CN110526921A - A kind of compound and its preparation method and application with anti-inflammatory effect - Google Patents
A kind of compound and its preparation method and application with anti-inflammatory effect Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
- C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
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Abstract
The present invention provides Formulas I compound represented or its salt.The experimental results showed that the compound of the present invention, especially compound 7 and compound 9, can effectively inhibit the emission levels of inflammatory factor IL-6；Compound 9 can inhibit the protein level of phosphorylation NF- κ B p65 protein level, phosphorylation Akt protein level, pSTAT3 protein level and adhesivemoleculeICAM1.The compounds of this invention has extraordinary application prospect on the drug of preparation treatment diseases associated with inflammation.
Description
Technical field
The invention belongs to pharmaceutical synthesis, and in particular to a kind of small point of miazines of pyrazolo [3,4-d] with anti-inflammatory effect
Sub- derivative and its preparation method and application.
Background technique
The occurrence and development of inflammation and most of diseases are closely related, including coronary heart disease, atherosclerosis, diabetes and
Tumour etc., inflammation have become a symbolic characteristic of a variety of diseases of the mankind.Tumor necrosis factor-alpha (TNF-α), leucocyte
The inflammatory factors such as interleukin -6 (IL-6), interleukin-1 ' beta ' (IL-1 β) play important work during inflammation occurrence and development
With.With deepening continuously for inflammatory signals pathway research, IL-6 has become the effective therapy target of active chronic inflammation, In
Play key player during inflammatory diseases.
Currently, having developed a variety of inhibitor for IL-6 signal cascade, including anti-IL-6, interleukin-6 receptor (IL-
6R) and the monoclonal antibody of solvable glycoprotein 130 (sgp130Fc), part of inhibitor are carrying out clinical test.It uses
Monoclonal antibody is often accompanied by the adverse reactions such as alpastic anemia and congestive heart failure.In contrast, small molecule
Closing object drug more usually has better bioavilability compared with monoclonal antibody drug and is conducive to the characteristics such as the manufacturing.
Therefore, the relevant micromolecular inhibitor of new IL-6 is developed with extraordinary prospect.
Pyrazolo [3,4-d] pyrimidines belong to nitrogenous fused ring compound, are the isosteres of purine, and by
Have similar structure to show multiple pharmacological effect with purine in it.Therefore, pyrazolo [3,4-d] pyrimidines
The always research hotspot of field of pharmacology.It has been reported that pyrazolo [3,4-d] pyrimidines have antibacterial, antiviral, anti-
Cancer, anti-diabetic, inhibits xanthine oxidase to treat a variety of pharmacological activity such as gout at anti-inflammatory, anti-hypertension.
But it is currently known pyrazolo [3,4-d] pyrimidines also generally existing anti-inflammatory efficiency on anti-inflammatory effect
Problem low, toxic side effect is strong.It is weighed very much so the relevant micromolecular inhibitor of the IL-6 for preparing more structure novels has
The meaning wanted.
Summary of the invention
The purpose of the present invention is to provide a kind of pyrazolo of structure novel [3,4-d] miazines small molecule derivatives.
The present invention provides a kind of Formulas I compound represented or its salt:
Wherein, R1ForWherein, W is selected from nothing, NH, CH2,O,S；R5Selected from H, C1-6 alkyl, C1-6 alkoxy,
Halogen, hydroxyl, carboxyl, amino, aryl, heteroaryl, saturated cyclic alkyls, saturated heterocyclyl；
R2Selected from halogen, amino, hydroxyl, carboxyl, by 0~5 R4The following group replaced: aryl, heteroaryl, saturated rings
Alkyl, saturated heterocyclyl, C1-6 alkyl, C1-6 alkoxy；R4Selected from hydroxyl, carboxyl, halogen, H, C1-6 alkyl, C1-6 alcoxyl
Base；
R3Selected from by 0~3 R6The following group replaced: C1-8 alkyl, C1-8 alkoxy, C2-8 alkenyl, C2-8 alkynyl；
R6Selected from hydroxyl, carboxyl, halogen.
Further,
R1For-WR5, wherein W is selected from NH or O, R5Selected from H, C1-4 alkyl, C1-4 alkoxy, halogen, hydroxyl, carboxyl；
R2ForWherein, M is CH or N, R4Selected from hydroxyl, carboxyl, halogen, H, C1-3 alkyl, C1-3 alcoxyl
Base；
R3Selected from C1-4 alkyl.
Further,
R1It is selected from
R2It is selected from
R3It is selected from
Further,
The compound is selected from one of flowering structure:
The present invention also provides a kind of methods for preparing above compound 1-9, the described method comprises the following steps:
(1) compound 10 is reacted with Iso-Propyl iodide or iodo-n-butane, obtains compound 11；
(2) compound 11 is reacted with sodium methoxide, obtains compound 12；
(3) compound 12 is reacted with 3- hydroxy benzenes pinacol borate or 4- hydroxy benzenes pinacol borate to get chemical combination
Object 1,2,3 or 4；
Or, the described method comprises the following steps:
(2 ') compound 11 is reacted with methylamine, obtains compound 13；
(3 ') compound 13 is reacted with 3- hydroxy benzenes pinacol borate or 4- hydroxy benzenes pinacol borate to get chemical combination
Object 5 or 6；
Or, the described method comprises the following steps:
(a) compound 14 is reacted with Iso-Propyl iodide, obtains compound 15；
(b) compound 15 is reacted with 3- hydroxy benzenes pinacol borate or 4- hydroxy benzenes pinacol borate to get chemical combination
Object 7 or 8；
Or, the described method comprises the following steps:
(a ') compound 16 is reacted with tertiary fourth hydrazine hydrochloride, obtains compound 17；
(b ') compound 17 and formamide, obtain compound 18；
(c ') compound 18 is reacted with N-bromosuccinimide, obtains compound 19；
(d ') compound 19 and 5- (4,4,5,5- tetramethyls -1,3,2- bis- dislike borine -2- base) pyrimidine -3- alcohol reaction, i.e.,
Obtain compound 9；
The structure of above compound 10 isThe structure of compound 11 isCompound 12
Structure isIn compound 11 and compound 12, R3For
The structure of compound 13 isWherein R3For
The structure of compound 14 isThe structure of compound 15 is
The structure of compound 16 isThe structure of compound 17 isThe structure of compound 18 isThe structure of compound 19 is
Further,
In step (1), the reaction is carried out under the action of potassium carbonate, and reaction dissolvent is organic solvent, reaction temperature
Degree is 60~100 DEG C, and the reaction time is 2.0~5.0 hours；
In step (2), the reaction dissolvent be organic solvent, reaction temperature be 60~100 DEG C, the reaction time be 2.0~
5.0 hour；
In step (2 '), the reaction dissolvent be organic solvent, reaction temperature be 60~100 DEG C, the reaction time be 2.0~
5.0 hour；
In step (3) or (3 '), the reaction is in PdCl2It is carried out under the action of dppf and potassium carbonate, reaction dissolvent
For the mixed solution of Isosorbide-5-Nitrae-dioxane and water, reaction temperature is 60~100 DEG C, and the reaction time is 2.0~5.0 hours；
In step (a), the reaction is carried out under the action of potassium carbonate, and reaction dissolvent is organic solvent, reaction temperature
Degree is 60~100 DEG C, and the reaction time is 2.0~5.0 hours；
In step (b), the reaction is in PdCl2It is carried out under the action of dppf and potassium carbonate, reaction dissolvent is Isosorbide-5-Nitrae-
The mixed solution of dioxane and water, reaction temperature are 60~100 DEG C, and the reaction time is 2.0~5.0 hours；
In step (a '), the reaction is carried out under the action of triethylamine, and reaction dissolvent is organic solvent, reaction temperature
Degree is 60~100 DEG C, and the reaction time is 2.0~5.0 hours；
In step (b '), the reaction temperature is 100~200 DEG C, and the reaction time is 2.0~5.0 hours；
In step (c '), the reaction dissolvent be organic solvent, reaction temperature be 60~100 DEG C, the reaction time be 2.0~
5.0 hour；
In step (d '), the reaction is in PdCl2It is carried out under the action of dppf and potassium carbonate, reaction dissolvent is Isosorbide-5-Nitrae-
The mixed solution of dioxane and water, reaction temperature are 60~100 DEG C, and the reaction time is 2.0~5.0 hours.
Further,
In step (1), the reaction dissolvent is MeCN, and reaction temperature is 100 DEG C, and the reaction time is 3 hours；
In step (2), the reaction dissolvent is methanol, and reaction temperature is 65 DEG C, and the reaction time is 3.5 hours；
In step (2 '), the reaction dissolvent is ethyl alcohol, and reaction temperature is 75 DEG C, and the reaction time is 3.5 hours；
In step (3) or (3 '), the reaction dissolvent is that the mixing of Isosorbide-5-Nitrae-dioxane and water that volume ratio is 4:1 is molten
Liquid, reaction temperature are 100 DEG C, and the reaction time is 2 hours；
In step (a), the reaction dissolvent is MeCN, and reaction temperature is 80 DEG C, and the reaction time is 3 hours；
In step (b), the reaction dissolvent is that volume ratio is Isosorbide-5-Nitrae-dioxane of 4:1 and the mixed solution of water, reaction
Temperature is 100 DEG C, and the reaction time is 2 hours；
In step (a '), the reaction dissolvent is ethyl alcohol, and reaction temperature is 78 DEG C, and the reaction time is 3 hours；
In step (b '), the reaction temperature is 180 DEG C, and the reaction time is 5 hours；
In step (c '), the reaction dissolvent is methanol, and reaction temperature is 100 DEG C, and the reaction time is 2 hours；
In step (d '), the reaction dissolvent is that volume ratio is Isosorbide-5-Nitrae-dioxane of 4:1 and the mixed solution of water, reaction
Temperature is 100 DEG C, and the reaction time is 2 hours.
The present invention also provides above compounds or its salt to prepare the purposes on interleukin-6 inhibitor.
The present invention also provides above compounds or its salt to prepare the purposes on anti-inflammatory drug, it is preferable that described anti-inflammatory
Drug can reduce phosphorylation NF- κ B p65 protein level, phosphorylation Akt protein level, pSTAT3 protein level and glue
The protein level of attached molecule ICAM-1.
The present invention also provides a kind of pharmaceutical composition, described pharmaceutical composition is with above compound or its salt for work
Property ingredient, in addition pharmaceutically acceptable auxiliary material is made.
The experimental results showed that the compound of the present invention, especially compound 7 and compound 9, can effectively inhibit inflammation because
The emission levels of sub- IL-6；Compound 9 can inhibit phosphorylation NF- κ B p65 protein level, phosphorylation Akt protein level, phosphoric acid
Change the protein level of STAT3 protein level and adhesivemoleculeICAM1.The compounds of this invention is in preparation treatment diseases associated with inflammation
There is extraordinary application prospect on drug.
Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The specific embodiment of form by the following examples remakes 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 example below.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.
Detailed description of the invention
Apoptosis result-streaming figure of Fig. 1 compound 7.
Inhibition-streaming figure of Fig. 2 compound 7 to the A549 cell cycle.
Influence-streaming figure of Fig. 3 compound 9 to A549 Apoptosis.
Inhibition-streaming figure of Fig. 4 compound 9 to the A549 cell cycle.
Fig. 5 compound on inflammation factor IL-6 level inhibiting effect (N=3, * P < 0.05, * * P <
0.01, * * * P < 0.001vs Medium group).
Fig. 6 .Western blotting method detection IL-6 upstream and downstream associated signal paths protein expression level (* p <
0.05, * * p < 0.01, * * * p < 0.001vs IL-4group).
Fig. 7 .Western blotting method detects ICAM-1 protein expression level, (* p < 0.05, * * p <
0.01, * * * p < 0.001vs IL-4group).
Specific embodiment
The raw materials used in the present invention and equipment are known product, as obtained by purchase commercial product.
Wherein, part Experiment reagent source is as shown in table 1:
1. part Experiment reagent of table
Key instrument is as follows:
(1) thin layer chromatography board: 60F245 type, 0.2mm, Merck, Germany
(2) column chromatography silica gel: silica gel H-GF254,200-300 mesh, Haiyang Chemical Plant, Qingdao
(3) mass spectrometer: Q-TOF spectrometer, ESI ionization source, German Bruker
(4) Nuclear Magnetic Resonance: AV II-400MHz, AV II-600MHz or AV II-800MHz, TMS are internal standard, Germany
Bruker。
The present invention uses following routes, has been respectively synthesized target compound of the invention:
(1) pyrazolo [3,4-d] pyrimidine derivatives synthetic route one:
Reagent and condition: i) Iso-Propyl iodide or iodo-n-butane, K2CO3, MeCN, 100 DEG C, 3h, yield: 11a:85%,
11b:83%；ii)CH3ONa,CH3OH, 65 DEG C, 3.5h, yield: 12a:73%, 12b:80%；Iii) methylamine, ethyl alcohol, 75 DEG C,
3.5h, Yield:69%；Iv) pinacol borate, PdCl2dppf,K2CO3,1,4- dioxane/water (4:1), 100 DEG C, 2h,
Yield :~73%-85%.(2) pyrazolo [3,4-d] pyrimidine derivatives synthetic route two:
Reagent and condition: i) Iso-Propyl iodide, K2CO3, MeCN, 80 DEG C, 3h, yield: 83%；Ii) pinacol borate,
PdCl2dppf,K2CO3,1,4- dioxane/water (4:1), 100 DEG C, 2h, Yield :~46%-85%.
(3) pyrazolo [3,4-d] pyrimidine derivatives synthetic route three:
Reagent and condition: i) tertiary fourth hydrazine hydrochloride, triethylamine, ethyl alcohol, 78 DEG C, 3h, yield: 96%；Ii) formamide, 180
DEG C, 5h, yield: 91%；Iii) N-bromosuccinimide, MeCN, 100 DEG C, 2h, yield: 89%；Iv) pinacol borate,
PdCl2dppf,K2CO3,1,4- dioxane/water (4:1), 100 DEG C, 2h, Yield:47%.
The synthesis path of target compound of the present invention is largely divided into 3 kinds: 1. with the chloro- 1H- pyrazolo [3,4-d] of the bromo- 4- of 3-
Pyrimidine is starting material；2. using bromo- 4- amino -1H- pyrazolo [3,4-d] pyrimidine of 3- as starting material；3. sub- with 2- ethyoxyl
Methylmalononitrile and tertiary fourth hydrazine hydrochloride are starting material.Work as R3When to replace isopropyl or normal-butyl, synthesis is using classics
It is alkylated substitution reaction, Iso-Propyl iodide or iodo-n-butane are sent out under the catalysis of potassium carbonate with pyrido [3,4-d] pyrimidine ring
Raw nucleophilic substitution obtains alkyl-substituted key intermediate.Work as R3When replacing for tert-butyl, due to tert-butyl steric hindrance
It is larger, it cannot react to obtain product with iodo-tert-butane by above-mentioned synthesis path.To in R3Position introduces tert-butyl, needs
It to be introduced before closed loop, referring to the synthetic method of Todorovic etc., using pyrazole ring as female ring synthesizing pyrazole simultaneously [3,4-d] pyrimidine
Derivative: 2- ethoxy methylene malononitrile and the reaction of tertiary fourth hydrazine hydrochloride first generates 1- tert-butyl -5- amino -1H- pyrazoles -
4- nitrile, then obtains R with formamide again3For pyrazolo [3,4-d] pyrimidine mother nucleus structure of tert-butyl, first two steps yield is all
Higher (about 90%), reacts to obtain key intermediate with N- bromo-succinimide (NBS) later, first three step comprehensive yied is about
75%.Different substituted aryl R are introduced on heterocycle2The suzuki coupling reaction of Shi Caiyong classics, using potassium carbonate and palladium chloride
Pinacol borate is replaced to react with aryl as catalyst.
The following are the synthesis steps of particular compound of the present invention:
The preparation of embodiment 1, the compounds of this invention
Chloro- 1H- pyrazolo [3,4-d] pyrimidine (11a) of the bromo- 4- of 1- normal-butyl -3-: 3- is added in 250mL round-bottomed flask
Chloro- 1H- pyrazolo [3, the 4-d] pyrimidine 10 (1g, 4.3mmol) of bromo- 4- and the dry acetonitrile solution of 150mL, stirring and dissolving, successively plus
Enter potassium carbonate (1.19g, 8.6mmol) and iodo normal-butyl 3mL (dropwise addition), is stirred at room temperature 10 minutes, temperature is then risen to 80
DEG C reflux.The reaction was continued about 3h, TLC monitoring show end of reaction (solvent: CH2Cl2/CH3OH=95/5).Liquid cooling to be reacted
But after, 50mL water and CH is added2Cl2(100mL × 3) extraction, merges organic phase, appropriate anhydrous sodium sulfate is added and dries, filters,
Concentration.Crude product separates (eluant, eluent: CH using silica gel column chromatography2Cl2) obtain the bromo- 4- of white solid product 1- normal-butyl -3-
Chloro- 1H- pyrazolo [3,4-d] pyrimidine 1.06g, yield: 85%.1H NMR (600MHz, CDCl3): δ 0.93 (t, J=7.4,
3H), 1.31 (m, 2H), 1.90 (m, 2H), 4.44 (t, J=7.2,2H), 8.71 (s, 1H)13C NMR(151MHz,CDCl3): δ
13.69,19.97,31.61,48.11,112.36,119.27,153.87,155.22,155.31.
Chloro- 1H- pyrazolo [3,4-d] pyrimidine (11b) of the bromo- 4- of 1- isopropyl -3-: the method for being combined to 11a according to alkyl:
Chloro- 1H- pyrazolo [3,4-d] pyrimidine (1g, 4.3mmol) of the bromo- 4- of 10 3- of compound participates in reaction and obtains white solid product 1-
Chloro- 1H- pyrazolo [3,4-d] the pyrimidine 0.98g of the bromo- 4- of isopropyl -3-, yield: 83%.1H NMR (600MHz, CDCl3): δ
1.56 (d, J=6.7,6H), 5.18 (hept, J=6.7,1H), 8.70 (s, 1H)13C NMR(151MHz,CDCl3): δ
22.11,50.80,112.55,119.04,153.06,154.93,155.19.
Bromo- 4- methoxyl group -1H- pyrazolo [3,4-d] pyrimidine (12a) of 1- normal-butyl -3-: by the bromo- 4- of 1- normal-butyl -3-
Chloro- 1H- pyrazolo [3,4-d] pyrimidine 11a (0.5g, 1.74mmol) is dissolved in the dry CH of 50mL3In OH solution, 15mL 2M is added
CH3ONa solution.The mixed liquor about reacts 3.5h at 65 DEG C, and TLC monitoring reaction terminates (solvent: CH2Cl2/CH3OH=95/
5).It is cooled to room temperature to reaction solution, 30mL water and CH is added2Cl2(150mL × 3) extraction, merges organic phase, is added appropriate anhydrous
Sodium sulphate dries, filters, and concentration, silica gel column chromatography separates (eluant, eluent: CH2Cl2) obtain white solid product 1- normal-butyl -3-
Bromo- 4- methoxyl group -1H- pyrazolo [3,4-d] pyrimidine 0.36g, yield: 73%.HRMS-ESI(m/z)calcdfor[M+H]+,
285.0273；found,285.1153.
Bromo- 4- methoxyl group -1H- pyrazolo [3,4-d] pyrimidine (12b) of 1- isopropyl -3-: using synthesis compound 12a's
Method, reacted, purifying obtains 12b, yield: 80%.1H NMR(600MHz,CDCl3): δ 1.53 (d, J=6.7,6H), 4.15
(s, 3H), 5.11 (hept, J=6.7,1H), 8.48 (s, 1H)13C NMR(151MHz,CDCl3)δ22.19,50.16,
54.62,103.19,117.72,154.56,155.69,164.03.
This process of aryl is introduced in pyrazolo [3,4-d] pyrimidine ring position parent nucleus C9 involved in the present invention, is adopted
It is reacted with classical Suzuki coupling.Concrete operation method is for synthesizing compound (1).
1- normal-butyl -3- (3- phenolic group) -4- methoxyl group -1H- pyrazolo [3,4-d] pyrimidine (1): 75mL 1,4- is being housed
The bromo- 4- methoxyl group -1H- pyrrole of 1- normal-butyl -3- is sequentially added in the 100mL round-bottomed flask of dioxane/water (4:1) mixed solution
Azoles simultaneously [3,4-d] pyrimidine 12a (0.20g, 0.70mmol), 3- hydroxy benzenes pinacol borate (0.23g, 1.05mmol), K2CO3
(0.19g, 1.4mmol), PdCl2Dppf (0.13g, 0.18mmol), is stirred well to is completely dissolved at room temperature.Reaction is placed in
Oil bath pan is heated to 100 DEG C of reflux, reacts about 2h, and TLC monitoring reaction terminates (solvent: CH2Cl2/CH3OH=95/5), molten
Liquid becomes black by orange-yellow.After reaction solution is cooling, 30mL water and CH is added2Cl2(100mL × 3) extraction, merges organic phase,
Appropriate anhydrous sodium sulfate is added to dry, filter, is concentrated, silica gel column chromatography separates (eluant, eluent: CH2Cl2/CH3OH=100/1-
100/3) faint yellow solid product 1- normal-butyl -3- (3- phenolic group) -4- methoxyl group -1H- pyrazolo [3,4-d] pyrimidine is obtained
0.16g, yield: 73.9%.1H NMR(600MHz,CDCl3): δ 0.93 (t, J=7.4,3H), 1.36 (m, 2H), 1.95 (m,
2H), 4.16 (s, 3H), 4.49 (t, J=7.2,2H), 6.91 (dd, J=8.0,2.3,1H), 7.33 (t, J=7.9,1H),
7.56 (s, 1H), 7.63 (d, J=7.7,1H), 8.58 (s, 1H)13C NMR(151MHz,CDCl3):δ13.82,20.12,
31.95,47.59,54.62,100.44,115.71,116.04,121.34,129.85,133.91,144.04,155.02,
155.64,156.11,164.42.HRMS-ESI(m/z)calcd for[M+H]+,299.1430；found,299.1505.
1- normal-butyl -3- (4- phenolic group) -4- methoxyl group -1H- pyrazolo [3,4-d] pyrimidine (2): pass through Suzuki
1 synthetic method of compound, yield: 85% are shown in coupling reaction, concrete operations.1H NMR(600MHz,DMSO):δ0.88(t,
), J=7.4,3H 1.25 (m, 2H), 1.84 (m, 3H), 4.10 (s, 3H), 4.40 (t, J=6.9,2H), 6.87 (d, J=8.7,
2H), 7.84 (d, J=8.6,2H), 8.57 (s, 1H), 9.71 (s, 1H)13C NMR(151MHz,DMSO):δ13.86,19.76,
31.51,46.73,54.75,99.35,115.66,123.39,130.13,143.71,155.34,155.75,158.45,
164.00.HRMS-ESI(m/z)calcd for[M+H]+,299.1430；found,299.1514.
1- isopropyl -3- (3- phenolic group) -4- methoxyl group -1H- pyrazolo [3,4-d] pyrimidine (3): pass through Suzuki
1 synthetic method of compound, yield: 77% are shown in coupling reaction, concrete operations.1H NMR(600MHz,DMSO):δ1.52(d,
), J=6.7,6H 4.11 (s, 3H), 5.16 (hept, J=6.8,1H), 6.82 (ddd, J=8.1,2.4,1.1,1H), 7.28
(t, J=7.7,1H), 7.46 (d, J=1.0,1H), 7.47 (s, 1H), 8.60 (s, 1H), 9.57 (s, 1H)13C NMR
(151MHz,DMSO):δ21.78,48.80,54.37,99.40,115.00,115.60,119.13,129.38,133.29,
142.80,154.49,154.73,157.31,163.44.HRMS-ESI(m/z)calcd for[M+H]+,285.1273；
found,285.1331.
1- isopropyl -3- (4- phenolic group) -4- methoxyl group -1H- pyrazolo [3,4-d] pyrimidine (4): pass through Suzuki
1 synthetic method of compound, yield: 83% are shown in coupling reaction, concrete operations.1H NMR(600MHz,DMSO):δ1.51(d,
), J=6.7,6H 4.10 (s, 3H), 5.13 (m, 1H), 6.87 (d, J=8.6,2H), 7.83 (d, J=8.6,2H), 8.57 (s,
1H),9.72(s,1H).13C NMR(151MHz,DMSO):δ21.77,48.64,54.28,99.15,115.17,123.08,
129.69,143.03,154.41,154.66,157.94,163.49.HRMS-ESI(m/z)calcd for[M+H]+,
285.1273；found,285.1327.
Bromo- 4- methylamino -1H- pyrazolo [3,4-d] pyrimidine (13) of 1- isopropyl -3-: 0.5g (1.83mmol) 1- isopropyl
Chloro- 1H- pyrazolo [3,4-d] the pyrimidine 11b of the bromo- 4- of base -3- is dissolved in 100mL ethanol solution, and lower addition 40% is stirred at room temperature
Methylamine water solution 0.8mL, is then heated to reflux, about 3.5h, TLC monitoring reaction terminate (solvent: PE/EtOAc=4:
1.5).It is cooled to room temperature to reaction solution, 30mL water and EtOAc (100mL × 3) extraction merge organic phase, appropriate anhydrous sulphur are added
Sour sodium dries, filters, and concentration, silica gel column chromatography separating purification (eluant, eluent: PE/EtOAc=100/5-100/10) obtains white
Bromo- 4- methylamino -1H- pyrazolo [3,4-d] the pyrimidine 0.34g of solid product 1- isopropyl -3-, yield: 69%.HRMS-ESI
(m/z)calcd for[M+H]+,270.0276；found,270.0354.
1- isopropyl -3- (3- phenolic group) -4- methylamino -1H- pyrazolo [3,4-d] pyrimidine (5): pass through Suzuki
1 synthetic method of compound, yield: 75% are shown in coupling reaction, concrete operations.1H NMR(600MHz,DMSO):δ1.45(d,
), J=6.8,6H 2.93 (d, J=4.7,3H), 4.96 (hept, J=6.6,1H), 5.53 (d, J=4.8,1H), 6.74 (dd, J
=8.1,1.6,1H), 6.86 (d, J=8.5,1H), 7.25 (t, J=7.8,1H), 7.39 (s, 1H), 8.22 (s, 1H), 9.55
(s,1H).13C NMR(151MHz,DMSO):δ22.38,27.79,45.29,100.11,113.66,115.15,118.98,
119.83,130.03,136.26,148.81,151.13,156.69,157.80.HRMS-ESI(m/z)calcd for[M+H]+,
284.1433；found,284.1511.
1- isopropyl -3- (4- phenolic group) -4- methylamino -1H- pyrazolo [3,4-d] pyrimidine (6): pass through Suzuki
1 synthetic method of compound, yield: 84% are shown in coupling reaction, concrete operations.1H NMR(600MHz,DMSO):δ1.46(d,
), J=6.7,6H 2.94 (d, J=4.7,3H), 5.03 (m, 1H), 6.25 (dd, J=9.1,4.5,1H), 6.91 (d, J=8.5,
2H), 7.45 (d, J=8.5,2H), 8.29 (s, 1H), 9.74 (s, 1H)13C NMR(151MHz,DMSO):δ21.80,27.93,
47.92,97.63,115.91,123.95,129.58,143.08,152.64,155.14,157.20,157.81.HRMS-ESI
(m/z)calcdfor[M+H]+,284.1433；found,284.1506.
Bromo- 4- amino -1H- pyrazolo [3,4-d] pyrimidine (15) of 1- isopropyl -3-: the method for being combined to 11a according to alkyl
15 are obtained, yield: 83%.HRMS-ESI(m/z)calcd for[M+H]+,256.0120；found,256.0197.
1- isopropyl -3- (3- phenolic group) -4- amino -1H- pyrazolo [3,4-d] pyrimidine (7): pass through Suzuki
1 synthetic method of compound, yield: 76% are shown in coupling reaction, concrete operations.1H NMR(600MHz,DMSO):δ1.48(d,
), J=6.7,6H 5.05 (m, 1H), 6.87 (dd, J=8.2,1.4,1H), 7.07 (s, 1H), 7.08 (d, J=2.9,1H),
7.34 (t, J=8.0,1H), 8.23 (s, 1H), 9.70 (s, 1H)13C NMR(151MHz,DMSO):δ21.78,48.04,
97.35,114.92,115.69,118.85,130.28,134.38,143.26,153.21,155.41,157.83,
158.01.HRMS-ESI(m/z)calcd for[M+H]+,270.1277；found,270.1357.
1- isopropyl -3- (4- phenolic group) -4- amino -1H- pyrazolo [3,4-d] pyrimidine (8): pass through Suzuki
1 synthetic method of compound, yield: 85% are shown in coupling reaction, concrete operations.1H NMR(600MHz,DMSO):δ1.47(d,
), J=6.7,6H 5.03 (hept, J=6.7,1H), 6.92 (d, J=8.5,2H), 7.47 (d, J=8.5,2H), 8.21 (s,
1H),9.76(s,1H).13C NMR(151MHz,DMSO):δ21.79,47.92,97.33,115.89,123.85,129.59,
143.46,153.13,155.27,157.88,158.08.HRMS-ESI(m/z)calcd for[M+H]+,270.1277；
found,270.1360.
1- tert-butyl -5- amino -1H- pyrazoles -4- nitrile (17): by tertiary fourth hydrazine hydrochloride (8g, 64.20mmol) and triethylamine
(6.50g, 64.20mmol) is dissolved in 500mL dehydrated alcohol, and it is sub- that 2- ethyoxyl is then slowly added dropwise into reaction flask under stiring
Methylmalononitrile (7.83g, 64.20mmol).It is heated to 78 DEG C to flow back, after about 3h, TLC monitoring reaction terminates (solvent: PE/
EtOAc=1:1).It is cooled to room temperature to reaction solution, is concentrated under reduced pressure and removes solvent, 100mL water and CH is added2Cl2(300mL×3)
Extraction merges organic phase, and appropriate anhydrous sodium sulfate drying is added, is concentrated to get orange-yellow sticky solid.The crude product that will be obtained
It is dissolved in 60mL ethyl acetate-hexane (1:9) mixed solution, ultrasound about 5min, static, observation has a large amount of crystalline precipitates to generate,
It filters (ethyl acetate-hexane mixed solution is washed 3 times), drying obtains orange solid product 1- tert-butyl -5- amino -1H- pyrrole
Azoles -4- nitrile 10.11g, yield: 96%.1H NMR(600MHz,DMSO):δ1.50(s,9H),6.22(s,2H),7.44(s,
1H).13C NMR(151MHz,DMSO):δ28.22,58.75,74.54,115.23,138.00,150.71.HRMS-ESI(m/z)
calcd for[M+Na]+,187.1062；found,187.0968.
1- tert-butyl -4- amino -1H- pyrazolo [3,4-d] pyrimidine (18): the tertiary fourth of 1- is added in 100mL round-bottomed flask
Base -5- amino -1H- pyrazoles -4- nitrile (5g, 30.47mmol), formamide solution 80mL, mixed liquor are placed in oil bath pan and are heated to 180
DEG C, after about 5h, TLC monitoring reaction terminates (solvent: PE/EtOAc=1:1).After reaction solution is cooling, be added 50mL water and
CH2Cl2(200mL × 3) extraction, merges organic phase, and anhydrous sodium sulfate dries, filters, and is concentrated, and silica gel column chromatography separating purification (is washed
De- agent: PE/EtOAc=4/1-1/1) obtain white solid product 1- tert-butyl -4- amino -1H- pyrazolo [3,4-d] pyrimidine
5.82g, yield: 91%.1H NMR (600MHz, DMSO): δ=1.69 (s, 9H), 8.03 (s, 1H), 8.14 (s, 1H)13C
NMR (151MHz, DMSO): δ 28.76,59.22,101.42,130.03,152.71,154.71,158.16.HRMS-ESI (m/
z)calcd for[M+H]+,192.1171；found,192.1254.
Bromo- 4- amino -1H- pyrazolo [3,4-d] pyrimidine (19) of 1- tert-butyl -3-: 1- tert-butyl -4- amino -1H- is weighed
18 3g (15.70mmol) of pyrazolo [3,4-d] pyrimidine is dissolved in 250mL anhydrous acetonitrile, and lower addition N- bromo succinyl is stirred at room temperature
Imines (NBS, 4.19g, 23.55mmol) is until be completely dissolved.It is heated to 100 DEG C of reflux about 2h, TLC monitoring reaction terminates (exhibition
Open agent: CH2Cl2/CH3OH=95/5), it is cooled to room temperature to reaction solution, methylene chloride/water extracts (× 3), merges organic phase, fits
Amount anhydrous sodium sulfate dries, filters, and is concentrated, silica gel column chromatography separating purification (eluant, eluent: CH2Cl2) obtain white solid product 1-
Bromo- 4- amino -1H- pyrazolo [3,4-d] the pyrimidine 4.22g of tert-butyl -3-, yield: 89%.1H NMR(600MHz,DMSO):δ
1.67(s,9H),8.20(s,1H).13C NMR(151MHz,DMSO):δ28.60,60.60,100.45,115.28,153.51,
155.63,157.50.HRMS-ESI(m/z)calcd for[M+H]+,270.0276；found,270.0355.
1- tert-butyl -3- (5- hydroxyl -3- pyridyl group) -4- amino -1H- pyrazolo [3,4-d] pyrimidine (9): pass through
Suzuki coupling reaction, concrete operations are detailed in 1 synthetic method of compound, the bromo- 4- amino -1H- pyrazoles of 1- tert-butyl -3-
And [3,4-d] pyrimidine (1.45g, 5.43mmol) participates in reaction and obtains gray solid product 1- tert-butyl -3- (5- hydroxyl -3- pyrrole
Piperidinyl) -4- amino -1H- pyrazolo [3,4-d] pyrimidine 0.58g, yield: 47%.Wherein solvent: CH2Cl2/CH3OH=10/
1, eluant, eluent: CH2Cl2/CH3OH=100:1-100:5.1H NMR (600MHz, DMSO): δ 1.75 (s, 9H), 7.38 (dd, J=
), 2.5,2.0,1H 8.20 (d, J=2.7,1H), 8.25 (s, 1H), 8.29 (d, J=1.8,1H), 10.16 (s, 1H)13C NMR
(151MHz,DMSO)δ29.16,60.32,99.29,121.91,130.11,138.39,139.21,140.03,154.11,
154.47,155.17,158.73.HRMS-ESI(m/z)calcd for[M+H]+,285.1386；found,285.1457.
By high resolution mass spectrum, nuclear magnetic resonance spectroscopy, carbon-13 nmr spectra analysis means, aforementioned identification data are obtained, are led to
Analysis data are crossed, confirm that the structure of above compound 1-9 obtained is as follows:
Compound 1-9 has been prepared in the results show, the present invention.
Below by way of experimental example prove the compounds of this invention the utility model has the advantages that
Inflammatory factor IL-6 is the effective therapy target of active chronic inflammation, is played the part of emphatically during inflammatory diseases
Want role.The present invention induces human lung carcinoma cell using IL-4 using uridine as positive control referring to the method for M ü ller et al.
Level-off under the IL-6 of A549 release IL-6 cellular inflammation model evaluation synthesis compound.And to the cell inhibitory effect of compound
The bioactivity such as activity, Apoptosis and period are evaluated.
One, experimental method
1 experimental material and instrument:
Some materials needed for 1.1 experiments and reagent are listed in table 2.
2. part Experiment material of table and reagent
1.2 Bioexperiment key instruments:
(1)CO2Incubator: Thermo311 type, Thermo, the U.S.
(2) fluorescence inverted microscope: TE2000-U type, Nikon, Japan
(3) flow cytometer: FACS Valibur, BD company, the U.S.
(4) automatically scanning formula multi-function microplate reader: Multiskan Sky, Thermo, the U.S.
(5) 4 DEG C/- 20 DEG C of refrigerators: HXC-158, company, Haier, China
(6) -80 DEG C of ultra low temperature freezers: Forma, Thermo, the U.S.
(7) agarose gel electrophoresis instrument: E-Gel PowerSnap, Invitrogen, the U.S.
(8) ultrasonic cell disruption instrument: JYD-650, the letter Instrument Ltd. in Shanghai, China
(9) constant-temperature table: SPH-211B-GZ, Shanghai generation is plain to test Co., Ltd, China
(10) full automatic gel imaging system: GelDoc, Bio-Rad company, the U.S.
(11) desk-top micro refrigerated centrifuge: 22R, Beckman, the U.S.
(12) high speed low temperature centrifugal machine: D37520, Heraeus, Germany
(13) cell viability analyzer: Vi-Cell, Beckman, the U.S.
(14) circulation constant temperature water bath: ZSXH-618/625, Shanghai ZHICHENG Anaiytical Instrument Manufacturing Co., Ltd.
1.3 supramolecular structures and property research characterize instrument:
(1) X-ray single crystal diffraction is analyzed: German Bruker APEX-II CCD diffractometerGraphite list
The Mo-K alpha ray of color.
(2) heat analysis (DSC/TGA): Dutch 1 synchronous solving of METTLER TOLEDO TGA/DSC
(3) alternating temperature polycrystal powder X-ray diffraction analysis (VT-PXRD): Dutch Panalytical-Empyrean type X- is penetrated
Line diffractometer
(4) scanning electron microscope (SEM): high-resolution INSPECTF50 type scanning electron microscope
(5) uv-visible absorption spectra is analyzed: UV-3600 spectrophotometer
2 cell culture
There to be class people's alveolar epithelium A549 cell culture in RPMI1640 culture medium, and supplement and 10% tire ox blood is added
(FBS) and 1% mycillin clearly.It is dispensed into culture bottle, tightens bottle cap, slightly turned round after moderately tightening, in favor of CO2Into
Enter, by cell incubation in 37 DEG C, 5%CO2Incubator.Wherein the purchase of A549 cell is in Chinese Academy of Sciences's cell bank (China).
3 cell growth inhibition assays (MTT)
Attached cell is digested with 0.25% trypsase, is matched with the RPMI1604 culture solution containing 10% fetal calf serum
At individual cells suspension, it is inoculated in 96 orifice plates with the density of every 2000 cells in hole, every pore volume 100ul.Culture plate is moved
Enter CO2In incubator, in 37 DEG C, 5%CO2Under the conditions of, it is incubated for 24 hours.The drug of various concentration is added in every hole, is arranged 3
Multiple holes then proceed to culture 72 hours.Thiazole bromide blue tetrazolium (MTT) 20 μ L is added by the every hole of operation manual, continues incubation 4
Hour, culture is terminated, careful inhale abandons culture supernatant in hole.490nm wavelength is selected, each hole is set up on enzyme-linked immunosorbent assay instrument
Receipts value records result.Using the time as horizontal axis, absorbance value is longitudinal axis drawing.And with Bliss method calculation of half inhibitory concentration
(IC50).Each experiment is at least repeated 3 times.
4 cell cycles detected (Flow Cytometry)
Cell cycle determination uses flow cytometer detection method, and PI first (propidium iodide) label dyeing is then thin by streaming
Born of the same parents' instrument detection PI is to measure the period profile of cell colony.A549 cell is added the pancreatin containing EDTA and is digested, wait digest
Afterwards, the DMEM containing 10%FBS is added and terminates digestion, gently blow and beat cell, cell suspension is collected into centrifuge tube.1000rpm/
Min is centrifuged 10 minutes, abandons supernatant.The ethyl alcohol of the 70-80% of 5ml pre-cooling is added dropwise, is vortexed to mix cell, 4 DEG C were protected from light
Night.1000-1500rpm/min is centrifuged 10 minutes, abandons supernatant.Then it is successively cleaned cell 2 times with PBS, the PBS solution containing PI
To remove all ethyl alcohol.Cell is resuspended in 0.5mL PI/RNase dyeing liquor, room temperature is protected from light incubation 30 minutes.Gently blow and beat
It mixes, filters into streaming pipe, 4 DEG C are kept in dark place sample, and flow cytometer detects within an hour.
5 Apoptosis detect (Flow Cytometry)
6 orifice plates culture A549 cell, the compound and DMSO for being separately added into various concentration are as blank control.24 hours
Afterwards, cell is collected using 0.25% trypsase without EDTA, PBS solution is rinsed 2 times.Then using Apoptosis detection examination
Agent box is dyed to specifications.It is analyzed later with flow cytomery.
6 enzyme-linked immunosorbent assays (ELISA)
Using the concentration of double-antibody sandwich (Sandwich) method test sample target protein.Respectively by sample or various concentration
Standard items are added in corresponding aperture according to 100 holes μ L/, seal reacting hole with sealing plate film (transparent), are incubated at room temperature 120 minutes.PBST
Board-washing 5 times, and last time is placed on thick blotting paper and pats dry.100 hole μ L/ of biotinylated antibody is added, is sealed instead with sealing plate film
Ying Kong is incubated at room temperature 60 minutes.It board-washing 5 times, is equally patted dry on the thick blotting paper of last time.Horseradish peroxidase-labeled is added
100 hole μ L/ Streptavidin (HRP-Streptavidin), sealing plate film (white) seal reacting hole, and room temperature is protected from light incubation 20
Minute.It board-washing 5 times, pats dry for the last time.100 hole μ L/ of color developing agent TMB solution is added, sealing plate film (white) seals reacting hole, room
Temperature is protected from light incubation 20 minutes, until the color change of highly significant occur in standard items and sample.50 hole μ L/ of terminate liquid is added, mixes
Measure absorbance value at 450nm immediately afterwards.IL-6 is tested to specifications using specific ELISA kit.
7 Western blot analysis (Western Blotting)
1) preparation of SDS--PAGE gel
The preparation of SDS--PAGE gel is for preparing 10%Tris- glycine SDS-PAGE: according to dual distilled water
3.18ml, 30% polyacrylamide solution 2.66ml, 1.5M Tris-HCl solution (pH 8.8) 2ml, 10% ammonium persulfate
0.08ml, 10% lauryl sodium sulfate (SDS) solution 0.08ml, tetramethyl diethylamine (TEMED) solution (are added) before use
It is prepared by the method for 0.0032ml.
2) SDS--PAGE electrophoresis
The gel slab prepared is fixed on electrophoretic apparatus, and baffle below is opened.The sweet ammonia of Tris-- is added
Sour running buffer working solution excludes the bubble of gel bottom, is loaded, then connects to power supply in a predetermined sequence.Power supply is set
Set: 100V, 400A close power supply and take out glass plate stopping electrophoresis after about 2 hours when bromophenol blue reaches separation gel bottom.
3) transferring film
It is sequentially placed into the transferring film clip after porous gasket soaks, accurate filter paper two is opened, separation gel, pvdf membrane, accurate filter paper
Two；It needs to be aligned between film, filter paper and gel and cannot have bubble.Clip is put into transferring film electrophoresis tank, is added in electrophoresis tank
Enter 4 DEG C of CAPS buffer, open condensation tap water and blender, connection power supply carries out transferring film.Power supply setting: 50V, 400mA.
Transferring film terminates after about 2.5 hours.
4) immunostaining
Pvdf membrane after transferring film is slightly soaked in pure methanol solution, places into TBS buffer and rinses.Closing buffering is added
Liquid (TBST) is placed in shaking table room temperature and closes 2 hours.TBST solution is washed film (4 × 10min), and 1:300 or 1:500 diluted one is added
Anti- (being dissolved in Block buffer), is then placed on shaking table and is incubated at room temperature 2 hours.TBST solution is washed film (3 × 5min), is added 1:
1000 diluted secondary antibodies (being dissolved in Block buffer) are placed in after the shaking table gently shaken is incubated at room temperature 1.5 hours with TBST solution
It washes film (3 × 5min), ECL chemical luminescence reagent kit dyes progress X-ray film exposure in 1 minute or sweeps figure.
8 statistical analysis
All experimental results carry out statistical analysis using 19.0 software of SPSS, withIt indicates, it is poor between t inspection comparative group
It is different.
Two, experimental result
1, influence of the mtt assay measurement the compounds of this invention to A549 cell inhibitory effect
Using mtt assay, test respectively the compound effects of various concentration in human lung cancer cell A549's cell for 24 hours after cell
Inhibiting rate, compound concentration are respectively 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM.Compound IC50Value is listed in the table below (table
3).As can be seen that compound 7, compound 8, compound 9 show certain cell inhibitory effect activity, drug effect for 24 hours after
Measure IC50Value is respectively 41.01 μM, 44.81 μM, 69.62 μM.
3. compound effects of table are in the IC of A549 cell50Value
2, Apoptosis and cell cycle determination (Flow Cytometry)
Cell cycle determination has been carried out to compound 7 and compound 9 and has had detected Apoptosis situation (Fig. 1-4, table 4-
5).Experiment selects human lung cancer cell A549's cell as detection system, and control group is blank control (DMSO), and compound test is dense
Degree be respectively 5 μM, 10 μM, 15 μM, compound effects in human lung cancer cell A549's cell for 24 hours, after 48h, 72h use fluidic cell
Art is detected.In Apoptosis result figure, the cell in quadrant Q1 is non-viable non-apoptotic cell, and quadrant Q2 is non-viable apoptotic cell,
Cell in the region quadrant Q3 is viable apoptotic cell, and the cell in quadrant Q4 represents living cells.
From test result as can be seen that influence of the compound 9 to cell is smaller, when compound concentration is 15 μM, 72h causes
The total apoptosis of cell also only has 15.66%.
In addition, compared with the control group, compound 7, which has, more significantly to lure when compound effects are after A549 cell 72h
It leads the effect of A549 Apoptosis and shows dose dependent (Fig. 1).Prompt compound 7 to the antiproliferative activity of A549 cell
May be related with the ability that it is induced cell apoptosis, show that compound 7 there may be certain anticancer activity.
Influence of 4. compound 7 of table to A549 Apoptosis
Influence of 5. compound 9 of table to A549 Apoptosis
3, influence of the ELISA method measurement compound to IL-4 induction A549 cell secretion IL-6 level
The horizontal measurement result of inflammatory factor IL-6 is shown in Fig. 5 under compound effects, and compound concentration is respectively 1 μM, 2.5 μM, 5
μM, 10 μM.
Experimental result shows that after the compound 7,9 of various concentration acts on cellular inflammation model for 24 hours, IL-6 level has
It reduces to a certain degree, wherein the inhibitory effect of compound 9 is most strong.Compound 7 and 9 all shows good dose dependent, with
The increase of concentration its anti-inflammatory activity gradually increase.After concentration reaches 5 μM, the efficiency that compound lowers IL-6 is better than positive right
According to medicine uridine (uridine), start to show preferable antiphlogistic effects.
Apoptosis and the cycle experimental result of binding compounds are it is found that suppression of the compounds of this invention to IL-6 emission levels
System is not influenced by Apoptosis.
4, inflammation associated signal paths probe into (Western Blotting)
The phosphorylation NF- κ B p65 protein level after compound effects, pSTAT3 (Ser727) albumen are tested respectively
Horizontal and phosphorylation Akt (Ser473) protein level detects crucial adjusting in these accesses using Western blotting method
The variation of the factor.Wherein β-ACTIN is used as internal reference.The present invention has also surveyed the albumen table of adhesivemoleculeICAM1 after compound effects
It reaches.
The results show that after the effect of compound 9 compared with the control group, medicine group phosphorylation NF- κ B p65 protein level, phosphoric acid
Changing STAT3 (Ser727) protein level and phosphorylation Akt (Ser473) protein level reduces (Fig. 6).AdhesivemoleculeICAM1
Protein level is also in that dose dependent reduces (Fig. 7).Therefore, the significant anti-inflammatory activity that the compounds of this invention 9 is shown, as
It is a kind of it is potential treatment diseases associated with inflammation drug have extraordinary application prospect.
To sum up, a kind of pyrazolo [3,4-d] miazines small molecule derivative has been prepared in the present invention the experiment has found that this
The compound 7 and compound 9 of invention, especially compound 9, can effectively inhibit the emission levels of inflammatory factor IL-6, chemical combination
Object 9 can inhibit phosphorylation NF- κ B p65 protein level, phosphorylation Akt protein level, pSTAT3 protein level and adherency
The protein level of molecule ICAM-1.Therefore, the compounds of this invention has very good on the drug of preparation treatment diseases associated with inflammation
Application prospect.
Claims (10)
1. Formulas I compound represented or its salt:
Wherein, R1ForWherein, W is selected from nothing, NH, CH2,O,S；R5Selected from H, C1-6 alkyl, C1-6 alkoxy, halogen,
Hydroxyl, carboxyl, amino, aryl, heteroaryl, saturated cyclic alkyls, saturated heterocyclyl；
R2Selected from halogen, amino, hydroxyl, carboxyl, by 0~5 R4Replace following group: aryl, heteroaryl, saturated cyclic alkyls,
Saturated heterocyclyl, C1-6 alkyl, C1-6 alkoxy；R4Selected from hydroxyl, carboxyl, halogen, H, C1-6 alkyl, C1-6 alkoxy；
R3Selected from by 0~3 R6The following group replaced: C1-8 alkyl, C1-8 alkoxy, C2-8 alkenyl, C2-8 alkynyl；R6Choosing
From hydroxyl, carboxyl, halogen.
2. compound according to claim 1 or its salt, it is characterised in that:
R1For-WR5, wherein W is selected from NH or O, R5Selected from H, C1-4 alkyl, C1-4 alkoxy, halogen, hydroxyl, carboxyl；
R2ForWherein, M is CH or N, R4Selected from hydroxyl, carboxyl, halogen, H, C1-3 alkyl, C1-3 alkoxy；
R3Selected from C1-4 alkyl.
3. compound according to claim 2 or 3 or its salt, it is characterised in that:
R1It is selected from
R2It is selected from
R3It is selected from
4. compound according to claim 1 or its salt, it is characterised in that: the compound is selected from one of flowering structure:
5. it is a kind of prepare benefit require 4 described in compound method, it is characterised in that:
It the described method comprises the following steps:
(1) compound 10 is reacted with Iso-Propyl iodide or iodo-n-butane, obtains compound 11；
(2) compound 11 is reacted with sodium methoxide, obtains compound 12；
(3) compound 12 reacted with 3- hydroxy benzenes pinacol borate or 4- hydroxy benzenes pinacol borate to get compound 1,
2,3 or 4；
Or, the described method comprises the following steps:
(2 ') compound 11 is reacted with methylamine, obtains compound 13；
(3 ') compound 13 is reacted with 3- hydroxy benzenes pinacol borate or 4- hydroxy benzenes pinacol borate to get compound 5
Or 6；
Or, the described method comprises the following steps:
(a) compound 14 is reacted with Iso-Propyl iodide, obtains compound 15；
(b) compound 15 react with 3- hydroxy benzenes pinacol borate or 4- hydroxy benzenes pinacol borate to get compound 7 or
8；
Or, the described method comprises the following steps:
(a ') compound 16 is reacted with tertiary fourth hydrazine hydrochloride, obtains compound 17；
(b ') compound 17 and formamide, obtain compound 18；
(c ') compound 18 is reacted with N-bromosuccinimide, obtains compound 19；
(d ') compound 19 and 5- (4,4,5,5- tetramethyls -1,3,2- bis- dislike borine -2- base) pyrimidine -3- alcohol reaction are to get change
Close object 9；
The structure of above compound 10 isThe structure of compound 11 isThe structure of compound 12
ForIn compound 11 and compound 12, R3For
The structure of compound 13 isWherein R3For
The structure of compound 14 isThe structure of compound 15 is
The structure of compound 16 isThe structure of compound 17 isThe structure of compound 18 isThe structure of compound 19 is
6. according to the method described in claim 5, it is characterized by:
In step (1), the reaction is carried out under the action of potassium carbonate, and reaction dissolvent is organic solvent, and reaction temperature is
60~100 DEG C, the reaction time is 2.0~5.0 hours；
In step (2), the reaction dissolvent is organic solvent, and reaction temperature is 60~100 DEG C, and the reaction time is 2.0~5.0 small
When；
In step (2 '), the reaction dissolvent is organic solvent, and reaction temperature is 60~100 DEG C, and the reaction time is 2.0~5.0
Hour；
In step (3) or (3 '), the reaction is in PdCl2It is carried out under the action of dppf and potassium carbonate, reaction dissolvent is Isosorbide-5-Nitrae-
The mixed solution of dioxane and water, reaction temperature are 60~100 DEG C, and the reaction time is 2.0~5.0 hours；
In step (a), the reaction is carried out under the action of potassium carbonate, and reaction dissolvent is organic solvent, and reaction temperature is
60~100 DEG C, the reaction time is 2.0~5.0 hours；
In step (b), the reaction is in PdCl2It is carried out under the action of dppf and potassium carbonate, reaction dissolvent is Isosorbide-5-Nitrae-dioxy six
The mixed solution of ring and water, reaction temperature are 60~100 DEG C, and the reaction time is 2.0~5.0 hours；
In step (a '), the reaction is carried out under the action of triethylamine, and reaction dissolvent is organic solvent, and reaction temperature is
60~100 DEG C, the reaction time is 2.0~5.0 hours；
In step (b '), the reaction temperature is 100~200 DEG C, and the reaction time is 2.0~5.0 hours；
In step (c '), the reaction dissolvent is organic solvent, and reaction temperature is 60~100 DEG C, and the reaction time is 2.0~5.0
Hour；
In step (d '), the reaction is in PdCl2It is carried out under the action of dppf and potassium carbonate, reaction dissolvent is Isosorbide-5-Nitrae-dioxy
The mixed solution of six rings and water, reaction temperature are 60~100 DEG C, and the reaction time is 2.0~5.0 hours.
7. according to the method described in claim 6, it is characterized by:
In step (1), the reaction dissolvent is MeCN, and reaction temperature is 100 DEG C, and the reaction time is 3 hours；
In step (2), the reaction dissolvent is methanol, and reaction temperature is 65 DEG C, and the reaction time is 3.5 hours；
In step (2 '), the reaction dissolvent is ethyl alcohol, and reaction temperature is 75 DEG C, and the reaction time is 3.5 hours；
In step (3) or (3 '), it is Isosorbide-5-Nitrae-dioxane of 4:1 and the mixed solution of water that the reaction dissolvent, which is volume ratio, instead
Answering temperature is 100 DEG C, and the reaction time is 2 hours；
In step (a), the reaction dissolvent is MeCN, and reaction temperature is 80 DEG C, and the reaction time is 3 hours；
In step (b), the reaction dissolvent is that volume ratio is Isosorbide-5-Nitrae-dioxane of 4:1 and the mixed solution of water, reaction temperature
It is 100 DEG C, the reaction time is 2 hours；
In step (a '), the reaction dissolvent is ethyl alcohol, and reaction temperature is 78 DEG C, and the reaction time is 3 hours；
In step (b '), the reaction temperature is 180 DEG C, and the reaction time is 5 hours；
In step (c '), the reaction dissolvent is methanol, and reaction temperature is 100 DEG C, and the reaction time is 2 hours；
In step (d '), the reaction dissolvent is that volume ratio is Isosorbide-5-Nitrae-dioxane of 4:1 and the mixed solution of water, reaction temperature
It is 100 DEG C, the reaction time is 2 hours.
8. benefit requires the described in any item compounds of 1-4 or its salt preparing the purposes on interleukin-6 inhibitor.
9. benefit requires the described in any item compounds of 1-4 or its salt preparing the purposes on anti-inflammatory drug, it is preferable that described anti-
Scorching drug can reduce phosphorylation NF- κ B p65 protein level, phosphorylation Akt protein level, pSTAT3 protein level and
The protein level of adhesivemoleculeICAM1.
10. a kind of pharmaceutical composition, it is characterised in that: described pharmaceutical composition is with claim 1-4 described in any itemization
Closing object or its salt is active constituent, in addition pharmaceutically acceptable auxiliary material is made.
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Cited By (1)
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|---|---|---|---|---|
| CN110548032A (en) * | 2019-08-07 | 2019-12-10 | 四川大学华西医院 | Application of pyrazolopyrimidine compound in preparation of drugs for preventing/treating tumors |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005007085A2 (en) * | 2003-06-20 | 2005-01-27 | The Regents Of The University Of California | Pyrazolo pyrimidine derivatives and methods of use thereof |
| WO2017161344A1 (en) * | 2016-03-17 | 2017-09-21 | The Regants Of The University Of California | Compositions and methods for treating parasitic diseases |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005007085A2 (en) * | 2003-06-20 | 2005-01-27 | The Regents Of The University Of California | Pyrazolo pyrimidine derivatives and methods of use thereof |
| WO2017161344A1 (en) * | 2016-03-17 | 2017-09-21 | The Regants Of The University Of California | Compositions and methods for treating parasitic diseases |
Non-Patent Citations (2)
| Title |
|---|
| BETH APSEL，等: "Targeted polypharmacology: discovery of dual inhibitors of tyrosine and phosphoinositide kinases", 《NATURE CHEMICAL BIOLOGY》 * |
| 王璐阳: "PI3K/Akt/AP-1通路对高糖培养血管平滑肌细胞增殖及CRP、IL-6、TNF-α表达的影响", 《河北医科大学硕士学位论文》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110548032A (en) * | 2019-08-07 | 2019-12-10 | 四川大学华西医院 | Application of pyrazolopyrimidine compound in preparation of drugs for preventing/treating tumors |
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