CN110483550A - One kind derivative of rutaecarpin containing trimethoxyphenyl and its application - Google Patents
One kind derivative of rutaecarpin containing trimethoxyphenyl and its application Download PDFInfo
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Abstract
The present invention modifies and optimizes to evodia rutaecarpa alkaloid compound, benzene trimethoxy is introduced into rutaecarpin skeleton, rutaecarpin and its derivative are linked by amido bond, carry out the experiment of inside and outside antitumor activity screening, a series of derivatives of rutaecarpin containing trimethoxyphenyl are synthesized, the antitumor drug candidate for filtering out high-efficiency low-toxicity, has widened the range of existing anticancer compound, can be used as lead compound and continues to optimize；The compound of the present invention toxic side effect is small simultaneously, and while inhibiting growth of cancer cells, to human normal cell's unrestraint activity, medication is safer.
Description
Technical field
The present invention relates to field of medicinal chemistry, and in particular to one kind rutaecarpin containing trimethoxyphenyl derivative, its system
Preparation Method and application thereof.
Background technique
Cancer is one of major causes of death in the world today.In the past few decades, chemotherapy is considered as improving
Tumor patient survival rate most efficient method.However, many chemotherapeutics in anticancer therapy have weak curative effect, toxic side effect big
The disadvantages of, it is restricted its clinical application.Therefore, the anticancer drug for finding high-efficiency low-toxicity is that the maximum that human health faces is chosen
War and urgent need.
Rutaecarpin (evodiamine) is isolated indoles quinoline from Rutaceae Evodia (Euodia) plant
Oxazolone Alkaloid compound.Rutaecarpin is a kind of faint yellow acicular crystal, not soluble in water, is soluble in methylene chloride, chloroform,
Dissolve in the organic solvents such as methanol, ethyl acetate.There is inhibiting effect to kinds of tumor cells.Rutaecarpin has antitumor thin
Born of the same parents' proliferation, the formation and invasion for inhibiting tumour cell micro-pipe, inducing apoptosis of tumour cell and necrosis, enhance cell autophagy, are good
Good topoisomerase enzyme inhibitor.Studies have shown that rutaecarpin is to cervical cancer cell, human leukemia cell, liver cancer cells, black
Plain oncocyte, stomach cancer cell, colon cancer cell etc. have certain inhibiting effect, and pharmacology activity research is also very deep, right
The research of rutaecarpin Anticancer Effect and Mechanism has gradually become a hot spot.Its mechanism of action may be to inhibit PI3K/Akt/
Caspase, Fas-L/NF- κ B signal access etc..With going deep into rutaecarpin pharmacological research, rutaecarpin is caused
The great interest of domestic and international scientist, has carried out the synthetic work to rutaecarpin derivative.Purpose is to obtain active more preferable, poison
The antitumor candidate compound that property is lower, property is more stable.
The prior art reports structure optimization and the structure activity study that system has been carried out to rutaecarpin, develops a system
Arrange rutaecarpin derivative with anti-tumor activity.Sheng Chun spring etc. (CN101787025A, CN103992336A,
CN105524061A, CN105418610A) by introducing substituent group on the N of rutaecarpin and in rutaecarpin skeleton
10- introducing hydroxyls have obtained a series of having antitumor and antifungal activity rutaecarpin derivative；Creep is preferably etc.
(CN107365322A) skeleton transition principle is utilized, structural modification is carried out to the skeleton of rutaecarpin, has obtained a series of having
The rutaecarpin derivative of anti-tumor activity；Luo Haibin etc. (107141288 A of CN) is by the substitution on rutaecarpin phenyl ring
Base carries out structural modification, has obtained a series of rutaecarpin derivatives with anti-tumor activity；Lee reaches Swarm etc. for rutaecarpin
N-13 position split nitrogen mustard derivatives, obtained a series of rutaecarpin derivatives with anti-tumor activity.
The growth and existence of tumour are both needed to provide sufficient oxygen and nutrient by blood vessel, and exclude metabolic waste, therefore, swell
Tumor blood-vessels target therapy is come into being, and a kind of effective ideas of cancer therapy is become.According to the difference of mechanism of action, tumor vessel
Targeted drug (vascular targeted agent, VTA) can be divided into 2 kinds: neovascularization inhibitor and vascular disrupting agents.Mesh
Before, many that all there is benzene trimethoxy group just in the tumor vessel disrupting agent structure of clinical test, such as CA4P and its similar
Object OXi4503 and AVE8062, colchicine analogue ZD6126, BNC-105 and CKD-516, benzene trimethoxy group is to tumour medicine
Object design has important researching value.
The present inventor modifies and optimizes to evodia rutaecarpa alkaloid compound, and benzene trimethoxy is introduced rutaecarpin bone
Frame links rutaecarpin and its derivative by amido bond, carries out the experiment of inside and outside antitumor activity screening, has synthesized a series of
The derivative of rutaecarpin containing trimethoxyphenyl filters out the antitumor drug candidate of high-efficiency low-toxicity.
Summary of the invention
The technical problems to be solved by the present invention are: a kind of derivative of rutaecarpin containing trimethoxyphenyl is provided,
It can be used as anticancer drug, and have lesser bio-toxicity for human normal cell line.
The first aspect of the invention is to provide a kind of 12 compound of formula and its pharmaceutically acceptable salt:
Wherein: R is selected from H, halogen, nitro, CN, C1-C6Alkyl, C1-C6Alkoxy, C1-C6Halogenated alkyl, C1-C6Alkyl halide
Oxygroup；
R1Selected from H, halogen, nitro, CN, C1-C6Alkyl, C1-C6Alkoxy, C1-C6Halogenated alkyl, C1-C6Haloalkoxy
Base.
Preferably, R is selected from H, halogen, C1-C4Alkyl, C1-C4Alkoxy；It is highly preferred that R is selected from H, methoxyl group；
Preferably, R1Selected from H, halogen, nitro, C1-C4Alkyl, C1-C4Alkoxy；It is highly preferred that R1Selected from H, methyl,
Cl, Br, nitro.
Preferably, 12 compound of formula is selected from following compound:
Another aspect of the present invention provides a kind of method of 12 compound of preparation formula, and synthetic route is as follows:
Specific reaction step is as follows:
Step a: being cooled to 0-5 DEG C for agitating solution of the anil (1) in 12% hydrochloric acid solution, is then added dropwise sub-
Sodium nitrate solution reaction, TLC are detected after the reaction was completed, solution are added in sodium sulfite aqueous solution at room temperature, then heats
To after 80 DEG C, 2 hours, concentrated hydrochloric acid is added, is stirred to react at 100 DEG C 2 hours, is cooled to room temperature, benzene is settled out from solution
Hydrazine hydrochloride derivative (2)；
Step b: the chloro- 1- hydroxyl -1- fourth of 4- is added in phenyl hydrazine hydrochloride salt derivative (2) in the solution in 30% ethanol solution
Sodium sulfonate and Na2HPO4, which stirs 5-7 hours (preferably 6 hours) at 70 DEG C, after ethanol evaporation, mixture dichloro
Then pH is adjusted to 7-8 with saturated sodium bicarbonate, and is cooled to 4 DEG C by methane and chloroform, molten from this during this period
It is settled out tryptamine hydrochloride in liquid, then potassium carbonate is abolished into hydrochloride, is extracted with ethyl acetate, obtains tryptamine derivatives (3)；
Step c: being dissolved in carboxylate solution for tryptamine derivatives (3) and be heated to 60 DEG C and be stirred to react, TLC monitoring reaction
After completely, intermediate 4 is obtained after revolving formic acid removal ethyl ester, the CH that intermediate 4 is dissolved in2Cl2POCl is added in solution3And at 0-5 DEG C
Lower stirring 6 hours, is then removed under reduced pressure solvent, by silica gel column purification, obtains intermediate 5；
Step d: 2 hydroxybenzoic acid derivative (10) is dissolved in CH2Cl2Thionyl chloride is added in solution and is stirred at 40 DEG C
Reaction 0.5-2h is mixed, solvent is then removed under reduced pressure；Residue is added to the CH of intermediate 52Cl2In solution, and stir at room temperature
Mix reaction 22-26 hours；After completion of the reaction, solvent is removed under reduced pressure, crude product purified by silica gel column purification obtains intermediate 11；
Step e: intermediate 11 is dissolved in n,N-Dimethylformamide, by its ice bath to 0 DEG C of backward interior addition sodium hydride
And it stirs 20 minutes；Then it will be put into above-mentioned reaction after 3,4,5- trimethoxy-benzoyl chlorides again, ice bath stirring reaction, TLC
Solid is precipitated after suitable quantity of water is added into solution after completion of the reaction in detection reaction, after suction filtration that solid is pure by silica gel column chromatography
Change, obtains final product 12；
Wherein, described R, R1As previously described.
In one embodiment, in step a: the molar ratio of anil (1), sodium nitrite and sodium sulfite is 1:
1-1.2:2-4 preferably 1:1-1.1:3；
In one embodiment, in step b: the chloro- 1- hydroxyl -1- fourth sodium sulfonate of phenyl hydrazine hydrochloride salt derivative (2), 4- and
Na2HPO4Molar ratio be 1:1-1.2:0.03-0.07, preferably 1:1-1.1:0.05；
In one embodiment, in step c: the molar ratio volume ratio of tryptamine derivatives (3) and Ethyl formate is 1:1-
3mmol/mL, preferably 1:2mmol/mL；Tryptamine derivatives (3) and POCl3Molar ratio volume ratio be 10:0.5-2mmol/
ML, preferably 10:1mmol/mL；
In one embodiment, in step d: the molar ratio of 2 hydroxybenzoic acid derivative (10) and compound 5 is 1:
0.8-1.5, preferably 1:1；The molar ratio of 2 hydroxybenzoic acid derivative (10) and thionyl chloride is 1:1-3, preferably 1:2；
In one embodiment, in step e: the molar ratio of intermediate 11 and 3,4,5- trimethoxy-benzoyl chlorides is 1:
1-1.5 preferably 1:1.2；The reaction time of step e is 20-30h, preferably 23-25h.
Another aspect of the present invention provides a kind of pharmaceutical composition, it includes 12 compound represented of formula or its pharmaceutically may be used
The salt and pharmaceutically acceptable carrier of receiving.
Another aspect of the present invention is related to a kind of 12 compound of formula or comprising its pharmaceutical composition in preparation treating cancer
Purposes in drug；Preferably, the cancer is selected from liver cancer, lung cancer, gastric cancer, colon cancer, breast cancer；More preferably gastric cancer.
Definition:
" alkyl ", which refers to, to be only made of carbon and hydrogen atom, and degree of unsaturation is not contained.In some embodiments, alkyl has
There are 1 to 6 or 1 to 4 carbon atom.Representative alkyl include but is not limited to methyl, ethyl, n-propyl, normal-butyl, n-pentyl and
N-hexyl, isopropyl, sec-butyl, isobutyl group, tert-butyl, isopentyl etc..
" alkoxy " refers to that " alkyl " is connected by oxygen atom with parent molecule, determines as described above wherein " alkyl " has
Justice.
" halogenated alkyl " refers to wherein all hydrogen moieties or all by selected from fluoro base, chloro base, bromo base and iodine
The alkyl of the halogen displacement of Dai Ji.The example of halogenated alkyl includes CF3, CF2CF3, CF2CF2CF3, CFCl2, CF2Cl etc..
Compared with prior art, the beneficial effects of the present invention are:
The present invention provides a new class of derivatives of rutaecarpin containing trimethoxyphenyl, have widened existing anticancer compound
Range, can be used as lead compound and continue to optimize.The compound of the present invention toxic side effect is small, is inhibiting the same of growth of cancer cells
When, to human normal cell's unrestraint activity, medication is safer.
Detailed description of the invention
Fig. 1 is the inhibiting effect that compound 12h forms HGC-27 cell colony.
Fig. 2 be compound 12h processing 48h inhibit HGC-27 cell migration: (A) take pictures under the microscope gained various concentration
The healing of scratch wound；(B) HGC-27 cell migration rate and compound 12h negative correlation.Error bas show
The SD, " * * " p < 0.01, compared with the control group.
Fig. 3 is that the influence after compound 12h handles 48h, to HGC-27 cellular morphology (uses 33258 dyes of Hoechst
Cytochrome): cell dyeing figure A) shot under fluorescence microscope.(B) Apoptosis total amount and the concentration of compound 12h are in just
Correlativity.Error bas show the SD, " * * " p < 0.01, compared with the control group.
Fig. 4 is influence of the compound 12h to HGC-27 cell cycle distribution.(A) flow cytometry analysis showed, at 12h
The distribution in its period is affected after reason HGC-27 cell 48h.The hundred of cell cycle different phase are calculated using FlowJo software
Divide ratio.(B) histogram of 8 Software on Drawing cell G1/G0, G2/M, S phase percentage of Origin is utilized.
Fig. 5 is influence to its Apoptosis after compound 12h processing HGC-27 cell 48h.(A) FlowJo software is used
The amount of apoptotic cell is calculated, Q1, Q2, Q3, Q4 respectively represent cell fragment, viable apoptotic cell, non-viable apoptotic cell, normally live
Cell.(B) the concentration correlation of Apoptosis total amount and compound 12h.Errorbas show the SD,"**"p
< 0.01, compared with the control group.
Specific embodiment
The contents of the present invention are illustrated below by embodiment.In the present invention, following embodiment is in order to more preferable
Ground illustrates the present invention, is not for limiting the scope of the invention.
The synthesis of 1 compound 12a of embodiment
Agitating solution of the aniline (1a, 2.80g, 0.03mol) in 12% hydrochloric acid solution (27mL) is cooled to 0-5 DEG C.
Then sodium nitrite solution (2.16g, 0.031mol) is added dropwise and reacts 1 hour.After the reaction was completed, solution is added at room temperature
In sodium sulfite aqueous solution (11.34g, 0.09mol), it is then heated to 80 DEG C.After 2 hours, it is added concentrated hydrochloric acid (6mL), 100
It is stirred to react at DEG C 2 hours.It is cooled to room temperature, hydrazinobenzene hydrochloride salt (2a) is settled out from solution.Yield is 94%
The chloro- 1- hydroxyl-of 4- is added in hydrazinobenzene hydrochloride salt (2a, 2.90g, 0.02mol) in the solution in 30% ethanol solution
1- fourth sodium sulfonate (4.21g, 0.02mol) and Na2HPO4(0.14g, 0.001mol), the mixture stir 6 hours at 70 DEG C.
After ethanol evaporation, then pH is adjusted to 7-8 with saturated sodium bicarbonate by mixture methylene chloride and chloroform, and by its
4 DEG C are cooled to, is settled out tryptamine hydrochloride from the solution during this period.Potassium carbonate is abolished into hydrochloride again, uses ethyl acetate
Extraction, obtains tryptamines (3a).Yield is 61%.
Tryptamines (3a, 1.60g, 0.01mol) is dissolved in Ethyl formate (20mL) solution and is heated to 60 DEG C and stirring 6 hours.
Compound 4a is obtained after rotation formic acid removal ethyl ester.The CH that compound 4a is dissolved in2Cl2POCl is added in (30mL) solution3(1mL) simultaneously
It is stirred 6 hours at 0-5 DEG C.Then solvent is removed under reduced pressure.Pass through silicagel column (CH2Cl2/CH3OH=10:1) purification of crude product,
Obtain intermediate 5a；Yield is 88%；
2 hydroxybenzoic acid (10a, 0.69g, 5mmol) is dissolved in CH2Cl2Thionyl chloride is added in (25mL) solution
It (1.18g, 10mmol) and is stirred 1 hour at 40 DEG C.Then solvent is removed under reduced pressure.Residue is added to intermediate 5a
The CH of (0.85g, 5mmol)2Cl2In (40mL) solution, and it is stirred at room temperature 24 hours.After the reaction was completed, it is removed under reduced pressure molten
Agent, crude product purified by silica gel column (CH2Cl2) purifying, obtain intermediate 11a.Yield is 50%.
It weighs intermediate 11a (0.29g, 1mmol) to be dissolved in n,N-Dimethylformamide (DMF), by its ice bath to after 0 DEG C
Sodium hydride 0.05g is inwardly added and stirs 20 minutes；Then again by (0.28g, 1.2mmol) 3,4,5- trimethoxy-benzoyl chlorides
It puts into above-mentioned reaction, after ice bath stirring 24 hours, solid is precipitated after 100mL water is added into solution, leads to solid after suction filtration
Silica gel column chromatography (eluant, eluent: absolute dichloromethane) purifying is crossed, faint yellow solid (12a), yield 31.6% are obtained.1H NMR
(400MHz, DMSO) δ 7.82 (dd, J=7.7,1.3Hz, 1H), 7.75 (d, J=7.3Hz, 1H), 7.59 (d, J=8.2Hz,
1H), 7.53-7.48 (m, 1H), 7.41 (dd, J=11.3,4.1Hz, 1H), 7.37 (t, J=7.4Hz, 1H), 7.21-7.01
(m, 4H), 6.26 (s, 1H), 4.75 (dd, J=13.1,4.8Hz, 1H), 3.81 (s, 6H), 3.67 (s, 3H), 3.30 (dd, J=
12.4,3.6Hz, 1H), 3.08 (d, J=14.9Hz, 1H), 2.97-2.85 (m, 1H) .MS (ESI): 484.50 (C28H24N2O6,
[M+H+])。
The synthesis of embodiment 2-14 compound 12b-12n
The preparation method of reference compound 12a, with substituted or unsubstituted 2 hydroxybenzoic acid (compound 10), replace or
Unsubstituted aniline (compound 1) is used as raw material, and compound 12b-12n is prepared, shown in table specific as follows:
15 external activity test of embodiment
1.MTT method tests the compounds of this invention in vitro to the inhibitory activity of different cancer cells
The derivative of rutaecarpin containing trimethoxyphenyl that the present invention is synthesized, negative control medicine rutaecarpin and the positive
Comparison medicine 5 FU 5 fluorouracil (5-FU) is configured to the DMSO solution that concentration is 33333 μm of ol/L.By the tumour of adherent growth
Cell is digested to cell suspension with pancreatin, and with every hole 5 × 103The density of a cell is inoculated in 60 hole of centre of 96 orifice plates,
200 μ L PBS finally are added in the every hole in the outer ring of 96 orifice plates.It is placed in incubator and cultivates for 24 hours, configure drug concentration gradient later
It is the pastille culture medium of 128,64,32,16,8,4,2,1 μm of ol/L, the drug containing training of 200 μ L various concentrations is added into experimental port
Base is supported, 5 multiple holes are arranged in each concentration.6 multiple holes are set without cell, isometric culture medium is only added and makees blank control.
6 multiple holes are set and there was only cell, 3 ‰ DMSO is as solvent group.96 orifice plates are finally put into 37 DEG C, 5%CO2Incubator in
Continue to cultivate.After 48h, the concentration that 20 μ L are added in each hole in addition to outer ring is the MTT solution of 5mg/L, continues to be put into incubator
It is incubated for 4h.Culture medium, the first that the living cells of 200 μ L DMSO dissolution hole bottom is added in each hole and MTT is formed is sucked out after taking out plate
A ceremonial jade-ladle, used in libation crystallization, shaking table are protected from light oscillation 10min and promote dissolution, are placed in multi-function microplate reader the absorbance (OD measured under 490nm wavelength
Value).
Each drug under various concentration is calculated to the inhibiting rate of every kind of cell, with GraphPad Prism using above-mentioned formula
6.0 softwares calculate the IC of each compound50Value.Experiment repeats three times in parallel every time, IC50Value takes its average value ± standard deviation
Poor (SD).
2. cell colony is tested
Single cell suspension is made in HGC-27 cell with pancreatin digestive juice, and is connect with the even density of every 1000 cells in hole
Kind is in 6 orifice plates.After cultivating for 24 hours in carbon dioxide incubator, 12h (3,1.5,0.75 μm of ol/ containing various concentration are replaced
L fresh medium) as a control group, replaces a culture solution with the culture solution containing 3 ‰ DMSO every three days.After culture 9 days, go
Except culture solution, 15min is fixed with 4% paraformaldehyde, then with violet staining 15min, is discarded Crystal Violet Dye, cleaned 3 with PBS
It is secondary to be placed on drying in drying box, finally take pictures.
As shown in Fig. 1, drug 12h can be more clearly seen to the antiproliferative of HGC-27 cell by colony experiment
Effect.It acts on the 12h (3,1.5,0.75 μm of ol/L) of various concentration and control group is without drug but the effect containing 3 ‰ DMSO carries out pair
Than, it is possible to find with the raising of 12h concentration, the colony of violet staining is reduced therewith, or even to 1.5 μm of ol/L concentration when,
Visible colony almost without.Experimental result illustrates that drug 12h has preferable antiproliferative to gastric carcinoma cell lines HGC-27
Effect.
3. cell scratch experiment
Single cell suspension is made in HGC-27 cell using pancreatin digestive juice, and with every hole 1 × 105The density of a cell
It is inoculated in 6 orifice plates.After culture for 24 hours, with the substitute pipette tips of 200 μ L, every hole draws 3 straight lines in 6 orifice plates, is washed with PBS
It goes after floating cells to take pictures under inverted fluorescence microscope again.Then (3,1.5,0.75 μ of 12h containing various concentration are replaced
Mol/L fresh medium) is further continued for being put into incubator as a control group cultivating with the culture solution containing 3 ‰ DMSO.Continue to train
It after supporting 48h, discards culture medium and clean with PBS and remove the suspension cell die, then to taking pictures under inverted microscope, and calculating is moved
Shifting rate.
The healing of cell scratch is observed by inverted microscope, and wide by the scratch width of 0h and the scratch of 48h
Degree is compared, computation migration rate.As shown in Fig. 2 (A), we are this it appears that the scratch healing of dosing group is lower than not dosing
Control group.As shown in Fig. 2 (B), cell migration rate (control group: 66.9%, 0.75 μm of ol/L:64.3%, 1.5 μm of ol/L:
24.8%, 3 μm of ol/L:12.1%) with the raising of concentration, the mobility of cell is lower.The results show, compound 12h
It is able to suppress the migration of HGC-27 cell.
4.Hoechst 33258 is dyed
Single cell suspension is made in HGC-27 cell, and with every hole 1 × 104The density of a cell is inoculated in 6 orifice plates
On.After culture for 24 hours, simultaneously with the subsequent continuous culture 48h of the fresh cultured fluid exchange of 12h containing various concentration (3,1.5,0.75 μm of ol/L)
With containing 3 ‰ DMSO without drug culture solution as a control group.After 48h, discards culture and clean the suspension die of removing with PBS thin
Born of the same parents add 33258 dyeing liquor of Hoechst covering sample, room temperature avoid light place 3-5min.Then Hoechst 33258 is sucked
Dyeing liquor and with PBS cleaning sample 3 times, each 3-5min.It observes and takes pictures under finally directly taking to inverted fluorescence microscope.
By 33258 dyeing liquor of Hoechst, we can intuitively see the apoptosis of drug 12h effect HGC-27 cell
Situation.As shown in figure 3, control cell is dyed with Hoechst 33258 is presented round homogeneous nucleus, normal blue-fluorescence does not have
Morphological change, and the HGC-27 cells show of 12h processing goes out bright Chromatin condensation and karyorrhexis, this is Apoptosis
Mark.And the percentage that apoptotic cell accounts for total cell in shooting figure is calculated by Image J.Coloration result proves compound
12h can induce the apoptosis of HGC-27 cell, and certain dose dependent is presented.
5. cell cycle and Apoptosis
(1) the Flow cytometry cell cycle
Single cell suspension is made in HGC-27 cell, and with every hole 1 × 105The density of a cell is inoculated in 6 orifice plates
On.After culture for 24 hours, simultaneously with the subsequent continuous culture 48h of the fresh cultured fluid exchange of 12h containing various concentration (3,1.5,0.75 μm of ol/L)
With containing 3 ‰ DMSO without drug culture solution as a control group.It is thin to HGC-27 according to the illustration method of cell cycle detection kit
Born of the same parents are handled, and are dyed with propidium iodide (PI) to it.PI is a kind of dyestuff that fluorescence can be generated in conjunction with double-stranded DNA,
Its intensity for generating fluorescence is directly proportional to the content of DNA.HGC-27 cell is detected by flow cytometer, according to DNA
The distribution situation of content analyzes the cell cycle.
(2) Apoptosis by Flow Cytometry
Using various concentration 12h (3,1.5,0.75 μm of ol/L) to HGC-27 cell handle 48h and with contain 3 ‰ DMSO without medicine
Object culture solution is as a control group.Cell is handled according to the illustration method of cell apoptosis detection kit, is used in combination
The bis- coloring agents of AnnexinV-FITC and PI dye it.AnnexinV with green fluorescence probe FITC is capable of specificity
The phosphatidylserine PS on combination cell surface, and PI can dye non-viable non-apoptotic cell or apoptosis advanced stage loses cell membrane integrity
Cell.Treated, and sample can pass through the apoptosis degree of flow cytometer quantitative detection cell.
Results and discussion
As shown in Fig. 4 (A), the 12h of (3,1.5,0.75 μm of ol/L) and compareing containing 3 ‰ DMSO are detected under various concentration
Phase cell cycle G1 after group effect HGC-27 cell 48h, S phase, the distribution situation of G2 phase.As shown in Fig. 4 (B), no drug effect
The period profile of lower cell is G1/G0:55.89%, S:22.65%, G2/M:21.49%.When concentration is 0.75 μm of ol/L (G1/
G0:53.71%, S:23.58%, G2/M:22.28%), 1.5 μm of ol/L (G1/G0:49.32%, S:22.68%, G2/M:
27.41%), 3 μm of ol/L (G1/G0:3.04%, S:31.22%, G2/M:66.39%)；Pass through the period to various concentration point
The analysis of cloth data, the HGC-27 cell concentration in the G0/G1 phase are gradually decreased with the increase of concentration, and thin in the G2/M phase
Born of the same parents' amount increases with the increase of drug concentration.The experimental results showed that compound 12h can block the G2/M phase of HGC-27 cell.
As shown in figure 5, cell is by measuring difference in Flow cytometry under the bis- staining reagents of AnnexinV-FITC and PI
The apoptosis situation of cell under concentration；Apoptotic cell and normal cell are distinguished with cross in figure, wherein the lower left corner represents survivaling cell
Ratio, the upper left corner represent non-viable non-apoptotic cell ratio, and the lower right corner then represented with the upper right corner it is thin in early apoptosis and late apoptic
Born of the same parents.The percentage of cerebral apoptosis of control group is 4.53% under no drug effect, the apoptosis of cell under 0.75 μm of ol/L drug effect
Percentage is 6.66%, and the apoptosis percentage of cell is 13.63% under 1.5 μm of ol/L drug effects, under 3 μm of ol/L drug effects
The apoptosis percentage of cell is 17.88%；With the increase of drug concentration, the apoptosis degree of cell is gradually increased, and is showed pair
The concentration dependent of drug 12h.Meanwhile also illustrating the ability that drug 12h has induction HGC-27 Apoptosis.
Claims (9)
1. a kind of 12 compound of formula and its pharmaceutically acceptable salt:
Wherein: R is selected from H, halogen, nitro, CN, C1-C6Alkyl, C1-C6Alkoxy, C1-C6Halogenated alkyl, C1-C6Haloalkoxy
Base；
R1Selected from H, halogen, nitro, CN, C1-C6Alkyl, C1-C6Alkoxy, C1-C6Halogenated alkyl, C1-C6Halogenated alkoxy.
2. 12 compound of formula according to claim 1, it is characterised in that: R is selected from H, halogen, C1-C4Alkyl, C1-C4Alcoxyl
Base；It is highly preferred that R is selected from H, methoxyl group；
R1Selected from H, halogen, nitro, C1-C4Alkyl, C1-C4Alkoxy；It is highly preferred that R1Selected from H, methyl, Cl, Br, nitro.
3. 12 compound of formula according to claim 1 is selected from following compound:
4. a kind of preparation method of 12 compound of formula as described in any one of claims 1-3, reaction route are as follows:
Wherein, R, R1Definition, as described in claim any one of 1-3.
5. preparation method as claimed in claim 4, which comprises the steps of:
Step a: agitating solution of the anil (1) in 12% hydrochloric acid solution is cooled to 0-5 DEG C, nitrous acid is then added dropwise
Sodium solution reaction, TLC are detected after the reaction was completed, solution are added in sodium sulfite aqueous solution at room temperature, is then heated to 80
DEG C, after 2 hours, concentrated hydrochloric acid is added, is stirred to react 2 hours, is cooled to room temperature at 100 DEG C, phenylhydrazine salt is settled out from solution
Acid salt derivant (2)；
Step b: the chloro- 1- hydroxyl -1- fourth sulfonic acid of 4- is added in phenyl hydrazine hydrochloride salt derivative (2) in the solution in 30% ethanol solution
Sodium and Na2HPO4, which stirs 5-7 hours (preferably 6 hours) at 70 DEG C, after ethanol evaporation, mixture methylene chloride
And chloroform, pH is then adjusted to 7-8 with saturated sodium bicarbonate, and be cooled to 4 DEG C, during this period from the solution
It is settled out tryptamine hydrochloride, then potassium carbonate is abolished into hydrochloride, is extracted with ethyl acetate, tryptamine derivatives (3) are obtained；
Step c: being dissolved in carboxylate solution for tryptamine derivatives (3) and be heated to 60 DEG C and be stirred to react, and TLC monitors fully reacting
Afterwards, intermediate 4 is obtained after revolving formic acid removal ethyl ester, the CH that intermediate 4 is dissolved in2Cl2POCl is added in solution3And it is stirred at 0-5 DEG C
It mixes 6 hours, solvent is then removed under reduced pressure, by silica gel column purification, obtain intermediate 5；
Step d: 2 hydroxybenzoic acid derivative (10) is dissolved in CH2Cl2Thionyl chloride is added in solution and is stirred at 40 DEG C anti-
0.5-2h is answered, solvent is then removed under reduced pressure；Residue is added to the CH of intermediate 52Cl2In solution, and it is stirred at room temperature anti-
It answers 22-26 hours；After completion of the reaction, solvent is removed under reduced pressure, crude product purified by silica gel column purification obtains intermediate 11；
Step e: intermediate 11 is dissolved in n,N-Dimethylformamide, its ice bath to 0 DEG C of backward interior addition sodium hydride and is stirred
It mixes 20 minutes；Then it will be put into above-mentioned reaction after 3,4,5- trimethoxy-benzoyl chlorides again, ice bath stirring reaction, TLC detection
Reaction is precipitated solid after suitable quantity of water is added into solution, purifies solid by silica gel column chromatography after suction filtration after completion of the reaction,
Obtain final product 12.
6. preparation method as claimed in claim 5, it is characterised in that:
In step a: the molar ratio of anil (1), sodium nitrite and sodium sulfite is 1:1-1.2:2-4, preferably 1:1-
1.1:3；
In step b: the chloro- 1- hydroxyl -1- fourth sodium sulfonate of phenyl hydrazine hydrochloride salt derivative (2), 4- and Na2HPO4Molar ratio be 1:1-
1.2:0.03-0.07 preferably 1:1-1.1:0.05；
In step c: the molar ratio volume ratio of tryptamine derivatives (3) and Ethyl formate is 1:1-3mmol/mL, preferably 1:
2mmol/mL；Tryptamine derivatives (3) and POCl3Molar ratio volume ratio be 10:0.5-2mmol/mL, preferably 10:1mmol/
mL；
In step d: the molar ratio of 2 hydroxybenzoic acid derivative (10) and compound 5 is 1:0.8-1.5, preferably 1:1；2- hydroxyl
The molar ratio of yl benzoic acid derivative (5) and thionyl chloride is 1:1-3, preferably 1:2；
In step e: intermediate 11 and 3, the molar ratio of 4,5- trimethoxy-benzoyl chlorides are 1:1-1.5, preferably 1:1.2；Step
The reaction time of rapid b is 20-30h, preferably 23-25h.
7. a kind of pharmaceutical composition, it includes described in any item 12 compounds of formula of claim 1-3 or its is pharmaceutically acceptable
Salt and pharmaceutically acceptable carrier.
8. described in any item 12 compounds of formula of claim 1-3 or pharmaceutical composition as claimed in claim 7 are treated in preparation
Purposes in the drug of cancer.
9. purposes as claimed in claim 8, which is characterized in that the cancer is selected from liver cancer, lung cancer, gastric cancer, colon cancer, mammary gland
Cancer；More preferably gastric cancer.
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