambogic acid medicament has lower toxicity and more preferable tumor-targeting.Dissolubility
Experiment shows, (gamlogic acid is water insoluble, reports about 0.018mg/ml), the gambogic acid chemical combination after modified with folic acid compared with prototype
Thing water solublity significantly improves (about 1.4-20mg/ml), convenient to pharmaceutical dosage form preparation and vascular drug delivery etc..
Under the mediation of cancerous cell targeted molecular folic acid drives, the folacin coupled gambogic acid medicament of the present invention is in body
Can quickly enter the tumor cell of folacin receptor high expressed；Preferably short chain ethylene glycol and derivant cross structure thereof can improve medicine
Thing water solublity, can reduce Conjugate Molecules size again simultaneously thus improve its cancerous tissue penetrance.
Compared with prior art, the present invention has the following advantages and beneficial effect: by external MTT test experience, finds
It has inhibition more carefully to tumor cell line (HeLa and HepG2 cell), stronger 4-10 times than gambogic acid medicament, but
Low more than 6 times to the toxicity of normal liver cell LO2.The gambogic acid compounds of this modified with folic acid has folacin receptor mediated targeted
The characteristic of tumor, the problem that the low and easy Multidrug resistance of gambogic acid cancer therapy drug selectivity can be overcome, reach by apoptosis,
The approach such as induction autophagy remove the effect of cancerous cell.
Accompanying drawing explanation
Fig. 1 is the synthesis signal that the present invention has the tumor-targeting gambogic acid compounds compound of logical formula I structure
Figure.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited to this.
Embodiment 1
1) preparation of folic acid-ethylene glycol: take folic acid (1.0g, 2.3mmol) and be dissolved in the DMSO of 40ml, dehydrant (N,
N'-dicyclohexylcarbodiimide, DCC, 0.47g, 2.3mmol) and catalyst (to dimethylamino naphthyridine, DMAP, 0.29g,
2.3mmol) under effect, 0 DEG C of stirring 4h；Then folic acid equimolar is pressed than adding ethylene glycol (HOCH2CH2OH), raise from ice bath
Temperature is to room temperature 25 DEG C, and lucifuge is stirred overnight 24h；Crossing and filter by-product, filtrate concentrates, ice ether sedimentation recrystallization or prepare liquid
Phase purification, lyophilizing i.e. obtains folic acid-ethylene glycol coupled product (HOCH2CH2OCO-FA)。
2) preparation of folic acid-ethylene glycol-gamlogic acid: take gamlogic acid (0.15mmol) and be dissolved in the DMSO of 60ml, in dehydration
Under agent DCC (3.0mmol) and catalyst DMAP (3.0mmol) effect, 0 DEG C of stirring 6h；Then gamlogic acid mol ratio 1:3 is pressed extraordinarily
Enter folic acid-ethylene glycol coupled product (HOCH2CH2OCO-FA), from ice bath, liter high-temperature is to room temperature 25 DEG C, and lucifuge is stirred overnight.
Filtrate concentrates, and ice ether or recrystallisation from isopropanol chromatograph or prepare liquid phase purification, and lyophilizing can obtain cancerous cell targeting rattan
Yellow acid compounds GA-COOCH2CH2OCO-FA (yield about 65%).
1HNMR (500MHz, DMSO): 3.6 and about 4.2 is the peak of hydrogen on ethylene glycol, and other peaks are gamlogic acid and folic acid
The peak of upper hydrogen；1.24-1.46 (GA:C24-H, C25-H, 6H), 1.56-2.16 (GA:C19-H, C20-H, C21-H, C34-H,
C35-H, C36-H, C39-H, C40-H, 21H), 2.26-2.66 (GA:C22-H, C26-H, C29-H, 6H, FA:C27-H, 2H),
2.96 (GA:C11-H, 1H), 3.23 (GA:C31-H, 2H), 4.45-4.63 (FA:C13-H, C24-H, 3H), 5.27 (GA:C37-
H, 1H), 5.84 (GA:C32-H, 1H), 6.04 (GA:C3-H, 1H), 6.71 (GA:C27-H, 1H), 6.84-6.92 (GA:C4-H,
C10-H,2H；FA: C16-H on phenyl ring, C17-H, 2H；), 7.61 (FA: C18-H on phenyl ring, C19-H, 2H).
IR: significantly increase 1728cm in infrared spectrum-1Ester carbonyl group peak, illustrate that gamlogic acid and folic acid are by ethylene glycol
On ester bond connects.
Embodiment 2
1) preparation of folic acid-ethylene glycol 282: take folic acid (1.0g, 2.3mmol) and be dissolved in the DMSO of 40ml, at dehydrant
Under the effect of (DCC, 0.47g, 2.3mmol) and catalyst (DMAP, 0.29g, 2.3mmol), 0 DEG C of stirring 4h；Then folic acid etc. is pressed
Mol ratio adds ethylene glycol 282 (HOCH2(CH2OCH2)5CH2OH), from ice bath, liter high-temperature is to room temperature 25 DEG C, and lucifuge is stirred
Night 24h；Crossing and filter by-product, filtrate concentrates, and ice ether sedimentation recrystallization or preparation liquid phase purification, lyophilizing i.e. obtains folic acid-second two
Alcohol 282 coupled product (HOCH2(CH2OCH2)5CH2OCO-FA)。
2) preparation of folic acid-ethylene glycol 282-gamlogic acid: take gamlogic acid (0.15mmol) and be dissolved in the DMSO of 60ml, de-
Under water preparation DCC (3.0mmol) and catalyst DMAP (3.0mmol) effect, 0 DEG C of stirring 6h；Then gamlogic acid mol ratio 1:3 times is pressed
Add folic acid-ethylene glycol 282 coupled product (HOCH2(CH2OCH2)5CH2OCO-FA), from ice bath, high-temperature is risen to room temperature 25
DEG C, lucifuge is stirred overnight；Filtrate concentrates, and ice ether or recrystallisation from isopropanol chromatograph or prepare liquid phase purification, and lyophilizing can obtain
Obtain cancerous cell targeting gambogic acid compounds GA-COOCH2(CH2OCH2)5CH2OCO-FA。
1HNMR (500MHz, DMSO): 3.6 and about 4.2 is the strong peak of hydrogen on ethylene glycol 282, other peaks be gamlogic acid and
The peak of hydrogen on folic acid；1.24-1.46 (GA:C24-H, C25-H, 6H), 1.56-2.16 (GA:C19-H, C20-H, C21-H, C34-
H, C35-H, C36-H, C39-H, C40-H, 21H), 2.26-2.66 (GA:C22-H, C26-H, C29-H, 6H, FA:C27-H,
2H), 2.96 (GA:C11-H, 1H), 3.23 (GA:C31-H, 2H), 4.45-4.63 (FA:C13-H, C24-H, 3H), 5.27 (GA:
C37-H, 1H), 5.84 (GA:C32-H, 1H), 6.04 (GA:C3-H, 1H), 6.71 (GA:C27-H, 1H), 6.84-6.92 (GA:
C4-H,C10-H,2H；FA: C16-H on phenyl ring, C17-H, 2H；), 7.61 (FA: C18-H on phenyl ring, C19-H, 2H).
IR: significantly increase 1727cm in infrared spectrum-1Ester carbonyl group peak, illustrate that gamlogic acid and folic acid pass through ethylene glycol
282 connect with ester bond.
Embodiment 3
1) preparation of folic acid active ester (γ-NHS-FA): take folic acid (1.0g, 2.3mmol) and be dissolved in the anhydrous DMSO of 40ml
In, add that 0.5ml TEA triethylamine is mixed to be incorporated in room temperature lucifuge under water-less environment and be stirred overnight；Then 0.47g is mixed
(2.3mmol) DCC and 0.26g (2.3mmol) NHS, lucifuge stirring 24h, be filtered to remove by-product dicyclohexylurea, cryogenic vacuum
It is dried and removes DMSO and TEA, obtain folic acid active ester.
2) preparation of folic acid-ethanolamine: folic acid active ester clear-cut for vacuum is dissolved in the anhydrous DMSO/TEA of 1.5ml
In (volume ratio 2:1) mixed solution, take ethanolamine equimolar with folic acid active ester and add in mixed solution under anhydrous condition
Overnight, the clear-cut reactant liquor of vacuum i.e. obtains folacin coupled ethanolamine (HOCH in reaction2CH2NHCO-FA)。
3) preparation of folic acid-ethanolamine-gamlogic acid: take gamlogic acid (0.15mmol) and be dissolved in the anhydrous DMSO of 60ml,
Under dehydrant DCC (3.0mmol) and catalyst DMAP (3.0mmol) effect, 0 DEG C of stirring 6h；Then gamlogic acid mol ratio 1:3 is pressed
Add folacin coupled ethanolamine (HOCH again2CH2NHCO-FA), from ice bath, liter high-temperature is to room temperature 25 DEG C, and lucifuge is stirred overnight.
Filtrate concentrates, and ice ether or recrystallisation from isopropanol chromatograph or prepare liquid phase purification, and lyophilizing can obtain cancerous cell targeting rattan
Yellow acid compounds GA-COOCH2CH2NHCO-FA。
1HNMR (500MHz, DMSO): 3.6 and about 4.2 is 2-CH on ethanolamine2The peak of-upper hydrogen, other peaks are Resina garciniae
The peak of hydrogen in acid and folic acid；1.24-1.46 (GA:C24-H, C25-H, 6H), 1.56-2.16 (GA:C19-H, C20-H, C21-H,
C34-H, C35-H, C36-H, C39-H, C40-H, 21H), 2.26-2.66 (GA:C22-H, C26-H, C29-H, 6H, FA:C27-
H, 2H), 2.96 (GA:C11-H, 1H), 3.23 (GA:C31-H, 2H), 4.45-4.63 (FA:C13-H, C24-H, 3H), 5.27
(GA:C37-H, 1H), 5.84 (GA:C32-H, 1H), 6.04 (GA:C3-H, 1H), 6.71 (GA:C27-H, 1H), 6.84-6.92
(GA:C4-H, C10-H, 2H；FA: C16-H on phenyl ring, C17-H, 2H；), 7.61 (FA: C18-H on phenyl ring, C19-H, 2H),
8.07 (NH, broad peaks).
IR: significantly increase 1500-1650cm in infrared spectrum-1Amide peak and 1727cm-1Ester carbonyl group peak, explanation
Gamlogic acid and folic acid are connected by amido link and the ester bond of ethylene glycol amine.
Embodiment 4
1) preparation of folic acid-ethylene glycol: take folic acid (1.0g, 2.3mmol) and be dissolved in the DMSO of 40ml, dehydrant (N,
N'-dicyclohexylcarbodiimide, DCC, 0.47g, 2.3mmol) and catalyst (to dimethylamino naphthyridine, DMAP, 0.29g,
2.3mmol) under effect, 0 DEG C of stirring 4h；Then folic acid equimolar is pressed than adding ethylene glycol (HOCH2CH2OH), raise from ice bath
Temperature is to room temperature 25 DEG C, and lucifuge is stirred overnight 24h；Crossing and filter by-product, filtrate concentrates, ice ether sedimentation recrystallization or prepare liquid
Phase purification, lyophilizing i.e. obtains folic acid-ethylene glycol coupled product (HOCH2CH2OCO-FA)。
2) preparation of folic acid-ethylene glycol-neogambogic acid: take neogambogic acid (0.15mmol) and be dissolved in the DMSO of 60ml,
Under dehydrant DCC (3.0mmol) and catalyst DMAP (3.0mmol) effect, 0 DEG C of stirring 6h；Then neogambogic acid mol ratio is pressed
1:3 times adds folic acid-ethylene glycol coupled product (HOCH2CH2OCO-FA), from ice bath, liter high-temperature is to room temperature 25 DEG C, and lucifuge is stirred
Mix overnight.Filtrate concentrates, and ice ether or recrystallisation from isopropanol chromatograph or prepare liquid phase purification, and lyophilizing can obtain cancerous cell
Targeting gambogic acid compounds NGA-COOCH2CH2OCO-FA。
1HNMR (500MHz, DMSO): 3.6 and about 4.2 is the peak of hydrogen on ethylene glycol, and other peaks are gamlogic acid and folic acid
The peak of upper hydrogen；1.24-1.46 (GA:C24-H, C25-H, 6H), 1.56-2.16 (GA:C3-H, C19-H, C20-H, C21-H,
C34-H, C35-H, C36-H, C39-H, C40-H, 23H), 2.26-2.66 (GA:C22-H, C26-H, C29-H, 6H, FA:C27-
H, 2H), 2.96 (GA:C11-H, 1H), 3.23 (GA:C31-H, 2H), 4.45-4.63 (FA:C13-H, C24-H, 3H), 5.27
(GA:C37-H, 1H), 5.84 (GA:C32-H, 1H), 6.71 (GA:C27-H, 1H), 6.84-6.92 (GA:C10-H, 1H；FA: benzene
C16-H on ring, C17-H, 2H；), 7.61 (FA: C18-H on phenyl ring, C19-H, 2H).
IR: significantly increase 1728cm in infrared spectrum-1Ester carbonyl group peak, illustrate that neogambogic acid and folic acid pass through ethylene glycol
Ester bond connect on.
Embodiment 5: the preparation of tumor-targeting gambogic acid compounds injection
Take equivalent in the tumor-targeting gambogic acid compounds (GA-P62-via embodiment 1 preparation of gamlogic acid 20g
FA), it is dissolved in water for injection, adds sodium chloride 5.0g and stir, dilute hydrochloric acid regulation pH=5.0, it is subsequently adding 0.5%
Injection activated carbon, 60 DEG C of constant temperature 30min, after de-charcoal, filtrate injects water to 1000ml, the sterilised membrane filter mistake of 0.22 μm
Filter, 2ml/ props up and is sub-packed in glass song neck ampoule, sealing by fusing, 100 DEG C of flowing steam moist heat sterilization 40min, preservation of then labelling.
Embodiment 6: the preparation of tumor-targeting gambogic acid compounds lyophilized injectable powder
Take equivalent in neogambogic acid 20g via embodiment 4 preparation tumor-targeting gambogic acid compounds powder, add
Enter glucose for injection powder 25g to stir, aseptic subpackaged in glass song neck ampoule by 40mg/ bottle, seal then labeling
Sign and preserve.
Embodiment 7: the vascular stimulation tests of tumor-targeting gambogic acid compounds
Take the tumor-targeting gambogic acid compounds 10mg of water for injection 50ml dilution embodiment 1 preparation, experimentalist
Rabbit 6, random 3 groups, wherein normal saline compares, to the rabbit left auricular vein folacin coupled gamlogic acid injection of instillation, right
Auricular vein instillation same volume normal saline, has instiled in 3h.Take after having instiled the blood vessel at the 1cm of injection point lower end and
The heart, liver, spleen, lung, pancreas, kidney and cerebral tissue are fixed formalin solution, specimens paraffin embedding slices, and HE dyes, numeral pathology
Learn assay.
It is observed that the diet of rabbit every day after administration, hair, anus, breathing, central nervous system, extremity active state etc.
All normal, without poisoning manifestations.To about about 48h, the animal of execution, observe mucous membrane of rectum smooth, without exception；Remaining is retained
Rabbit is monitored day by day, appearance without exception.Put to death animal by the 7th day, observe body weight and with reference to " study of tcm new drug guide " to blood
Pipe stimulates and carries out classification.Histopathologic examination's result of Vascular stimulation test is that auricle, epidermis are without exception, corium blood
Endothelial cell is without swelling, and blood capillary tube wall is without hemorrhage, downright bad or inflammatory cell infiltration, and cartilage layers, chondrocyte are without hypertrophy
Or downright bad, chondrocyte marshalling；Liver, cardiac muscular tissue, cerebral tissue, pulmonary, kidney and pancreatic tissue are the most without exception.But rattan
The auricle epidermis of yellow acid class medicaments injection is substantially abnormal, and dermal vascular endotheliocytic swelling, blood capillary tube wall is hemorrhage, downright bad
Or have inflammatory cell infiltration district.Saline control group auricle epidermis is without exception, and dermal vascular endotheliocyte is without swelling, blood capillary
Tube wall without hemorrhage, downright bad or inflammatory cell infiltration, chondrocyte marshalling, without hypertrophy or necrosis, cartilage layers, chondrocyte without
Hypertrophy or necrosis.Cancerous cell targeting gambogic acid compounds medicine group and normal saline group nothing on pathological tissue is the poorest
Different.
Embodiment 8: Cytotoxicity tests evaluation experimental
GA and NGA and the external anticarcinogenic effect of folate conjugate (GA-P62-FA, GA-P282-FA, NGA-P62-FA) thereof
Evaluate.In view of gambogic acid compounds is broad spectrum anticancer cytotoxic agent, the present embodiment uses the folic acid that multiple cancerous tissue is originated
The corresponding coupled product that embodiment 1,2 and 4 is prepared by the tumor cell (HeLa, HepG2 and SK-OV-3) that receptor positive is expressed
GA-P62-FA, GA-P282-FA and NGA-P62-FA and prototype compound GA and NGA thereof carry out evaluating drug effect, simultaneously LO2 liver
Cell carries out Normocellular poison experiment test to it.
Take the logarithm the cell of trophophase, inoculate 2～10 × 10 according to the size of cell3Individual on 96 orifice plates, 24h to be grown
After, abandon supernatant, be then administered by following packet: cancerous cell sets not dosing group and dosing group (concentration 0.0625～5 μMs thin to cancer
Born of the same parents, concentration 0.5～100 μMs are to LO2 cell), cisplatin (Cisplatin) is positive drug reference, and gamlogic acid and neogambogic acid are made
Parent compound compares, corresponding coupled product GA-P62-FA, GA-P282-FA and the NGA-to embodiment 1,2 and 4 preparation
P62-FA carries out cytotoxicity detection.Often group sets 4～6 multiple holes, after cultivating 72h, abandons supernatant, adds 100 μ l containing 0.5mg/ml
MTT (tetrazolium) serum-free medium cultivate 4h, add 100 μ l DMSO (dimethyl sulfoxide), be positioned on micro-oscillating instrument
Vibration 10min, then it is placed in microplate reader detection OD value 570nm at.Normal cell system LO2 compares.Experiment all repeats 3 every time
Secondary.
Result shows, along with drug level increases, compares with corresponding not dosing matched group, cell-proliferation activity respectively under
Fall, illustrates that compound is the growing multiplication of concentration dependent anticancer.And the propagation of normal hepatic cell line LO2 cell is lived
Property does not has significant change, demonstrates that this compound has low toxicity characteristic (table 1) to normal cell.
The IC of the different cell of table 150Value (72h) and different compound IC50Ratio
Embodiment 9
The LD of tumor-targeting gambogic acid compounds50Test
Take the healthy mice 60 of 18-22g, 10/group, male and female half and half, fasting 12h before experiment, freely drinks water.By etc. rub
The gamlogic acid of your amount carries out mouse peritoneal drug administration by injection to different coupling group medicines, is administered by 0.2ml/10g body weight, dissolves solvent
For negative with reference to carrying out equal-volume administration, gamlogic acid sodium salt group is the comparison of former medicine, observes reaction symptom that each group of mice occur, dead
Die time record mortality number, repeatedly observe in first 24h, observe follow-up every day 3 times, Continuous Observation 12 days, calculate LD50's
General numerical value.Test result indicate that, folacin coupled gamlogic acid greatly reduces the toxicity of proto-drug, with gamlogic acid sodium salt ratio
Relatively (the LD of mouse mainline50=38.8mg/kg), the cancerous cell targeting gambogic acid compounds of part modified with folic acid
LD50Dramatically increase (the mouse mainline LD of G-P62-FA, G-P106-FA, G-P150-FA and G-P238-FA50It is respectively
75.12,74.6,81.21 and 92.4mg/kg, convert with Resina garciniae acid number).
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention are not by above-described embodiment
Limit, the change made under other any spirit without departing from the present invention and principle, modify, substitute, combine, simplify,
All should be the substitute mode of equivalence, within being included in protection scope of the present invention.
Claims (10)
1. a tumor-targeting gambogic acid compounds, it is characterised in that: this compound has a structure as shown in formula (I):
G-P-FA
(I)
Wherein, G is gambogic acid compounds；
P is HOCH2(CH2OCH2)nCH2OH or HOCH2(CH2OCH2)nCH2NH2, wherein n is 0 or natural number between 1～10；
FA is folic acid or folacin；
Described FA is with ester bond or amido link and P coupling, and P is with the carboxyl coupling of terminal hydroxy group Yu G.
A kind of tumor-targeting gambogic acid compounds the most according to claim 1, it is characterised in that: described gambogic acid
Compound is gamlogic acid, neogambogic acid, allogambogic acid, TH-2 or their mixture.
A kind of tumor-targeting gambogic acid compounds the most according to claim 1, it is characterised in that: described P's is average
Molecular weight is 61～600.
A kind of tumor-targeting gambogic acid compounds the most according to claim 1, it is characterised in that: the molecule of described P
Amount 61,62,105,106,149,150,237,238,281,282,325,326,413,414,501 or 502.
A kind of tumor-targeting gambogic acid compounds the most according to claim 1, it is characterised in that: described folic acid is similar to
Thing is 5-methyltetrahydrofolate, 5-formoxyl tetrahydrofolic acid, methotrexate or 5,10-methylene tetrahydrofolate.
The preparation method of a kind of tumor-targeting gambogic acid compounds the most according to claim 1, it is characterised in that bag
Include following operating procedure:
(1) take folic acid to be dissolved in a solvent, at dehydrant N, N'-dicyclohexylcarbodiimide or 1-(3-dimethylamino-propyl)-3-
Ethyl-carbodiimide hydrochloride, and catalyst is under dimethylamino naphthyridine effect, 0 DEG C of stirring reacts 1～6h；Then with folic acid
Equimolar, than adding oligomerization ethylene glycol, rises high-temperature from ice bath and is stirred overnight to room temperature, lucifuge；Cross and filter by-product, filter
Liquid concentrates, and ice ether sedimentation recrystallization or preparation liquid phase purification, lyophilizing i.e. obtains folacin coupled oligomerization ethylene glycol；
Or, the active ester of the active ester or folacin that take folic acid is dissolved in anhydrous DMSO/TEA, adds and folic acid
The amidized derivant in active ester equimolar oligomerization ethylene glycol one end, reacts under anhydrous condition overnight, and vacuum freeze-drying separates
Or chromatogram purification, obtain folacin coupled oligomerization ethylene glycol amination derivant；
Described oligomerization ethylene glycol is HOCH2(CH2OCH2)nCH2OH, the amidized derivant in described oligomerization ethylene glycol one end is
HOCH2(CH2OCH2)nCH2NH2), wherein n is 0 or natural number between 1～10；
(2) gambogic acid compounds is dissolved in a solvent, at dehydrant N, N'-dicyclohexylcarbodiimide or 1-(3-diformazan ammonia
Base propyl group)-3-ethyl-carbodiimide hydrochloride, and catalyst is under dimethylamino naphthyridine effect, 0 DEG C of stirring reacts 1～6h；
Then the amount pressing gambogic acid compounds mol ratio 1:1-3 adds step (1) gained folacin coupled oligomerization ethylene glycol or folic acid
Coupling oligomerization ethylene glycol amination derivant, rises high-temperature from ice bath and is stirred overnight to room temperature, lucifuge；Filtrate concentrates,
Ice ether or recrystallisation from isopropanol, chromatograph or prepare liquid phase purification, and lyophilizing can obtain structure cancer target as shown in formula (I)
Property gambogic acid compounds.
Preparation method the most according to claim 6, it is characterised in that: step (1) described a solvent is CH2Cl2, DMSO or
DMF；The time of described stirring reaction is 2.5h；Described room temperature is 25 DEG C；The volume of DMSO and TEA in described anhydrous DMSO/TEA
Ratio is 2:1；
Step (2) described a solvent is CH2Cl2, DMSO or DMF；The time of described stirring reaction is 2.5h；Described room temperature is 25
℃。
Preparation method the most according to claim 6, it is characterised in that: step (1) described folic acid, dehydrant and catalyst
Mol ratio is 1:1:1～1:30:30；Step (2) described gambogic acid compounds, dehydrant, the mol ratio of catalyst are 1:1:1
～1:25:25.
A kind of tumor-targeting gambogic acid compounds the most according to claim 1 or its pharmaceutically acceptable salt are in system
Purposes in the medicine of standby treatment Cancerous disease.
Purposes the most according to claim 9, it is characterised in that: described medicine contains the cancer target of therapeutically effective amount
Property gambogic acid compounds or its pharmaceutically acceptable salt and pharmaceutically acceptable carrier.
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