CN103405437B - PI3K small-molecule inhibitor and application thereof - Google Patents
PI3K small-molecule inhibitor and application thereof Download PDFInfo
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- CN103405437B CN103405437B CN201310387348.6A CN201310387348A CN103405437B CN 103405437 B CN103405437 B CN 103405437B CN 201310387348 A CN201310387348 A CN 201310387348A CN 103405437 B CN103405437 B CN 103405437B
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Abstract
The invention discloses a PI3K small-molecule inhibitor and an application thereof. The inhibitor is 5-[3-(2-thiophene)-2-propene-1-] 2, 4, 6 (1H, 3H, 5H)-barbituric acid and has the functions of effectively inhibiting the propagation of various tumor cells and inducing the apoptosis of the tumor cells. The PI3K small-molecule inhibitor newly developed by the invention has low toxicity for normal cells and has a remarkable inhibiting effect on the phosphorylation of AKT (Threonine Kinase) induced by PI3K and the activation of an AKT downstream protein, so that the propagation of the various tumor cells can be effectively inhibited, the apoptosis of the tumor cells can be induced, and furthermore, the PI3K small-molecule inhibitor is expected to be developed into a novel anti-tumor drug. The PI3K small-molecule inhibitor has in-vitro and in-vivo therapeutic effects for various tumors such as acute and chronic leukemia, multiple myeloma, lymphoma and the like.
Description
Technical field
The invention belongs to medicinal chemistry art, relate to PI3K inhibitor, be specifically related to a kind of PI3K micromolecular inhibitor and application thereof.
Background technology
Cancer is still one of main cause of the death of the mankind at present.Clinician and scientists are all being devoted to the improvement of tumor therapeuticing method, as improved surgical skill, refine X-ray therapy, researching and developing new chemicals, although these effort are all beneficial for the treatment development of cancer, but the treatment of tumor still has very large development space at present, find that novel low-toxicity antitumor drug that is efficient and high specific is the important directions that life scientists are studied.
Phosphatidylinositol-3-kinase (phosphatidylinositol 3-kinase, be called for short PI3K) be a kind of conclusive kinases affecting multiple path, these paths can regulate cell to increase, the cell activities such as apoptosis and survival, especially the PI3K of I type, comprise a P85 and regulate subunit and P110 catalytic subunit, all play an important role in these cell activities, PI3K is once be activated, will phosphorylation AKT(protein kinase B), the AKT be activated will activate the GSK that Function protein matter is transcribed successively, P70S6k, 4EBP1, and regulate the P27 of cell cycle, P21, cMyc, cyclinD1 etc., in addition, the AKT be activated can act on mitochondrial protein that is as apoptosis-induced in Bad etc. and anti-apoptotic equally.
Nearest research finds, PI3K signal path is the signal path of modal allosome sudden change in human tumor cell, and the excessive activation of this signal path and the generation of tumor, development, prognosis are closely related, and suppress PI3K activity, apoptosis appears in cancerous cell; Suppress PI3K signal can overcome the chemical drug resistance comprising the kinds of tumors such as leukemia, lymphoma, multiple myeloma, so be that the research and development of the antitumor drug of target spot are more and more more subject to people's attention with PI3K.The also positive exploitation of domestic and international many pharmaceuticals and scientific research institution is as the compound of target spot, and what have enters clinical trial.
Although reported chemical compound lot in prior art can suppress PI3K intracellular signaling, there is pharmaceutically active too strong, large and unstable in aqueous phase to normal cytotoxicity, limit the shortcoming of its application.Therefore need to study a kind of new low molecular organic depressant, obvious inhibitory action can be had to PI3K signal path, thus develop the effective antitumour medicine made new advances.
Summary of the invention
Goal of the invention of the present invention is to provide a kind of PI3K micromolecular inhibitor, and described inhibitor has the propagation that can suppress kinds of tumor cells, and induces the function of its apoptosis.
To achieve the above object of the invention, the technical solution used in the present invention is: a kind of PI3K micromolecular inhibitor, and described inhibitor is expressed by the following chemical structure formula:
。
The chemical name of PI3K micromolecular inhibitor disclosed by the invention is 5-[3-(2-thiophene)-2-propylene-1-] 2,4,6 (1H, 3H, 5H)-barbituratess, is called C96.
The present invention protects above-mentioned PI3K micromolecular inhibitor preparing the application in antitumor drug simultaneously.
Preferably, described tumor is following any one: leukemia, myeloma, lymphoma.
Principle of the present invention is: the new PI3K micromolecular inhibitor of the present invention's exploitation is mainly through suppressing PI3K/AKT signal path, and then the expression of T suppression cell cycle element, suppress the phosphorylation of AKT and the carrying out in T suppression cell cycle, thus can the propagation of effective inhibition tumor cell; And its apoptosis of active cell apoptosis enzyme induction.
The present invention is claimed a kind of antineoplastic pharmaceutical compositions simultaneously, and the active component of described antitumor drug is above-mentioned PI3K micromolecular inhibitor, also comprises pharmaceutically receptible carrier or adjuvant.
PI3K micromolecular inhibitor of the present invention can make preparation administration separately or with more than one acceptable carrier combination agent.Such as, solvent, diluent etc.Can oral dosage form, as tablet, capsule, dispersible powder, granule etc.; Also can injection-type administration, as lyophilized injectable powder.The various dosage forms of pharmaceutical composition of the present invention can be prepared according to the method known in pharmaceutical field.Can containing such as 0.05% ~ 90% wt. Active ingredient with carrier combinations in these pharmaceutical formulations, more common active component about between 15% ~ 60%.The compounds of this invention dosage can make 0.005 ~ 5000mg/kg/ days, also can exceed this dosage range according to the different using dosages of disease severity or dosage form.
Because technique scheme is used, the present invention compared with prior art has following advantages:
1. the present invention prepares PI3K micromolecular inhibitor by the method that area of computer aided drug screening and biology be combined with each other, and reacts simple to operation, product is easily purified, yield is high, stablizes;
2. the PI3K signal path micromolecular inhibitor researched and developed of the present invention is low to normal cytotoxicity, and have significant inhibitory action to the AKT phosphorylation of PI3K induction, the activation of AKT downstream albumen, therefore, it is possible to effectively suppress kinds of tumor cells propagation, inducing apoptosis of tumour cell, thus be expected to be developed to new antitumor drug.
Accompanying drawing explanation
Fig. 1 is the design sketch that in embodiment two, Compound C 96 suppresses multiple myeloma cells to grow;
Fig. 2 is the design sketch that in embodiment two, Compound C 96 suppresses leukemic cell growth;
Fig. 3 is the design sketch that in embodiment three, Compound C 96 induces multiple myeloma apoptosis;
Fig. 4 is the design sketch that in embodiment three, Compound C 96 induces multiple myeloma apoptosis;
Fig. 5 be in embodiment four Compound C 96 to the design sketch of the induction of the activation of multiple multiple myeloma cells apoptosis enzyme;
Fig. 6 is the induction of activation and the design sketch of compound concentration relation of Compound C 96 pairs of multiple myeloma cells apoptosis enzymes in embodiment four;
Fig. 7 is the induction of activation and the design sketch of compound effects time relationship of Compound C 96 pairs of multiple myeloma cells apoptosis enzymes in embodiment four;
Fig. 8 be in embodiment five Compound C 96 to the inhibitory action of the P-AKT stimulated by ICF I;
Fig. 9 be in embodiment five Compound C 96 to the design sketch of the P-AKT inhibitory action stimulated by ICF I and compound concentration;
Figure 10 is the inhibitory action of Compound C 96 to the P-AKT stimulated by ICF I and the design sketch of action time in embodiment five;
Figure 11 is that in embodiment five, Compound C 96 transfers to the inhibitory action on cell membrane to the P-AKT stimulated by ICF I;
Figure 12 is the action effect figure of Compound C 96 pairs of PI3K-AKT-mTOR signal path downstream associated protein in embodiment six;
Figure 13 be in embodiment six Compound C 96 to the action effect figure of other pathway associated proteins;
Figure 14 is the gross tumor volume comparison diagram of administration group and matched group in heteroplastic transplantation experiment in embodiment seven;
Figure 15 is the Mouse Weight comparison diagram of administration group and matched group in heteroplastic transplantation experiment in embodiment seven;
Figure 16 is in embodiment seven in heteroplastic transplantation experiment, the inhibitory action figure of Compound C 96 pairs of AKT phosphoric acid activations.
Detailed description of the invention
Below in conjunction with embodiment, the invention will be further described:
The synthesis of embodiment one: 5-[3-(2-thiophene)-2-propylene-1-] 2,4,6 (1H, 3H, 5H)-barbituratess (C96)
General line:
Specifically respectively walk reaction:
By compound
1(500mg, 4.46mmol, 1eq), malonic acid (695.9mg, 6.69mol, 1.5eq), morpholine (38.8mg, 0.45mmol, 0.1eq), be dissolved in the pyridine of 5mL, 80 DEG C of back flow reaction 24h, reactant liquor dichloromethane (DCM) dilution, 1M hydrochloric acid (HCl) washs, organic facies anhydrous sodium sulfate (NaSO
4) dry, concentrated, column chromatography (DCM+0.5% acetic acid (HAc)), obtains compound
4for faint yellow solid 634.0mg (92%);
1h NMR (400 MHz, CDCl
3) δ 10.75 (s, 1H), 7.89 (d,
j=15.6 Hz, 1H), 7.43 (d,
j=4.9 Hz, 1H), 7.30 (s, 1H), 7.08 (t,
j=4.0 Hz, 1H), 6.25 (d,
j=15.6 Hz, 1H).
By compound
4(20mg, 0.13mmol, 1.0eq) is dissolved in 0.5ml methanol (MeOH), instills two thionyl chloride (SOCl
2) stirred overnight at room temperature, after having reacted, revolve and steam removing methanol and thionyl chloride, obtain faint yellow solid compound
5, 21mg (100%).
By compound
5(38.2mg, 0.23mmol, 1.0eq) joins in anhydrous tetrahydro furan (THF), DIBAL-H (31.1mg, 0.27mmol, 1.2eq) is slowly instilled under-78 DEG C of conditions, the cancellation that adds water is reacted, and ethyl acetate (EA) extracts, anhydrous Na SO
4drying, column chromatography purification obtains compound
6for pale yellow oil 21.3mg (66.9%), place after spending the night and become solid;
1h NMR (400 MHz, CDCl
3) δ 7.16 (d,
j=2.4 Hz, 1H), 6.96 (d,
j=2.9 Hz, 2H), 6.74 (d,
j=15.7 Hz, 1H), 6.20 (dt,
j=15.7,5.8 Hz, 1H), 4.28 (dd,
j=5.7,1.1 Hz, 2H).
Prepare active MnO
2
By 9.6gKMnO
4be dissolved in 60mL water, reflux, slowly add containing 5g MnSO under stirring
4 .h
2the 15mL aqueous solution of O, 40% sodium hydroxide (NaOH) solution 11.7mL, insulated and stirred reacts about 1h; By reactant liquor through buchner funnel sucking filtration, filter cake washes with water, until eluate is water white transparency, filter cake oven drying, obtains brown ceramic powder 5g, is active MnO
2, powder is evenly fine and smooth, can be used for subsequent oxidation reaction.
By compound
6(21.3mg, 0.15mmol, 1.0eq), adds compound
6the active MnO of 8 equivalents
2, after stirring at room temperature a period of time, reacting liquid filtering, uses DCM washing leaching cake, and filtrate concentrated by rotary evaporation, obtains compound
7for yellow oil 16.4mg (84%);
1h NMR (400 MHz, CDCl
3) δ 9.62 (d,
j=7.7 Hz, 1H), 7.58 (d,
j=15.6 Hz, 1H), 7.50 (d,
j=4.9 Hz, 1H), 7.36 (d,
j=2.9 Hz, 1H), 7.11 (t,
j=4.2 Hz, 1H), 6.51 (dd,
j=15.6,7.7 Hz, 1H).
By compound
7(barbiturates joins grease for (100mg, 0.724mmol, 1eq) and barbiturates (92.6mg, 0.724mmol, 1eq) mix homogeneously
7in, add ethyl acetate 5 milliliters, then ethyl acetate be spin-dried for, obtain the homogeneous mixture of 8 and barbiturates), this mixture is put microwave oven microwave reaction (fiery 2min × 3 of middle height); In system, add ether after having reacted, filter, then use washed with diethylether three times after mixing, with hot wash several, to eluate clarification, filter cake is dry under infrared lamp, obtains Chinese red solid C96,89 mg (38.3%);
1h NMR (400 MHz, DMSO) δ 11.23 (s, 1H), 11.18 (s, 1H), 8.17 (dd,
j=15.0,12.0 Hz, 1H), 7.97 (d,
j=11.9 Hz, 1H), 7.91 (d,
j=15.2 Hz, 1H), 7.86 (d,
j=4.9 Hz, 1H), 7.52 (d,
j=3.2 Hz, 1H), 7.21 (dd,
j=4.9,3.8 Hz, 1H).
Embodiment two C96 suppresses the growth of multiple myeloma cells and leukaemia
Cultivate multiple multiple myeloma cells (LP1, RPMI-8226, U266, OPM2, KMS11, OCI-MY5, JJN3 cell) and leukaemia's (THP1, K562, AML2, NB4OCI-MY5, cell), with the C96 process 72h of variable concentrations, by MTS/PMS staining analysis of cells survival rate.
The design sketch that accompanying drawing 1 suppresses multiple myeloma cells to grow for C96, result shows that C96 presents concentration dependent to the inhibitory action that multiple myeloma is bred; Accompanying drawing 2 is the design sketch of C96 suppression leukemic cell growth, and result shows that the inhibitory action of C96 to Leukemia Cell Proliferation presents concentration dependent, presents concentration dependent to the inhibitory action of Leukemia Cell Proliferation.Reach a conclusion thus: C96 effectively can suppress the growth of multiple myeloma and leukaemia.
Embodiment three C96 induces the apoptosis of multiple myeloma cells
Cultivate LP1, OPM2, OCI-MY5, KMS11, RPMI-8226, JJN3 cell, with variable concentrations C96 process 24h, collecting cell, by the two dye of Annexin V-FITC and PI, with flow cytomery Annexin V
+ratio shared by cell.
Accompanying drawing 3,4 is the design sketch of C96 induction multiple myeloma apoptosis, and result shows: C96 effectively can induce the apoptosis of multiple myeloma, and apoptosis rate and C96 present concentration dependent.
Embodiment four C96 activates apoptosis of tumor cells enzyme
By multiple myeloma cell line cell LP1, OPM2, U266, KMS11, RPMI-8226, OCI-MY5 cell, through variable concentrations C96 process 24 hours, with the SDS protein lysate cracking containing positive vanadium sodium, get 30mg albumen after quantification of protein and carry out gel electrophoresis, detect the expression of PARP, caspase3, with GAPDH or actin as internal reference.
Accompanying drawing 5 is the design sketch of C96 to the induction of the activation of multiple multiple myeloma cells apoptosis enzyme, can prove that C96 effectively can induce the apoptosis of multiple myeloma; Accompanying drawing 6 is that C96 is to the inducing action of apoptotic proteins enzyme of LP1, OPM2, JJN3 cell and the relation of drug level, can find out that Pro-Caspase3 declines gradually with the rising of C96 concentration, and Cle-Caspase3 rises gradually along with the rising of C96 concentration, also there is identical phenomenon in PARP; Accompanying drawing 7 is that C96 is to the inducing action of apoptotic proteins enzyme of LP1, OPM2, JJN3 cell and the relation of action time.Composition graphs 6,7 shows that C96 effectively can induce the apoptotic proteins enzyme of multiple myeloma, and this inducing action and C96 present concentration and time dependence.
Embodiment five C96 suppresses AKT activation
By multiple myeloma cells LP1, OPM2, OCI-MY5, JJN3 cell, low serum (0.5%) overnight is cultivated, 15min is acted on IGFI after medicine (C96 or DMSO) processes 2 hours, collecting cell, total protein is extracted with the SDS protein lysate containing positive vanadium sodium, detect the expression of phosphorylation p-AKT and total AKT with Western blot, GAPDH is as internal reference.
Accompanying drawing 8 is the inhibitory action of C96 to the P-AKT stimulated by ICF I, and result shows that C96 can effectively suppress AKT phosphorylation; Accompanying drawing 9, accompanying drawing 10 are respectively C96 to the P-AKT inhibitory action stimulated by ICF I and the relation of compound concentration and action time, can find out that the suppression of C96 to the P-AKT stimulated by ICF I presents the dependency of action time and concentration.
Further with the OPM2 cell that C96 process is cultivated, immunofluorescence analysis is made with specificity p-AKT, AKT antibody, Laser Scanning Confocal Microscope is utilized to take immunofluorescence image, accompanying drawing 11 is C96 transfers on cell membrane inhibitory action to the P-AKT stimulated by ICF I, result shows that C96 can suppress p-AKT, and it can be suppressed to the gathering of cell membrane.
Embodiment six C96 acts on the associated protein in AKT downstream
By multiple myeloma cell line cell LP1, OPM2, JJN3 cell, through variable concentrations C96 process 24 hours, with the SDS protein lysate cracking containing positive vanadium sodium, get 30mg albumen and carry out gel electrophoresis, detect the expression of associated protein, GAPDH is as internal reference.
Accompanying drawing 12,13 is respectively the action effect figure of C96 to PI3K-AKT-mTOR signal path downstream associated protein and other pathway associated proteins; Can find out that C96 can make the minimizing as corresponding in p-mTOR, mTOR, p-P70S6K, P70S6K, p-4E-BP1,4E-BP1 of the downstream associated protein of AKT, also can make p-FOXO1 (FOXO3), the corresponding minimizing of BCL-2, p-GSK (ser9), along with C96 concentration raise and rise.
Embodiment seven C96 suppresses the growth of multiple myeloma and suppresses AKT phosphorylation in its body in mice multiple myeloma models
Myeloma cell strain JJN3 subcutaneous injection, until tumor tissues reach can touch time, random packet, is divided into matched group and administration group, start administration (100mg/kg/day), continuous 16 days.Measure tumor size every other day, every day weighs Mouse Weight, and accompanying drawing 14 is the gross tumor volume comparison diagram of administration group and matched group in above-mentioned heteroplastic transplantation experiment; Accompanying drawing 15 is the Mouse Weight comparison diagram of administration group and matched group in above-mentioned heteroplastic transplantation experiment; Result show C96 can effectively Tumor suppression growth but the body weight of mice is not significantly affected.
The tumor of laboratory animal is taken out, enter to shred, add the ultrasonication of lysate income, then centrifuging and taking supernatant, gets 30mg albumen and carries out gel electrophoresis, detects the expression of PARP, caspase3, GAPDH is as internal reference, accompanying drawing 16 is in above-mentioned heteroplastic transplantation experiment, the inhibitory action of Compound C 96 pairs of AKT phosphoric acid activations, shows that C96 effectively can suppress the AKT phosphorylation in animal tumor tissue.
Claims (1)
1. the application of compound in preparation PI3K micromolecular inhibitor of formula 1;
Formula 1.
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