Nitrogen substituent group phenyl pyrazoles xanthine oxidoreductase inhibitors and preparation and application
Technical field
The invention belongs to pharmaceutical chemistry technical fields, and in particular to a kind of oxidation of nitrogen substituent group phenyl pyrazoles xanthine is also
Reductase inhibitor and preparation and application.
Background technique
Uric acid is the final product that purine is metabolized in vivo, by hypoxanthine, xanthine xanthine oxidase (XO) work
With lower generation.Due to lacking urate oxidase in the purine metabolism approach of the mankind and primate, uric acid cannot be degraded into
It is highly soluble in the allantoin of water, the mankind can only be made easily to form hyperuricemia through kidney excretion overwhelming majority uric acid.
Hyperuricemia will lead to gout and renal insufficiency, and be further considered it is a kind of cause coronary heart disease because
Element.In addition, hyperuricemia, which is recognized as, has close relationship with the formation of adult human disease (such as vascular hypertension).Therefore,
Gout not only can be treated effectively for the treatment of hyperuricemia but also can effectively be prevented various related with diet
Disease.
Uric acid generation and uricosuric is inhibited to drain both approach currently, treatment hyperuricemia can be used.Uricosuric row
The promotor let out mainly includes Benzbromarone and the kind Lesinurad just listed.Although the Benzbromarone of low dosage reduces
The effect of blood uric acid is better than Allopurinol, but the urate crystals that such excretion of drug generates are easy to be deposited in urinary tract, thus
Lead to different degrees of injury of kidney.European clinical research also has been reported that Benzbromarone has the danger for causing hepatic injury, has removed
Some European market out.Lesinurad is listed in U.S.'s in October, 2015, and the clinical research of III phase shows: being applied alone or is combined yellow fast
Purine oxidoreductase inhibitors can effectively reduce serum Uric Acid Concentration, and can be used for treating allopurinol and do not tolerate patient or curative effect
Bad patient can even make response rate be increased to 90% when drug combination.
Xanthine oxidoreductase enzyme and the generation of hyperuricemia, gout are closely related, by inhibiting xanthine oxidation also
The bioactivity of protoenzyme can block the access that xanthine oxidase turns to hypoxanthine and hypoxanthine is oxidized to uric acid, effectively drop
Low serum uric acid level reaches the occurrence and development for preventing and treating hyperuricemia and gout.Therefore xanthine oxidoreductase
Enzyme inhibitor is the important target spot of present hyperuricemia drug development.The medicine for acting on xanthine oxidoreductase enzyme is listed
Object includes Allopurinol, Febuxostat, Topiroxostat.Allopurinol passes through renal metabolism in vivo, can generate different degrees of bad
Reaction, such as the fash and super quick syndrome of hepatotoxicity wind agitation, acute renal failure, aplastic anemia, different severity;Non- cloth
Sotan is as novel anti-gout drugs, by liver metabolism, can preferably avoid Allopurinol because caused by renal metabolism not
Good reaction, but it still has the common adverse reaction such as dysfunction of liver, fash, diarrhea, headache and arthralgia;Topiroxostat
In June, 2013 by Japan's approval listing, treated for gout or the hyperuricemia of non-patient with gout.It is in conceptual phase
Xanthine oxidoreductase inhibitors include selenazoles class, imidazoles and 2- (indoles -5- base) thiazoles etc..Such as 2- (3- cyano-
2- allyloxy-phenyl) -4- methyl selenazoles -5- carboxylic acid in vitro effects it is fine, IC50Value is 5.5nM, is mixed-type inhibitor;
1- hydroxy-4-methyl -2- phenyl -1- imidazole-5-carboxylic acid derivant IC50The range of value is from 0.003 μM to 1.2 μM, to xanthine
Oxidoreducing enzyme shows good inhibitory activity;2- (indoles -5- base) thiazole compound 2- (3- cyano -2- isobutyl group Yin
Diindyl -5- base) -4- methyl thiazole-5-carboxyl acid inhibitory effect it is best, IC50Value has reached 3.5nM, can be effectively reduced blood
In uric acid concentration.
Investigation display in the recent period, the gout disease incidence in the U.S. are 3.9%;In the generation crowd of hyperuricemia, young man
Just account for 21.4% (4,300,000).The incidence of hyperuricemia and gout is higher and higher, and the trend of rejuvenation is presented, special
It is not the south and coastal cities in China, the disease incidence in a middle-aged person is up to 8%.Therefore, efficient xanthine oxidation is developed also
Reductase inhibitor is the important directions for reducing blood uric acid levels, treating gout.
Summary of the invention
In order to solve the disadvantage that the above prior art and shortcoming, taken the primary purpose of the present invention is that providing a kind of nitrogen
For base phenyl pyrazoles xanthine oxidoreductase inhibitors.
Another object of the present invention is to provide above-mentioned nitrogen substituent group phenyl pyrazoles xanthine oxidoreductase inhibitors
Preparation method.
A further object of the present invention is to provide above-mentioned nitrogen substituent group phenyl pyrazoles xanthine oxidoreductase inhibitors
Application in the preparation drugs such as inhibiting hyperuricemia or gout.
The object of the invention is achieved through the following technical solutions:
A kind of nitrogen substituent group phenyl pyrazoles xanthine oxidoreductase inhibitors, shown in structure such as formula (I):
Wherein, R1, R2It is independently represented each other hydrogen or the alkyl of C1-C6;Or R1、R2The nitrogen-atoms being connected with them
It is formed together the monocycle nitrogen heterocycle that carbon atom number is 3 to 6;
R3For nitro or cyano;
R4For hydrogen, methyl or trifluoromethyl.
Unless otherwise indicated, term of the invention has following meaning:
" hydrogen " refers to protium (1H), it is the major stable isotope of protium.
" cyano " expression-CN group.
" alkyl " indicates saturated fat alkyl, including straight chain and branched group.
In preferred formula (I), R1, R2It is independently represented each other hydrogen or the alkyl of C1-C4;Or R1、R2It is connected with them
The nitrogen-atoms connect is formed together the monocycle nitrogen heterocycle that carbon atom number is 3 to 6, wherein the heterocycle has 1-2
Hetero atom selected from the group below: O or N;
R3For nitro or cyano;
R4It is hydrogen, methyl or trifluoromethyl.
In preferred formula (I), R1, R2It is independently represented each other hydrogen, methyl, isopropyl or isobutyl group;Or R1、R2With
The nitrogen-atoms that they are connected is formed together piperidyl, pyrrole radicals or 2- methyl-pyrrole;
R3For nitro or cyano;
R4For hydrogen, methyl or trifluoromethyl.
In preferred formula (I), R1, R2It is independently represented each other hydrogen, methyl, isopropyl or isobutyl group;Or R1、R2With
The nitrogen-atoms that they are connected is formed together piperidyl, pyrrole radicals, 2- methyl-pyrrole or Lin Ji;
R3For nitro or cyano;
R4For hydrogen, methyl or trifluoromethyl.
Preferred formula (I) compound includes following described in any item compounds:
1- (3 '-cyano -4 '-piperidines-phenyl) -3- methyl pyrazole -4- formic acid,
1- (3 '-cyano -4 '-piperidines-phenyl)-pyrazoles -4- formic acid,
1- (3 '-nitros -4 '-piperidines-phenyl) -3- methyl pyrazole -4- formic acid,
1- (3 '-cyano -4 '-morpholine-phenyl) -3- methyl pyrazole -4- formic acid,
1- (3 '-nitros -4 '-morpholine-phenyl) -3- methyl pyrazole -4- formic acid,
1- (3 '-cyano -4 '-dimethylamino-phenyl)-pyrazoles -4- formic acid,
1- (3 '-cyano -4 '-nafoxidine-phenyl)-pyrazoles -4- formic acid,
1- (3 '-cyano -4 '-(N- methyl) isobutylamine-phenyl)-pyrazoles -4- formic acid,
- 4 formic acid of 1- (3 '-cyano -4 '-isobutylamine-phenyl)-pyrazoles,
1- (3 '-cyano -4 '-(2- methyl nafoxidine) phenyl)-pyrazoles -4- formic acid.
The preparation side of the above-mentioned nitrogen substituent group phenyl pyrazoles xanthine oxidoreductase inhibitors with formula (I) structural formula
Method includes the following steps:
(1) by the fluoro- 1- cyano of the bromo- 2- of 5- or nitrobenzene (II), nitrogenous (ring) alkane (HNR1R2) and inorganic base in organic phase
Middle heating reaction, obtains the bromo- 2- alkyl amine group -1- cyano of 5- or nitrobenzene (III);
(2) under nitrogen protection, by the bromo- 2- alkyl amine group -1- cyano of 5- or nitrobenzene, 1H- pyrazoles -4- formic acid esters or 3- first
Base -1H- pyrazoles -4- formic acid esters, CuI, K2CO3, (E)-N, N '-dimethyl -1,2- cyclohexyl diamine is coupled in DMF through C-N
Reaction, obtains 1- (3- cyano or nitro -4- alkyl amine group-phenyl)-pyrazoles -4- formic acid esters or 1- (3- cyano or nitro -4- alkane
Base Amino-phenyl) -3- methyl pyrazole -4- formic acid esters (IV);
(3) step (2) products therefrom (IV) obtains 1- (3- cyano or nitro -4- alkylamine after basic hydrolysis, acidification
Base-phenyl)-pyrazoles -4- formic acid or 1- (3- cyano or nitro -4- alkyl amine group-phenyl) -3- methyl pyrazole -4- formic acid (I).
The synthetic route chart of above-mentioned preparation step is as shown in Figure 1.Figure conditional is expressed as follows: a:K2CO3, DMF (DMSO);
B:CuI, K2CO3, (E)-N, N '-dimethyl -1,2- cyclohexyl diamine, DMF;C:1M NaOH, EtOH/THF, 1M HCl.
The compounds of this invention can be prepared with above-mentioned or similar above-mentioned preparation method, according to the difference of substituent group or
The difference of the position of substitution selects corresponding raw material.
Above-mentioned nitrogen substituent group phenyl pyrazoles xanthine oxidoreductase inhibitors are in preparation inhibiting hyperuricemia or gout
Application in drug.
The drug includes as formula (I) compound of effective component or its pharmaceutically acceptable salt, ester and pharmaceutically
Acceptable carrier.
" pharmaceutically acceptable salt " include formula (I) compound and organic acid, inorganic acid formed salt, or with gold
Belong to ion, the salt that organic base is formed, those of biological effectiveness and the property of parent compound salt can be retained.These salt packets
It includes:
(1) it is obtained by the free alkali of parent compound with inorganic acid or reacting for organic acid, inorganic acid is such as with acid at salt
(but being not limited to) hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid, sulfurous acid and perchloric acid etc., organic acid (but being not limited to) acetic acid, third
Acid, oxalic acid, malic acid, fumaric acid, hydroxybenzoic acid, methoxy benzoic acid, methanesulfonic acid, naphthalene -1- sulfonic acid, p-methyl benzenesulfonic acid, water
Poplar acid, citric acid, lactic acid, mandelic acid, succinic acid or malonic acid etc..
(2) acid proton being present in parent compound is replaced or given birth to organic base ligand chemical combination by metal ion
At salt, metal ion such as alkali metal ion, alkaline-earth metal ions or aluminium ion, organic base such as ethanol amine, diethanol amine, three second
Hydramine, tromethamine, piperidines, piperazine etc..
The invention has the following advantages and beneficial effects:
Gained formula (I) compound of the invention has the chemical structure different from known xanthine oxidase inhibitor;Such as exist
Demonstrated in following experimental example, they show excellent inhibiting effect to xanthine oxidase related with gout, and
In acute hyperuricemia mouse model, excellent inhibitory effect is shown;Therefore, they can be used for prevent and treat with
The relevant disease of xanthine oxidase, for example, hyperuricemia, heart failure, cardiovascular disease, hypertension, kidney diaseases, inflammation
Disease, arthropathy etc..
Detailed description of the invention
Fig. 1 is the synthetic route chart of nitrogen substituent group phenyl pyrazoles xanthine oxidoreductase inhibitors of the present invention;
Fig. 2 is nitrogen substituent group phenyl pyrazoles xanthine oxidoreductase inhibitors obtained by the embodiment of the present invention to acute height
The anti-trioxypurine of uricacidemia mouse model acts on test result figure.
Specific embodiment
Below with reference to embodiment, the present invention is described in further detail, and embodiments of the present invention are not limited thereto.
Embodiment 1
1- (3 '-cyano -4 '-piperidines-phenyl) -3- methyl pyrazole -4- formic acid (I1) synthesis
(1) the fluoro- 1- cyano benzene (II of the bromo- 2- of 5-1, 2.64g, 13mmol) and it is dissolved in DMSO (20mL), K is added2CO3
(5.4g, 39mmol), piperidines (3.3g, 39mmol) react 6h at 100 DEG C, and TLC tracks fully reacting.Reaction solution is cooled to room
The dilution of 100mL water is added in temperature, and ethyl acetate (100mL × 3) extraction, saturated common salt water washing, anhydrous magnesium sulfate is dry, decompression
Solvent is evaporated off, obtains the bromo- 2- piperidines-benzonitrile (III of yellow liquid 5-1) 3.3g, yield 95.7%.
(2)N2Under protection, 3- methyl-1 H- pyrazoles -4- methyl formate (0.14g, 1.0mmol), CuI (19.1mg,
0.1mmol)、K2CO3(0.29g,2.1mmol)、III1(0.318g, 1.2mmol), (E)-N, N '-dimethyl -1,2- cyclohexyl
Diamines (28.5mg, 0.2mmol) and DMF (3mL) are added in bis- neck bottle of 25mL, are reacted for 24 hours at 110 DEG C, are cooled to room temperature, are added
The dilution of 10mL water, is extracted with ethyl acetate (15mL × 3), and saturated salt solution (10mL) washing, anhydrous magnesium sulfate is dry, and decompression is steamed
Except solvent, silica gel column purification (VEthyl acetate: VPetroleum ether=6:1), obtain yellow solid 1- (3 '-cyano -4 '-piperidines-phenyl) -3- methyl -
Pyrazoles -4- methyl formate (IV1) 0.15g, yield 46.2%.
(3)IV1(0.15g 0.46mmol) dissolves in THF (24mL) and the mixed solution of ethyl alcohol (24mL), and 1M is added
NaOH aqueous solution 1mL, flow back 1h, is cooled to room temperature, and it is 2-3 that 1M HCL aqueous solution, which is added, and adjusts pH, is diluted with water, and solid is precipitated,
It filtering, filter cake is washed with water to neutrality, and it is dry, obtain white solid 1- (3 '-cyano -4 '-piperidines-phenyl) -3- methyl pyrazole -4-
Formic acid (I1) 0.12g, yield 81.3%.Product appraising datum is as follows:
1H NMR(400MHz,DMSO-d6) δ: 12.50 (s, 1H), 8.89 (s, 1H), 8.17 (d, J=2.7Hz, 1H),
8.04 (dd, J=9.0,2.7Hz, 1H), 7.22 (d, J=9.1Hz, 1H), 3.17-3.05 (m, 4H), 2.40 (s, 3H), 1.66
(m,4H),1.55(m,2H);HRMS(ESI,m/z):[M+H]+,Calcd.311.1503,Found 311.1511。
Embodiment 2
1- (3 '-cyano -4 '-piperidines-phenyl)-pyrazoles -4- formic acid (I2) synthesis
(1) with embodiment 1.
(2)N2Under protection, 1H- pyrazoles -4- Ethyl formate (0.22g, 1.6mmol), CuI (30mg, 0.16mmol), K2CO3
(0.45g,3.3mmol)、III1(0.5g, 1.9mmol), (E)-N, N '-dimethyl -1,2- cyclohexyl diamine (45mg,
It 0.3mmol) is added in bis- neck bottle of 25mL with DMF (3mL), 110 DEG C of reactions for 24 hours, are cooled to room temperature, and the dilution of 10mL water is added, uses
Ethyl acetate (15mL × 3) extraction, saturated salt solution (10mL) washing, anhydrous magnesium sulfate is dry, evaporating solvent under reduced pressure, silicagel column
Purify (VEthyl acetate: VPetroleum ether=4:1), obtain white solid 1- (3 '-cyano -4 '-piperidines-phenyl)-pyrazoles -4- Ethyl formate (IV2)
0.28g, yield 55.0%.
(3)IV2(0.28g, 0.86mmol) dissolves in THF (24mL) and the mixed solution of ethyl alcohol (24mL), and 1M is added
NaOH aqueous solution 4mL, flow back 1h, is cooled to room temperature, and it is 2-3 that 1M HCL aqueous solution, which is added, and adjusts pH, is diluted with water, and solid is precipitated,
It filtering, filter cake is washed with water to neutrality, and it is dry, obtain white solid 1- (3 '-cyano -4 '-piperidines-phenyl)-pyrazoles -4- formic acid (I2)
0.18g, yield 71.3%.Product appraising datum is as follows:
1H NMR(400MHz,DMSO-d6) δ: 12.66 (s, 1H), 9.00 (s, 1H), 8.22 (d, J=2.7Hz, 1H),
8.08 (dd, J=9.1,2.8Hz, 1H), 8.06 (s, 1H), 7.25 (d, J=9.1Hz, 1H), 3.14 (m, 4H), 1.67 (m,
4H),1.60-1.51(m,2H);HRMS(ESI,m/z):[M+H]+,Calcd.297.1346,Found 297.1348。
Embodiment 3
1- (3 '-nitros -4 '-piperidines-phenyl) -3- methyl pyrazole -4- formic acid (I3) synthesis
(1) the fluoro- 1- nitrobenzene (II of the bromo- 2- of 5-2, 3.3g, 15mmol) and it is dissolved in DMSO (20mL), K is added2CO3(6.2g,
45mmol), piperidines (3.8g, 45mmol), reacts 4h at 100 DEG C, and TLC tracks fully reacting.Reaction solution is cooled to room temperature, is added
The water of 100mL dilutes, and ethyl acetate (100mL × 3) extraction, saturated common salt water washing, anhydrous magnesium sulfate is dry, removes under reduced pressure molten
Agent obtains the bromo- 2- piperidines -1- nitrobenzene (III of orange liquid 5-3) 4.2g, yield 98.2%.
(2)N2Under protection, 3- methyl-1 H- pyrazoles -4- methyl formate (0.14g, 1.0mmol), CuI (19.1mg,
0.1mmol)、K2CO3(0.29g,2.1mmol)、III3(0.34g, 1.2mmol), (E)-N, N '-dimethyl -1,2- cyclohexyl two
Amine (28.5mg, 0.2mmol) and DMF (3mL) are added in bis- neck bottle of 25mL, are reacted for 24 hours at 110 DEG C, are cooled to room temperature, are added
The dilution of 10mL water, is extracted with ethyl acetate (15mL × 3), and saturated salt solution (10mL) washing, anhydrous magnesium sulfate is dry, and decompression is steamed
Distillation solvent, silica gel column purification (VEthyl acetate: VPetroleum ether=6:1), obtain orange solids 1- (3 '-nitros -4 '-piperidines-phenyl) -3- first
Base-pyrazoles -4- methyl formate (IV3) 0.2g, yield 58.1%.
(3) by IV3(0.17g, 0.49mmol) dissolves in THF (6mL) and the mixed solution of ethyl alcohol (5mL), and 1M is added
NaOH aqueous solution 1mL, flow back 1h, is cooled to room temperature, and it is 2-3 that 1M HCL aqueous solution, which is added, and adjusts pH, is diluted with water, and solid is precipitated,
It filtering, filter cake is washed with water to neutrality, and it is dry, obtain orange solids 1- (3 '-nitros -4 '-piperidines-phenyl) -3- methyl pyrazole -4-
Formic acid (I3) 76mg, yield 49.4%.Product appraising datum is as follows:
1H NMR(400MHz,DMSO-d6) δ: 12.52 (s, 1H), 8.94 (s, 1H), 8.28 (d, J=2.6Hz, 1H),
8.06 (dd, J=9.0,2.6Hz, 1H), 7.40 (d, J=9.1Hz, 1H), 2.98 (t, 4H), 2.42 (s, 3H), 1.60 (m,
4H),1.54(m,2H);HRMS(ESI,m/z):[M+H]+,Calcd.331.1401,Found 331.1404。
Embodiment 4
1- (3 '-cyano -4 '-morpholine-phenyl) -3- methyl pyrazole -4- formic acid (I4) synthesis
(1) by II1(2.0g, 10mmol) is dissolved in DMSO (20mL), and K is added2CO3(4.1g, 30mmol), morpholine
(2.6g, 30mmol), 4h is reacted at 120 DEG C, and TLC tracks fully reacting.Reaction solution is cooled to room temperature, and the water dilution of 100mL is added,
Ethyl acetate (100mL × 3) extraction, saturated common salt water washing, anhydrous magnesium sulfate is dry, and evaporating solvent under reduced pressure obtains orange liquid
The bromo- 2- morpholine-benzonitrile (III of 5-4) 2.6g, yield 97.3%.
(2)N2Under protection, 3- methyl-1 H- pyrazoles -4- methyl formate (0.14g, 1.0mmol), CuI (19.1mg,
0.1mmol)、K2CO3(0.29g,2.1mmol)、III4(0.32g, 1.2mmol), (E)-N, N '-dimethyl -1,2- cyclohexyl two
Amine (28.5mg, 0.2mmol) and DMF (3mL) are added in bis- neck bottle of 25mL, are reacted for 24 hours at 110 DEG C, are cooled to room temperature, are added
The dilution of 10mL water, is extracted with ethyl acetate (15mL × 3), and saturated salt solution (10mL) washing, anhydrous magnesium sulfate is dry, and decompression is steamed
Except solvent, silica gel column purification (VEthyl acetate: VPetroleum ether=4:1), obtain yellow solid 1- (3 '-cyano -4 '-morpholine-phenyl) -3- methyl -
Pyrazoles -4- methyl formate (IV4) 0.3g, yield 46.0%.
(3) by IV4(0.25g, 0.77mmol) dissolves in THF (30mL) and the mixed solution of ethyl alcohol (20mL), and 1M is added
NaOH aqueous solution 2mL, flow back 1h, is cooled to room temperature, and it is 2-3 that 1M HCL aqueous solution, which is added, and adjusts pH, is diluted with water, and solid is precipitated,
It filtering, filter cake is washed with water to neutrality, and it is dry, obtain white solid 1- (3 '-cyano -4 '-morpholine-phenyl) -3- methyl pyrazole -4-
Formic acid (I4) 0.19g, yield 80.7%.Product appraising datum is as follows:
1H NMR(400MHz,DMSO-d6) δ: 12.52 (s, 1H), 8.93 (s, 1H), 8.24 (d, J=2.6Hz, 1H),
8.11 (dd, J=9.0,2.6Hz, 1H), 7.28 (d, J=9.1Hz, 1H), 3.77 (t, 4H), 3.17 (t, 4H), 2.41 (s,
3H);HRMS(ESI,m/z):[M+Na]+,Calcd.335.1115,Found 335.1114。
Embodiment 5
1- (3 '-nitros -4 '-morpholine-phenyl) -3- methyl pyrazole -4- formic acid (I5) synthesis
(1) by II2(3.0g, 13.6mmol) is dissolved in DMSO (20mL), and K is added2CO3(6.2,45mmol), morpholine
(3.9g, 45mmol), 4h is reacted at 120 DEG C, and TLC tracks fully reacting.Reaction solution is cooled to room temperature, and the water dilution of 100mL is added,
Ethyl acetate (100mL × 3) extraction, saturated common salt water washing, anhydrous magnesium sulfate is dry, and evaporating solvent under reduced pressure obtains orange liquid
The bromo- 2- piperidines -1- nitrobenzene (III of 5-5) 3.4g, yield 97.6%.
(2)N2Under protection, 3- methyl-1 H- pyrazoles -4- methyl formate (0.14g, 1.0mmol), CuI (19.1mg,
0.1mmol)、K2CO3(0.29g,2.1mmol)、III5(0.34g, 1.2mmol), (E)-N, N '-dimethyl -1,2- cyclohexyl two
Amine (28.5mg, 0.2mmol) and DMF (3mL) are added in bis- neck bottle of 25mL, are reacted for 24 hours at 110 DEG C, are cooled to room temperature, are added
The dilution of 10mL water, is extracted with ethyl acetate (15mL × 3), and saturated salt solution (10mL) washing, anhydrous magnesium sulfate is dry, and decompression is steamed
Except solvent, silica gel column purification (VEthyl acetate: VPetroleum ether=4:1), obtain orange solids 1- (3 '-nitros -4 '-morpholine-phenyl) -3- methyl -
Pyrazoles -4- methyl formate (IV5) 0.16g, yield 46.2%.
(3) by IV5(0.14g, 0.41mmol) dissolves in THF (8mL) and the mixed solution of ethyl alcohol (4mL), and 1M is added
NaOH aqueous solution 1mL, flow back 1h, is cooled to room temperature, and it is 2-3 that 1M HCL aqueous solution, which is added, and adjusts pH, is diluted with water, and solid is precipitated,
It filtering, filter cake is washed with water to neutrality, and it is dry, obtain orange solids 1- (3 '-nitros -4 '-morpholine-phenyl) -3- methyl pyrazole -4-
Formic acid (I5) 0.10g, yield 74.9%.Product appraising datum is as follows:
1H NMR(400MHz,DMS-d6) δ: 12.54 (s, 1H), 8.95 (s, 1H), 8.31 (s, 1H), 8.10 (d, J=
9.0Hz, 1H), 7.44 (d, J=9.0Hz, 1H), 3.68 (t, 4H), 2.99 (t, 4H), 2.41 (s, 3H);HRMS(ESI,m/z):
[M+Na]+,Calcd.355.1013,Found 355.1018。
Embodiment 6
1- (3 '-cyano -4 '-dimethylamino-phenyl)-pyrazoles -4- formic acid (I6) synthesis
(1) by II1(1.0g, 5mmol) is dissolved in DMF (15mL), and Cs is added2CO3(4.8g, 15mmol), dimethylamine
(0.7g, 15mmol), reacts 12h at room temperature, and TLC tracks fully reacting.The water that 80mL is added dilutes, ethyl acetate (80mL ×
3) it extracts, saturated common salt water washing, anhydrous magnesium sulfate is dry, and evaporating solvent under reduced pressure, silica gel column purification (petroleum ether) obtains faint yellow
The bromo- 2- methyl amine-benzonitrile (III of solid 5-6) 0.73g, yield 64.9%.
(2)N2Under protection, 1H- pyrazoles -4- Ethyl formate (0.28g, 2.0mmol), CuI (38.2mg, 0.2mmol),
K2CO3(0.58g,4.2mmol)、III6(0.54g, 2.4mmol), (E)-N, N '-dimethyl -1,2- cyclohexyl diamine (57mg,
0.4mmol) it is added in bis- neck bottle of 25mL with DMF (6mL), 110 DEG C of reactions for 24 hours, are cooled to room temperature, the dilution of 25mL water is added,
It is extracted with ethyl acetate (25mL × 3), saturated salt solution (25mL) washing, anhydrous magnesium sulfate is dry, evaporating solvent under reduced pressure, silica gel
Column purification (VEthyl acetate: VPetroleum ether=6:1), obtain faint yellow solid 1- (3 '-cyano -4 '-dimethylamino-phenyl)-pyrazoles -4- formic acid
Ethyl ester (IV6) 0.37g, yield 65.0%.
(3) by IV6(0.3g, 1.05mmol) dissolves in THF (15mL) and the mixed solution of ethyl alcohol (5mL), and 1M is added
NaOH aqueous solution 2mL, flow back 1h, is cooled to room temperature, and it is 2-3 that 1M HCL aqueous solution, which is added, and adjusts pH, is diluted with water, and solid is precipitated,
It filtering, filter cake is washed with water to neutrality, and it is dry, obtain white solid 1- (3 '-cyano -4 '-dimethylamino-phenyl)-pyrazoles -4- formic acid
(I6) 0.25g, yield 91.4%.Product appraising datum is as follows:
1H NMR(400MHz,DMSO-d6) δ: 12.63 (s, 1H), 8.97 (s, 1H), 8.13 (d, J=2.7Hz, 1H),
8.04 (s, 1H), 8.01 (dd, J=9.2,2.7Hz, 1H), 7.14 (d, J=9.3Hz, 1H), 3.02 (s, 6H);HRMS(ESI,
m/z):[M+Na]+,Calcd.279.0852,Found 279.0855。
Embodiment 7
1- (3 '-cyano -4 '-nafoxidine-phenyl)-pyrazoles -4- formic acid (I7) synthesis
(1) by II1(1.0g, 5mmol) is dissolved in DMF (15mL), and K is added2CO3(2.0g, 15mmol), nafoxidine
(1.0g, 15mmol), reacts 6h at 86C, and TLC tracks fully reacting.The water that 80mL is added dilutes, ethyl acetate (80mL ×
3) it extracts, saturated common salt water washing, anhydrous magnesium sulfate is dry, and evaporating solvent under reduced pressure obtains the bromo- 2- nafoxidine-of orange solids 5-
Benzonitrile (III7) 1.1g, yield 97.3%.
(2)N2Under protection, 1H- pyrazoles -4- Ethyl formate (0.28g, 2.0mmol), CuI (38.2mg, 0.2mmol),
K2CO3(0.58g,4.2mmol)、III7(0.6g, 2.4mmol), (E)-N, N '-dimethyl -1,2- cyclohexyl diamine (57mg,
It 0.4mmol) is added in bis- neck bottle of 25mL, is reacted at 110 DEG C for 24 hours with DMF (6mL), be cooled to room temperature, the dilution of 25mL water is added,
It is extracted with ethyl acetate (25mL × 3), saturated salt solution (25mL) washing, anhydrous magnesium sulfate is dry, evaporating solvent under reduced pressure, silica gel
Column purification (VEthyl acetate: VPetroleum ether=6:1), obtain white solid 1- (3 '-cyano -4 '-nafoxidine-phenyl)-pyrazoles -4- formic acid second
Ester (IV7) 0.23g, yield 37.1%.
(3) by IV7(0.15g, 0.48mmol) dissolves in THF (10mL) and the mixed solution of ethyl alcohol (5mL), and 1M is added
NaOH aqueous solution 1mL, flow back 1h, is cooled to room temperature, and it is 2-3 that 1M HCL aqueous solution, which is added, and adjusts pH, is diluted with water, and solid is precipitated,
It filtering, filter cake is washed with water to neutrality, and it is dry, obtain white solid 1- (3 '-cyano -4 '-nafoxidine-phenyl)-pyrazoles -4- formic acid
(I7) 0.1g, yield 73.4%.Product appraising datum is as follows:
1H NMR(400MHz,DMSO-d6) δ: 12.62 (s, 1H), 8.91 (s, 1H), 8.00 (d, J=0.5Hz, 1H),
7.99 (s, 1H), 7.91 (dd, J=9.3,2.8Hz, 1H), 6.87 (d, J=9.4Hz, 1H), 3.54 (m, 4H), 2.08-1.78
(m,4H);HRMS(ESI,m/z):[M+Na]+,Calcd.305.1009,Found 305.1000。
Embodiment 8
1- (3 '-cyano -4 '-(N- methyl) isobutylamine-phenyl)-pyrazoles -4- formic acid (I8) synthesis
(1) by II1(1.0g, 5mmol) is dissolved in DMSO (15mL), is added N- methyl-isobutyl amine (1.3g, 15mmol),
12h is reacted at 78 DEG C, TLC tracks fully reacting.Reaction solution is cooled to room temperature, and the water dilution of 80mL, ethyl acetate (80mL is added
× 3) it extracts, saturated common salt water washing, anhydrous magnesium sulfate is dry, and evaporating solvent under reduced pressure obtains the bromo- 2- of yellow liquid 5- (N- methyl)
Isobutylamine-benzonitrile (III8) 1.1g, yield 97.3%.
(2)N2Under protection, 1H- pyrazoles -4- Ethyl formate (0.28g, 2.0mmol), CuI (38.2mg, 0.2mmol),
K2CO3(0.58g,4.2mmol)、III8(0.64g, 2.4mmol), (E)-N, N '-dimethyl -1,2- cyclohexyl diamine (57mg,
It 0.4mmol) is added in bis- neck bottle of 25mL with DMF (6mL), 110 DEG C of reactions for 24 hours, are cooled to room temperature, and the dilution of 25mL water is added, uses
Ethyl acetate (25mL × 3) extraction, saturated salt solution (25mL) washing, anhydrous magnesium sulfate is dry, evaporating solvent under reduced pressure, silicagel column
Purify (VEthyl acetate: VPetroleum ether=8:1), obtain white solid 1- (3 '-cyano -4 '-(N- methyl) isobutylamine-phenyl)-pyrazoles -4-
Ethyl formate (IV8) 0.18g, yield 27.6%.
(3) by IV8(0.15g, 0.46mmol) dissolves in THF (5mL) and the mixed solution of ethyl alcohol (5mL), and 1M is added
NaOH aqueous solution 1mL, flow back 1h, is cooled to room temperature, and it is 2-3 that 1M HCL aqueous solution, which is added, and adjusts pH, is diluted with water, and solid is precipitated,
It filtering, filter cake is washed with water to neutrality, and it is dry, obtain white solid 1- (3 '-cyano -4 '-(N- methyl) isobutylamine-phenyl) -1H-
Pyrazoles -4- formic acid (I8) 0.13g, yield 90.4%.Product appraising datum is as follows:
1H NMR(400MHz,DMSO-d6) δ: 12.59 (s, 1H), 8.95 (s, 1H), 8.08 (d, J=2.8Hz, 1H),
8.02 (s, 1H), 7.97 (dd, J=9.3,2.8Hz, 1H), 7.15 (d, J=9.3Hz, 1H), 3.24 (d, J=7.5Hz, 2H),
3.02 (s, 3H), 2.06-1.89 (m, 1H), 0.84 (d, J=6.6Hz, 6H);HRMS(ESI,m/z):[M+Na]+,
Calcd.321.1322,Found 321.1328。
Embodiment 9
1- (3 '-cyano -4 '-isobutylamine-phenyl)-pyrazoles -4- formic acid (I9) synthesis
(1) by II1(1.0g, 5mmol) is dissolved in DMSO (15mL), 70 DEG C of addition N- isobutylamine (1.1g, 15mmol)
Lower reaction 5h, TLC track fully reacting.Reaction solution is cooled to room temperature, and the water dilution of 80mL, ethyl acetate (80mL × 3) extraction is added
It takes, saturated common salt water washing, anhydrous magnesium sulfate is dry, and evaporating solvent under reduced pressure obtains the bromo- 2- isobutylamine-benzene first of yellow liquid 5-
Nitrile (III9) 1.1g, yield 86.7%.
(2)N2Under protection, 1H- pyrazoles -4- Ethyl formate (0.28g, 2.0mmol), CuI (38.2mg, 0.2mmol),
K2CO3(0.58g,4.2mmol)、III9(0.61g, 2.4mmol), (E)-N, N '-dimethyl -1,2- cyclohexyl diamine (57mg,
It 0.4mmol) is added in bis- neck bottle of 25mL, is reacted at 110 DEG C for 24 hours with DMF (6mL), be cooled to room temperature, the dilution of 25mL water is added,
It is extracted with ethyl acetate (25mL × 3), saturated salt solution (25mL) washing, anhydrous magnesium sulfate is dry, evaporating solvent under reduced pressure, silica gel
Column purification (VEthyl acetate: VPetroleum ether=8:1), obtain white solid 1- (3 '-cyano -4 '-isobutylamine-phenyl)-pyrazoles -4- formic acid second
Ester (IV9) 0.17g, yield 27.1%.
(3) by IV9(0.15g, 0.48mmol) dissolves in THF (5mL) and the mixed solution of ethyl alcohol (5mL), and 1M is added
NaOH aqueous solution 1mL, flow back 1h, is cooled to room temperature, and it is 2-3 that 1M HCL aqueous solution, which is added, and adjusts pH, is diluted with water, solid analysis
Out, it filtering, filter cake is washed with water to neutrality, and it is dry, obtain white solid 1- (3 '-cyano -4 '-isobutylamine-phenyl)-pyrazoles -4-
Formic acid (I9) 0.12g, yield 88.3%.Product appraising datum is as follows:
1H NMR(400MHz,DMSO-d6) δ: 8.79 (s, 1H), 7.94 (s, 1H), 7.91 (d, J=2.7Hz, 1H), 7.83
(dd, J=9.2,2.7Hz, 1H), 6.84 (d, J=9.3Hz, 1H), 6.35 (t, J=5.9Hz, 1H), 2.97 (t, J=6.5Hz,
2H), 1.83 (m, 1H), 0.84 (d, J=6.6Hz, 6H);HRMS(ESI,m/z):[M+Na]+,Calcd.307.1165,Found
307.1172。
Embodiment 10
1- (3 '-cyano -4 '-(2- methyl-tetrahydro pyrroles) phenyl)-pyrazoles -4- formic acid (I10) synthesis
(1) by II1(1.0g, 5mmol) is dissolved in DMSO (15mL), addition 2- methyl-tetrahydro pyrroles (1.3g,
15mmol), 5h is reacted at 70 DEG C, TLC tracks fully reacting.Reaction solution is cooled to room temperature, and the water dilution of 80mL, ethyl acetate is added
(80mL × 3) extraction, saturated common salt water washing, anhydrous magnesium sulfate is dry, and evaporating solvent under reduced pressure obtains the bromo- 2- (first of yellow liquid 5-
Base-nafoxidine)-benzonitrile (III10) 1.28g, yield 96.7%.
(2)N2Under protection, 1H- pyrazoles -4- Ethyl formate (0.28g, 2.0mmol), CuI (38.2mg, 0.2mmol),
K2CO3(0.58g,4.2mmol)、III10(0.63g, 2.4mmol), (E)-N, N '-dimethyl -1,2- cyclohexyl diamine (57mg,
It 0.4mmol) is added in bis- neck bottle of 25mL, is reacted at 110 DEG C for 24 hours with DMF (6mL), be cooled to room temperature, the dilution of 25mL water is added,
Ethyl acetate (25mL × 3) extraction, saturated salt solution (25mL) washing, anhydrous magnesium sulfate is dry, evaporating solvent under reduced pressure, silicagel column
Purify (VEthyl acetate: VPetroleum ether=8:1), obtain white solid 1- (3 '-cyano -4 '-(2- methyl-tetrahydro pyrroles) phenyl)-pyrazoles -4-
Ethyl formate (IV10) 0.4g, yield 61.7%.
(3) by IV10(0.2g, 0.6mmol) dissolves in THF (5mL) and the mixed solution of ethyl alcohol (5mL), and 1M NaOH is added
Aqueous solution 1.2mL, flow back 1h, is cooled to room temperature, and it is 2-3 that 1M HCL aqueous solution, which is added, and adjusts pH, is diluted with water, and solid is precipitated, and takes out
Filter, filter cake is washed with water to neutrality, dry, obtains white solid 1- (3 '-cyano -4 '-(2- methyl-tetrahydro pyrroles) phenyl)-pyrazoles -
4- formic acid (I10) 0.11g, yield 61.9%.Product appraising datum is as follows:
1H NMR(400MHz,DMSO-d6) δ: 12.54 (s, 1H), 8.89 (s, 1H), 8.01 (d, J=1.9Hz, 1H),
8.01 (s, 1H), 7.92 (dd, J=9.4,2.8Hz, 1H), 6.94 (d, J=9.5Hz, 1H), 4.26 (m, 1H), 3.86-3.39
(m, 2H), 2.19-1.93 (m, 2H), 1.93-1.57 (m, 2H), 1.14 (d, J=6.1Hz, 3H);HRMS(ESI,m/z):[M+
H]+,Calcd.297.1346,Found 297.1343。
The activity rating of above embodiments products therefrom:
(1) to the inhibitory activity in-vitro evaluation of xanthine oxidase
1. solution is prepared
Buffer: 10 × PBS (pH 7.4) is diluted to 1 × PBS.Except special instruction, meaning PBS is referred both in reaction system
1×PBS。
Substrate: weighing 15.2mg xanthine, is added 45mL PBS ultrasound dissolution, then plus PBS be settled to 200mL to get
The substrate solution of 0.5mmol/L.
Enzyme solution: under ice bath, 10.2 μ L xanthine oxidoreductase enzyme mother liquors are diluted to get to 0.5 μ g/100 with the PBS of 20mL
The enzyme solution of μ L.
Untested compound: required compound (embodiment products therefrom) is accurately weighed, is configured to the molten of 1mmol/L with DMSO
Liquid reserve, is protected from light storage in 20 DEG C.It is diluted to required concentration with PBS using preceding, DMSO content will control within 5% to guarantee
It is on enzyme activity without influence.
2. measurement
The above-mentioned PBS solution prepared, sample or blank solution (blank solution is PBS solution), 100 μ L enzyme solutions are successively added
Enter 96 orifice plates, be incubated for 3min at 37 DEG C in microplate reader, substrate starting reaction is then added into the microwell plate hatched,
It is primary every 1min reading at 292nm, 10min is read altogether, and every group of experiment is measured in parallel three times.By test chemical combination at various concentrations
The initial velocity of object is converted into the inhibiting rate percentage (%) based on the initial velocity in the presence of no inhibitor, calculates IC50Value, as a result
As shown in table 1.According to the present invention, develop such compound: the compound is shown for xanthine oxidase in nM water
Flat IC50Value.
Inhibitory activity of the 1 embodiment products therefrom compound of table to xanthine oxidase
"-" indicates inhibitor under 100nM/L concentration, and inhibiting rate does not reach 50%.
As seen from the results in Table 1,1) part of compounds shows apparent inhibition xanthine oxidoreductase enzyme activity sexuality, such as
Compound I2、I7、I8、I9And I10, these compounds show the IC in nM level for xanthine oxidoreductase enzyme50Value;2) press down
The result of XOR enzymatic activity processed shows obviously structure-activity relationship: it is active that pyrazole ring C-3 replaces modification to press down compound enzyme
Influence it is most important, such as compound I1、I3、I4And I5It is that methyl replaces, has apparent reduction to the inhibitory activity of enzyme;Phenyl ring
The compound I replaced on C-3' for nitro3And I5Inhibiting enzyme activity, compared on phenyl ring C-3' be cyano replace compound I5Obviously
It reduces.C-4' replaces modification is active on the suppression of compound enzyme to influence also important, to replace with piperidyl compound on phenyl ring
I2Activity is best.
(2) to the inhibitory activity interior evaluating of xanthine oxidase
1) experimental method:
The SPF grade ICR mouse adaptable fed of 18-22g is after a week, random to be grouped, and is divided into Normal group (Control
Group, physiological saline), model control group (Vehicle group, Oteracil Potassium 300mgkg-1+ hypoxanthine 500mgkg-1), sun
Property control group (Febuxostat (Febuxostat) group, Oteracil Potassium 300mgkg-1+ hypoxanthine 500mgkg-1+ Fei Busuo
Smooth 5mgkg-1), untested compound (embodiment products therefrom) administration group (Oteracil Potassium 300mgkg-1+ hypoxanthine
500mg·kg-1+ untested compound 5mgkg-1), every group 8.
Mouse weight is weighed before experiment, gives model control group and each administration group subcutaneous injection Oteracil Potassium 300mgkg-1
+ Intraperitoneal injection of hypoxanthine 500mgkg-1, Normal group gives same amount of normal saline, measures serum uric acid level respectively after 1h
(being denoted as 1h blood uric acid), and to each medicine group, drug to be measured is given in stomach-filling respectively immediately, Normal group and model control group fill
Stomach is given equivalent solvent (0.5%CMC-Na solution), 1h after administration, 2h, 3h, 4h measure respectively uric acid level (be denoted as 2h respectively,
3h, 4h, 5h blood uric acid).Acquired results are statisticallyd analyze, as a result as shown in Figure 2 with the mapping of graphpad 5.0 with t-test.
2) experimental result
As shown in Figure 2: contrast model control group, I2And I8Mouse serum uric acid level (P < 0.05) can be effectively reduced.Especially
It is I8, the ability and Febuxostat of anti-trioxypurine be suitable.
If above-mentioned experiment is revealed, formula (I) compound of the present invention has excellent inhibiting effect to xanthine oxidase.
Therefore, the compound of the present invention can be used as treating and preventing disease relevant to people's xanthine oxidase, such as high lithemia
The drug of mass formed by blood stasis, heart failure, cardiovascular disease, hypertension, kidney diaseases, inflammation, arthropathy etc..
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.