CN110204529A - A kind of preparation method and purposes of deuterated compound in triazine class - Google Patents
A kind of preparation method and purposes of deuterated compound in triazine class Download PDFInfo
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Abstract
The invention discloses the preparation methods and purposes of the deuterated compound in triazine class as shown in formula (I) or its officinal salt.Deuterated compound in triazine class of the present invention can be used for preparing PI3K/mTOR inhibitor.
Description
Technical field
The present invention relates to field of medicinal chemistry, and in particular to a kind of system of deuterated compound in triazine class or its officinal salt
Preparation Method and its pharmaceutical composition are in Prevention and/or treatment and/or the proliferative diseases of adjuvant treatment PI3K zymogenesis
Drug in application.
Background technique
Phosphatidyl-inositol 3-kinase (phosphoinositide 3-kinase, PI3K) signal transduction path is human carcinomas
One of the system that topnotch is mutated in disease, while being also the key factor in the various other diseases of the mankind, participate in various diseases, packet
Include allergic contact dermatitis, rheumatoid arthritis, osteoarthritis, inflammatory bowel disease, chronic obstructive pulmonary disease, psoriasis, more
Hair property hardening, asthma, the obstacle for being related to diabetic complication and cardiovascular system inflammatory complication such as acute coronary syndrome.
PI3K is unique and conservative lipid within endothelial cells kinase families member, according to the special of its structure and phosphorylated substrate
Property is divided into I, II, III 3 seed types.The I class PI3K (p110 α, p110 β, p110 δ, p110 γ) of most study is usually logical at present
It crosses tyrosine kinase or g protein coupled receptor activation generates PIP3 combination downstream effect object.Under PI3K signal transduction pathway
Trip medium includes the mammal target (mTOR) of Akt (protein kinase B) and rapamycin.A kind of critical function of Akt is logical
The activity of peroxophosphoric acid TSC2 and other mechanism enhancing mTOR.MTOR is serine-Soviet Union relevant to PI3K family lipid kinase
Histidine kinase.MTOR participates in a variety of bioprocess, including cell growth, cell Proliferation, cell mobility and survival.MTOR is
Integrate growth factor and trophic signals with the translation of regulatory protein matter, nutrition intake, from thermophilic and mitochondrial function multi-functional kinases.
Phosphatidyl-inositol 3-kinase/protein kinase B/rapamycin target protein (PI3K/Akt/mTOR) signal path and thin
Born of the same parents' period, vascularization, tumour occur and invasion it is in close relations, for the access inhibitor also have in recent years it is different degrees of
Development, antineoplaston promise well.PI3K/mTOR double inhibitor has become one of the hot spot of anti-tumor drug research and development.
Bimiralisib is a kind of inhibitor of PI3K/mTOR specificity, is currently in the clinical II phase and studies.Its chemistry
Structure is as follows:
Deuterated modification is to improve a kind of very potential new drug development technology of pharmacokinetic properties.In the method,
Try by the way that one or more hydrogen atom D-atoms are replaced to slow down the drug metabolism of CYP mediation or be reduced undesirable
The generation of metabolin.Deuterium be it is safe and stable, do not have radioactive isotope, compared with hydrogen, deuterium can form stronger with carbon
Key.In some cases, the increased bond strength formed by deuterium can be with the ADME property of positive influence drug, as a result may be bright
Extend drug metabolism aobviously to recycle, the interaction between the generation and drug of reduction toxic metabolite, improve safety and obtain
Obtain more preferably curative effect.In the prior art, deuterated Bimiralisib compound has not been reported.
Summary of the invention
The purpose of the present invention is aiming at the shortcomings in the prior art, provide a kind of deuterated compound in triazine class or its is pharmaceutically acceptable
Salt.
The second aspect of the invention, provides a kind of pharmaceutical composition, and described pharmaceutical composition includes described in first aspect
Deuterated compound in triazine class.
The third aspect of the present invention provides the pharmaceutical composition as described in second aspect in Prevention and/or treatment
And/or the application in the drug of the proliferative diseases of adjuvant treatment PI3K/mTOR zymogenesis.
The fourth aspect of the invention provides deuterated compound in triazine class or its officinal salt described in one kind or contains deuterium
The pharmaceutical composition of substituted triazine compound inhibits the application in cancer cell growth in vitro.
The fifth aspect of the invention provides the preparation side of deuterated compound in triazine class or its officinal salt described in one kind
Method.
In order to achieve the above object, the present invention provides a kind of deuterated compound in triazine class or its officinal salt, chemistry knots
Shown in structure such as formula (I):
Wherein:
R1-R12 is independently represented each other H or D；
X represents N, CH or CD；
Preferably, deuterium is greater than natural deuterium isotopic content (0.015%) in the deuterium isotopic content of deuterium the position of substitution, preferably
Ground is greater than 30%, even more preferably greater than 50%, even more preferably greater than 75%, even more preferably greater than 95%, even more preferably greater than 98%.
It is highly preferred that the deuterated compound in triazine class is selected from:
Wherein, the officinal salt includes but is not limited to halogen acid salt, such as hydrofluoride, hydrochloride, hydrobromate, hydrogen
Iodate etc.；Inorganic acid salt, such as nitrate, perchlorate, sulfate, phosphate；Rudimentary alkyl sulfonate, as mesylate,
Fluoroform sulphonate, esilate etc.；Arylsulphonate, such as benzene sulfonate, P-TOLUENE SULFO ACID 99's salt；Acylate, as acetate,
Malate, fumarate, succinate, citrate, tartrate, oxalates, maleate etc.；Amino-acid salt, it is such as sweet
Propylhomoserin salt, trimethylglycine salt, arginine salt, ornithine salt, glutamate, aspartate.
In the second aspect of the present invention, a kind of pharmaceutical composition is provided, described pharmaceutical composition includes first aspect
The deuterated compound in triazine class or its officinal salt；The officinal salt include its crystal form, pharmaceutically acceptable salt,
Prodrug, hydrate or solvate.
In a preferred example, described pharmaceutical composition further includes at least one pharmaceutically acceptable carrier.
In another preferred example, the pharmaceutical composition is injection, wafer, tablet, pill, powder or granule.
In another preferred example, described pharmaceutical composition further includes at least one other anti-tumor drug, described
Other anti-tumor drug.
Preferably, the anti-tumor drug includes but is not limited to: taxol, vincaleukoblastinum, vincristine, mostly because of him
Match, cis-platinum, carboplatin, cyclophosphamide, Carmustine, Dacarbazine, dactinomycin D, daunorubicin, bleomycin, podophyllotoxin
Class, urine fluoropyrimidine, methotrexate (MTX), cytarabine, camptothecin, tamoxifen, Raloxifene, receptor or non-receptor tyrosine swash
Enzyme.
To realize above-mentioned third purpose, the present invention provides a kind of medicine groups comprising the deuterated compound in triazine class
Close application of the object in the drug of Prevention and/or treatment and/or the proliferative diseases for assisting in the treatment of PI3K zymogenesis.
Further, the proliferative diseases of the PI3K/mTOR zymogenesis are colorectal cancer, gastric cancer, breast cancer, lung
Cancer, liver cancer, prostate cancer, film gland cancer, thyroid cancer, bladder cancer, kidney, brain tumor, neck cancer, the cancer of CNS, glioblastoma,
Or myeloproliferative disease and leukaemia and lymph cancer.
To realize that above-mentioned 4th purpose, the present invention provide deuterated compound in triazine class or its officinal salt described in one kind
Or the pharmaceutical composition containing deuterated compound in triazine class inhibits the application in cancer cell growth in vitro.
To realize above-mentioned 5th purpose, the present invention provides the systems of a kind of deuterated compound in triazine class or its officinal salt
Preparation Method, comprising the following steps:
Specifically includes the following steps:
Step 1: compound A is dissolved in organic solvent, and B is added, and base reagent, under nitrogen protection, heating reaction obtain
Target product compound C.
Step 2: compound C and D are dissolved in organic solvent, water, base reagent and palladium catalyst are added, in nitrogen protection
Under, heating reaction obtains target product compound E.
Preferably, the organic solvent is n,N-Dimethylformamide, tetrahydrofuran, dimethyl sulfoxide, toluene, N- first
Base pyrrolidones, dioxane.
Preferably, the base reagent is K2CO3, Na2CO3, Cs2CO3, K3PO4, triethylamine, n,N-diisopropylethylamine.
The invention has the advantages that having synthesized a kind of deuterated compound in triazine class, such compound selects PI3K/mTOR enzyme
Selecting property inhibitory activity is preferable, while such compound also has preferable pharmacokinetic profile.Due to the change after deuterated
Closing object becomes difficult the metabolism of drug, and first pass effect reduces, and gets a promotion to liver microsomes stability.In such case
Under, thus it is possible to vary dosage simultaneously forms durative action preparation, can also improve applicability in the form of durative action preparation, for further exploitation
Selective PI3K/mTOR inhibitor provides may.
Specific embodiment
The specific embodiment of form by the following examples makees further specifically above content of the invention
It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to following embodiment.It is all to be based on above content of the present invention
The technology realized all belongs to the scope of the present invention.
Embodiment one
The synthesis of compound E1:
Step 1: compound 1 (10mmol) is dissolved in tetrahydrofuran, N-bromosuccinimide is added portionwise
(10mmol) reacts 3 hours at room temperature, is poured into water, is extracted with ethyl acetate, and organic phase 2N salt acid extraction 3 times, water phase is used
Sodium carbonate is extracted with ethyl acetate again after being tuned into neutrality, merges organic phase, and drying obtains crude product 2 after being spin-dried for and is directly used in next step.
Step 2: compound 2 (10mmol) is dissolved in tetrahydrofuran, and 1,1- dimethoxy-N, N '-dimethyl first is added
Amine (12mmol), 60 degree are spin-dried for after lower reaction 3 hours, and heptane is added, crystallizes under 0 degree, filter intermediate 3 is directly used in down
One step.
Step 3: under 0 degree of nitrogen protection, the anhydrous tetrahydrofuran solution of compound 3 (10mmol) is added dropwise to isopropyl
In the tetrahydrofuran solution of base magnesium chloride (12mmol), under 0 degree after reaction 30 minutes, reacts 20 minutes, be then added at room temperature
2- isopropoxy -4,4,5,5- tetramethyls -1,3,2- dioxaborolanes (13mmol), 60 degree are reacted 3 hours down, under ice bath
It is quenched, is extracted with ethyl acetate with saturated aqueous ammonium chloride, it is dry, it is crystallized under -20 degree after being spin-dried for heptane, obtains compound
4。
Step 4: compound 6 (1mmol) and sodium bicarbonate (2mmol) is soluble in water, compound 5 (1mmol) is added
Dichloromethane solution reacts 1 hour at room temperature, and organic phase is spin-dried for rear pillar and chromatographs to obtain compound 7.
Step 5: compound 8 (2mmol) is soluble in water, the dichloromethane solution of compound 7 (1mmol), room temperature is added
Lower reaction 1 hour, organic phase is spin-dried for rear pillar and chromatographs to obtain compound 9.
Step 6: compound 4 (1mmol) and compound 9 (1mmol) are dissolved in tetrahydrofuran (8mL), and potassium carbonate is added
Then (2mmol), water, tetra-triphenylphosphine palladium (0.05mmol), heated under nitrogen add HCl's to 60 degree of reactions to complete
The solution of dioxanes reacts under the conditions of 55 degree, completely after, diluted with the HCL aqueous solution and ethyl acetate of 5M, separate water phase, used
Water phase is adjusted to neutrality by saturated sodium bicarbonate, then is extracted with ethyl acetate, and merges organic phase, drying obtains crude product after being spin-dried for, through column
Product E 1 is obtained after chromatography.
1H NMR(300MHz,DMSO-d6)δ8.57(s,1H),6.94(s,2H),6.79(s,1H),3.73(m,4H),
3.61(m,8H)。
Embodiment two
The synthesis of compound E2:
Step 1: compound 2 (2mmol) is soluble in water, the dichloromethane solution of compound 1 (1mmol), room temperature is added
Lower reaction 1 hour, organic phase is spin-dried for rear pillar and chromatographs to obtain compound 3.
Step 2: compound 4 (2mmol) is soluble in water, the dichloromethane solution of compound 3 (1mmol), room temperature is added
Lower reaction 1 hour, organic phase is spin-dried for rear pillar and chromatographs to obtain compound 5.
Step 3: compound 5 (1mmol) and compound 6 (1mmol) are dissolved in tetrahydrofuran (8mL), and potassium carbonate is added
Then (2mmol), water, tetra-triphenylphosphine palladium (0.05mmol), heated under nitrogen add HCl's to 60 degree of reactions to complete
The solution of dioxanes reacts under the conditions of 55 degree, completely after, diluted with the HCL aqueous solution and ethyl acetate of 5M, separate water phase, used
Water phase is adjusted to neutrality by saturated sodium bicarbonate, then is extracted with ethyl acetate, and merges organic phase, drying obtains crude product after being spin-dried for, through column
Product E 2 is obtained after chromatography.
1H NMR(300MHz,DMSO-d6)δ8.57(s,1H),6.94(s,2H),6.79(s,1H),3.73(m,8H),
3.61(m,4H)。
Embodiment three
The synthesis of compound E3:
Step 1: compound 2 (2mmol) is soluble in water, the dichloromethane solution of compound 1 (1mmol), room temperature is added
Lower reaction 1 hour, organic phase is spin-dried for rear pillar and chromatographs to obtain compound 3.
Step 2: compound 4 (2mmol) is soluble in water, the dichloromethane solution of compound 3 (1mmol), room temperature is added
Lower reaction 1 hour, organic phase is spin-dried for rear pillar and chromatographs to obtain compound 5.
Step 3: compound 5 (1mmol) and compound 6 (1mmol) are dissolved in tetrahydrofuran (8mL), and potassium carbonate is added
Then (2mmol), water, tetra-triphenylphosphine palladium (0.05mmol), heated under nitrogen add HCl's to 60 degree of reactions to complete
The solution of dioxanes reacts under the conditions of 55 degree, completely after, diluted with the HCL aqueous solution and ethyl acetate of 5M, separate water phase, used
Water phase is adjusted to neutrality by saturated sodium bicarbonate, then is extracted with ethyl acetate, and merges organic phase, drying obtains crude product after being spin-dried for, through column
Product E 3 is obtained after chromatography.
1H NMR(300MHz,DMSO-d6)δ8.57(s,1H),6.94(s,2H),6.79(s,1H),3.73(m,4H),
3.61(m,4H)。
Example IV
The synthesis of compound E4:
Step 1: compound 2 (2.5mmol) and sodium bicarbonate (5mmol) is soluble in water, it is added compound 1 (1mmol)
Dichloromethane solution, at room temperature react 1 hour, organic phase is spin-dried for rear pillar and chromatographs to obtain compound 3.
Step 2: compound 3 (1mmol) and compound 4 (1mmol) are dissolved in tetrahydrofuran (8mL), and potassium carbonate is added
Then (2mmol), water, tetra-triphenylphosphine palladium (0.05mmol), heated under nitrogen add HCl's to 60 degree of reactions to complete
The solution of dioxanes reacts under the conditions of 55 degree, completely after, diluted with the HCL aqueous solution and ethyl acetate of 5M, separate water phase, used
Water phase is adjusted to neutrality by saturated sodium bicarbonate, then is extracted with ethyl acetate, and merges organic phase, drying obtains crude product after being spin-dried for, through column
Product E 4 is obtained after chromatography.
1H NMR(300MHz,DMSO-d6)δ8.57(s,1H),6.94(s,2H),6.79(s,1H),3.59(s,8H)。
Embodiment five
The synthesis of compound E7:
Step 1: compound 2 (4mmol) is soluble in water, the dichloromethane solution of compound 1 (1mmol), room temperature is added
Lower reaction 1 hour, organic phase is spin-dried for rear pillar and chromatographs to obtain compound 3.
Step 2: compound 3 (1mmol) and compound 4 (1mmol) are dissolved in tetrahydrofuran (8mL), and potassium carbonate is added
Then (2mmol), water, tetra-triphenylphosphine palladium (0.05mmol), heated under nitrogen add HCl's to 60 degree of reactions to complete
The solution of dioxanes reacts under the conditions of 55 degree, completely after, diluted with the HCL aqueous solution and ethyl acetate of 5M, separate water phase, used
Water phase is adjusted to neutrality by saturated sodium bicarbonate, then is extracted with ethyl acetate, and merges organic phase, drying obtains crude product after being spin-dried for, through column
Product E 7 is obtained after chromatography.
1H NMR(300MHz,DMSO-d6)δ8.57(s,1H),6.94(s,2H),6.79(s,1H),3.73(s,8H)。
Embodiment six
The synthesis of compound E9:
Step 1: compound 2 (4mmol) is soluble in water, the dichloromethane solution of compound 1 (1mmol), room temperature is added
Lower reaction 1 hour, organic phase is spin-dried for rear pillar and chromatographs to obtain compound 3.
Step 2: compound 3 (1mmol) and compound 4 (1mmol) are dissolved in tetrahydrofuran (8mL), and potassium carbonate is added
Then (2mmol), water, tetra-triphenylphosphine palladium (0.05mmol), heated under nitrogen add HCl's to 60 degree of reactions to complete
The solution of dioxanes reacts under the conditions of 55 degree, completely after, diluted with the HCL aqueous solution and ethyl acetate of 5M, separate water phase, used
Water phase is adjusted to neutrality by saturated sodium bicarbonate, then is extracted with ethyl acetate, and merges organic phase, drying obtains crude product after being spin-dried for, through column
Product E 9 is obtained after chromatography.
1H NMR(300MHz,DMSO-d6)δ8.57(s,1H),6.94(s,2H),6.79(s,1H)。
Embodiment seven
The synthesis of compound E10:
Step 1: compound 2 (4mmol) is soluble in water, the dichloromethane solution of compound 1 (1mmol), room temperature is added
Lower reaction 1 hour, organic phase is spin-dried for rear pillar and chromatographs to obtain compound 3.
Step 2: compound 3 (1mmol) and compound 4 (1mmol) are dissolved in tetrahydrofuran (8mL), and potassium carbonate is added
Then (2mmol), water, tetra-triphenylphosphine palladium (0.05mmol), heated under nitrogen add HCl's to 60 degree of reactions to complete
The solution of dioxanes reacts under the conditions of 55 degree, completely after, diluted with the HCL aqueous solution and ethyl acetate of 5M, separate water phase, used
Water phase is adjusted to neutrality by saturated sodium bicarbonate, then is extracted with ethyl acetate, and merges organic phase, drying obtains crude product after being spin-dried for, through column
Product E 10 is obtained after chromatography.
1H NMR(600MHz,DMSO-d6)δ8.27(s,1H),6.78(s,2H),5.97(s,1H),4.77(bs,2H)。
Embodiment eight
The synthesis of the hydrochloride of E4
Step 1: compound E4 (1mmol) is dissolved in acetone, and isopropanol (5M, 1.5mmol) solution of HCl, room is added
The lower stirring and crystallizing of temperature one hour, solid filter E4 hydrochloride.
Embodiment nine
The preparation of the hydrochloride monohydrate of E4
Step 1: compound E4 (1mmol) is dissolved in acetone, the 12M aqueous solution (1.5mmol) of HCl is added, at room temperature
Stirring and crystallizing one hour, solid filter E4 hydrochloride monohydrate.
Embodiment ten
The preparation of the mesylate of E4
Step 1: compound E4 (1mmol) is dissolved in acetone, is added methanesulfonic acid (1mmol), at room temperature stirring and crystallizing one
Hour, solid filter E4 mesylate.
Embodiment 11
The external zymetology inhibitory activity of the compounds of this invention
MTOR zymetology experimental method
1. agent formulation
1.1 1 times of kinase buffer liquids: 50mM HEPES, pH 7.5,10mM MgCl2, 1mM EGTA, 3mM MnCl2,
0.01%Tween-20,2mM DTT；
1.2 4 times of kinase solutions: being added mTOR kinases, prepare 4 times of kinase solutions in 1 times of kinase buffer liquid, final concentration of
2.5nM or 4nM；
1.3 2 times of substrates and ATP solution: substrate 4EBP1 and ATP are added in 1 times of kinase buffer liquid, it is molten to prepare 2 times of substrates
Liquid, final concentration of 10.8 μM or 6 μM of 4EBP1 final concentration of 50nM, ATP；
1.4 4 times of tester solution: 100 times of maximum concentration of tester solution, final concentration 10 are prepared using 100%DMSO
μM, 25 times are diluted with 4 times of the 100%DMSO concentration of gradient dilution 10, then with 1 times of kinase buffer liquid, obtains 4 times of tester
Solution；
The preparation of 1.5 detection liquid: the detection liquid for containing 2 times of concentration EDTA and 4EBP1 phospho-ABs, EDTA final concentration are prepared
For 8mM, the final concentration of 2nM of 4EBP phospho-AB.
2. experimental procedure
2.1 4 times of tester solution that 2.5 μ L of every hole addition are serially diluted into 384 orifice plates, multiple holes；
2.5 μ L, 4 times of kinase solutions are added in 2.2 every holes, and vibration mixes；
5 μ L, 2 times of substrates and ATP solution is added in 2.3 every holes, is incubated for 1 hour at room temperature；
2.4, which are eventually adding 10 μ L detection liquid, terminates reaction, and after sixty minutes, Envision reads data Lance signal
(665nM)。
3. data processing
Inhibiting rate %=(maximum value-sample value)/(maximum value-minimum value) * 100
Wherein " maximum value " is DMSO control wells reading, and " minimum value " is the control wells reading that kinases is not added.
It inputs GraphPad Prism 5.0 to map, obtains curve and IC50.
PI3K α zymetology experimental method
1. agent formulation
1.1 1 times of kinase buffer liquids: 50mM HEPES, pH 7.5,3mM MgCl2, 1mM EGTA, 100mM NaCl,
0.03%CHAPS, 2mM DTT；
1.2 4 times of kinase solutions: being added PI3K alpha kinase, prepare 4 times of kinase solutions in 1 times of kinase buffer liquid, final concentration of
1.65nM；
1.3 2 times of substrates and ATP solution: substrate PIP2 and ATP are added in 1 times of kinase buffer liquid, it is molten to prepare 2 times of substrates
Liquid, final concentration of 50 μM of PIP2, final concentration of 25 μM of ATP；
1.4 4 times of tester solution: 100 times of maximum concentration of tester solution, final concentration 10 are prepared using 100%DMSO
μM, 25 times are diluted with 4 times of the 100%DMSO concentration of gradient dilution 10, then with 1 times of kinase buffer liquid, obtains 4 times of tester
Solution；
1.5Kinase-Glo reagent reagent, is placed and is warming up to room temperature, for terminating reaction and generating detection letter
Number.
2. experimental procedure
2.1 4 times of tester solution that 2.5 μ L of every hole addition are serially diluted into 384 orifice plates；
2.5 μ L, 4 times of kinase solutions are added in 2.2 every holes, and vibration mixes；
5 μ L, 2 times of substrates and ATP solution is added in 2.3 every holes, is incubated for 1 hour at room temperature；
2.4, which are eventually adding 10 μ L detection liquid, terminates reaction, and after slowly shaking 15 minutes, Flexstation reads data
RLU。
3. data processing
Inhibiting rate %=(maximum value-sample value)/(maximum value-minimum value) * 100
Wherein " maximum value " is DMSO control wells reading, and " minimum value " is the control wells reading that kinases is not added.
It inputs GraphPad Prism 5.0 to map, obtains curve and IC50.
PI3K δ zymetology experimental method
1. agent formulation
1.1 1 times of kinase buffer liquids: 50mM HEPES, pH 7.5,3mM MgCl2, 1mM EGTA, 100mM NaCl,
0.03%CHAPS, 2mM DTT；
1.2 4 times of kinase solutions: being added PI3K δ kinases, prepare 4 times of kinase solutions in 1 times of kinase buffer liquid, final concentration of
5.7nM；
1.3 2 times of substrates and ATP solution: substrate PIP2 and ATP are added in 1 times of kinase buffer liquid, it is molten to prepare 2 times of substrates
Liquid, final concentration of 50 μM of PIP2, final concentration of 25 μM of ATP；
1.4 4 times of tester solution: 100 times of maximum concentration of tester solution, final concentration 10 are prepared using 100%DMSO
μM, 25 times are diluted with 4 times of the 100%DMSO concentration of gradient dilution 10, then with 1 times of kinase buffer liquid, obtains 4 times of tester
Solution；
1.5Kinase-Glo reagent reagent, is placed and is warming up to room temperature, for terminating reaction and generating detection letter
Number.
2. experimental procedure
2.1 4 times of tester solution that 2.5 μ L of every hole addition are serially diluted into 384 orifice plates；
2.5 μ L, 4 times of kinase solutions are added in 2.2 every holes, and vibration mixes；
5 μ L, 2 times of substrates and ATP solution is added in 2.3 every holes, is incubated for 1 hour at room temperature；
2.4, which are eventually adding 10 μ L detection liquid, terminates reaction, and after slowly shaking 15 minutes, Flexstation reads data
RLU。
3. data processing
Inhibiting rate %=(maximum value-sample value)/(maximum value-minimum value) * 100
Wherein " maximum value " is DMSO control wells reading, and " minimum value " is the control wells reading that kinases is not added.
It inputs GraphPad Prism 5.0 to map, obtains curve and IC50。
PI3K β, PI3K γ zymetology experimental method
1. agent formulation
1.1 1 times of kinase buffer liquids: 50mM HEPES, pH 7.5,3mM MgCl2, 1mM EGTA, 100mM NaCl,
0.03%CHAPS, 2mM DTT；
1.2 4 times of kinase solutions: it is separately added into PI3K β or PI3K γ kinases in 1 times of kinase buffer liquid, prepares 4 times of kinases
Solution, final concentration of PI3K β 4.8nM, PI3K γ 7.6nM；
1.3 2 times of substrates and ATP solution: substrate PIP2 and ATP are added in 1 times of kinase buffer liquid, it is molten to prepare 2 times of substrates
Liquid, final concentration of 50 μM of PIP2, final concentration of 25 μM of ATP；
1.4 4 times of tester solution: 100 times of maximum concentration of tester solution, final concentration 10 are prepared using 100%DMSO
μM, 25 times are diluted with 4 times of the 100%DMSO concentration of gradient dilution 10, then with 1 times of kinase buffer liquid, obtains 4 times of tester
Solution；
1.5ADP-Glo reagent reagent, is placed and is warming up to room temperature, for terminating reaction and generating detection signal.
2. experimental procedure
2.1 4 times of tester solution that 2.5 μ L of every hole addition are serially diluted into 384 orifice plates；
2.5 μ L, 4 times of kinase solutions are added in 2.2 every holes, and vibration mixes；
5 μ L, 2 times of substrates and ATP solution is added in 2.3 every holes, is incubated for 1 hour at room temperature；
Often also taking out 5 μ L reaction solutions is transferred to 384 new orifice plates in 2.4 384 orifice plates, new 384 orifice plates that reaction solution is added
In every hole 5 μ L ADP-Glo reagent are added, after slowly shaking 1 minute, balance 60 minutes, synergy reads data RLU.
3. data processing
Inhibiting rate %=(maximum value-sample value)/(maximum value-minimum value) * 100
Wherein " maximum value " is DMSO control wells reading, and " minimum value " is the control wells reading that kinases is not added.
It inputs GraphPad Prism 5.0 to map, obtains curve and IC50。
External the enzyme activity (the IC of 1. compound of table50, nM)
| Tester | PI3Kα | PI3K β, | PI3Kγ | PI3Kδ | mTOR |
| E4 | 36 | 678 | 731 | 451 | 91 |
| E9 | 40 | 684 | 728 | 460 | 95 |
| Bimiralisib | 38 | 685 | 721 | 465 | 94 |
Embodiment 12
External people's liver microsome enzyme stability experiment
(1) experimental procedure
Test-compound (will be shown in Table) under the following conditions with people's hepatomicrosome carries out total incubation, be added into incubation tube by
Examination compound makees liquid, and wink is from being placed in 37 DEG C of water-baths after mixing.Then the working solution that NADPH is added starts reaction.0,5,
15,30,60min, which takes out part Incubating Solution and is transferred in acetonitrile containging interior traget, terminates reaction.After albumen precipitation, 3,700rpm
It is centrifuged 10min, takes supernatant.Test-compound in supernatant is analyzed by LC-MS/MS method.It is being incubated for according to test-compound
Removing half-life period in system calculates external inherent clearance rate.Midazolam is incubated in parallel as positive control.Incubation conditions are total
Knot such as following table (content of incubation system organic solvent is no more than 1%), parallel to be incubated for 2 parts:
(2) data are analyzed
The ratio between analyte/internal standard peak area (Aanalyte/AIS) will be obtained by instrument, remaining percentage (%Control)
It is calculated by the ratio between non-zero time point sample and Aanalyte/AIS in zero moment sample.When by Ln (%Control) to being incubated for
Between map and carry out linear fit.Test-compound remove constant (k, min-1), remove it is half-life period (T1/2, min) and external
Inherent clearance rate (CLint, μ L min-1mg-1proteins) is calculated by following equation.
K=-slope
T1/2=0.693/k
CLint=k/Cprotein
Cprotein (mg mL-1) refers to the microsomal protein matter concentration in incubation system.
(3) 2 be the results are shown in Table
Stability test of 2 target compound of table to people's hepatomicrosome enzyme
It can be seen that deuterated compound in triazine class E4, E9 that the present invention is protected from the experimental result of table 1 to swash PI3K
Different subtype PI3K α, PI3K β, PI3K γ, PI3K δ and the mTOR of enzyme all have apparent inhibitory activity, with reference substance
Bimiralisib is compared, it is deuterated after compound inhibitory activity not less than even better than reference substance Bimiralisib.From table 2
Experimental result can be seen that it is deuterated after compound E4 and E9 to the stability of people's hepatomicrosome enzyme better than reference substance
Bimiralisib.It can be seen that the compound in triazine class after deuterated has preferable PI3K/mTOR inhibitory activity, while to liver
Dirty microsome shows higher stability, before the wide market that provides of exploitation of specificity PI3K/mTOR inhibitor
Scape.
The preferred embodiment of the present invention has been described in detail above, but the invention be not limited to it is described
Embodiment, those skilled in the art can also make various equivalent on the premise of not violating the inventive spirit of the present invention
Variation or replacement, these equivalent variation or replacement are all included in the scope defined by the claims of the present application.
Claims (9)
1. a kind of deuterated compound in triazine class as shown in formula (I) or its officinal salt,
It is characterized in that, wherein:
R1-R10 is independently represented each other H or D；
X represents N, CH or CD.
2. deuterated compound in triazine class according to claim 1 or its officinal salt, which is characterized in that described is pharmaceutically acceptable
Salt includes inorganic acid salt or acylate.
3. a kind of pharmaceutical composition, which is characterized in that described pharmaceutical composition contains deuterated triazine as described in claim 1
Class compound or pharmaceutically acceptable salt thereof.
4. pharmaceutical composition according to claim 3, which is characterized in that described pharmaceutical composition includes at least one pharmacy
Upper acceptable carrier.
5. pharmaceutical composition according to claim 3 or 4, which is characterized in that described pharmaceutical composition includes at least one
Other anti-tumor drug.
6. a kind of pharmaceutical composition as claimed in claim 5 is in Prevention and/or treatment and/or adjuvant treatment PI3K/
Application in the drug of the proliferative diseases of mTOR zymogenesis.
7. a kind of deuterated compound in triazine class as described in claim 1 or its officinal salt inhibit cancer cell growth in vitro
In application.
8. a kind of pharmaceutical composition as claimed in claim 5 inhibits the application in cancer cell growth in vitro.
9. a kind of preparation method of the deuterated compound in triazine class as shown in formula (I), which is characterized in that the following route of the method
(1) shown in:
Specifically includes the following steps:
Step 1: compound A is dissolved in organic solvent, and compound B is added, and base reagent, under nitrogen protection, heating react
To target product compound C.
Step 2: compound C and D are dissolved in organic solvent, and water, base reagent is added, and palladium catalyst adds under nitrogen protection
Thermal response obtains target product compound E.
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