CN110305125B - 5-pyrimidine-6-oxy-pyrazolopyridine derivative and preparation method and application thereof - Google Patents

5-pyrimidine-6-oxy-pyrazolopyridine derivative and preparation method and application thereof Download PDF

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CN110305125B
CN110305125B CN201910492351.1A CN201910492351A CN110305125B CN 110305125 B CN110305125 B CN 110305125B CN 201910492351 A CN201910492351 A CN 201910492351A CN 110305125 B CN110305125 B CN 110305125B
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李乾斌
胡高云
胡立庆
陈卓
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Shandong Xinhua Pharmaceutical Co Ltd
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Abstract

The invention relates to a 5-pyrimidine-6-oxygen-pyrazolopyridine derivative, a pharmaceutically available salt thereof, a preparation method and an application thereof, wherein the chemical structural formula (II) of the 5-pyrimidine-6-oxygen-pyrazolopyridine derivative is shown as the following formula:

Description

5-pyrimidine-6-oxy-pyrazolopyridine derivative and preparation method and application thereof
Technical Field
The invention relates to 5-pyrimidine-6-oxygen-pyrazolopyridine derivatives and pharmaceutically acceptable salts thereof, and also relates to a preparation method thereof and application thereof as a medicament, in particular to application of the derivatives and the pharmaceutically acceptable salts thereof as a medicament for treating pulmonary fibrosis.
Background
Pulmonary fibrosis is a fatal pulmonary disease, the pathological features of which are damage and abnormal proliferation of alveolar epithelial cells, deposition of extracellular matrix and proliferation and activation of fibroblasts, and is characterized by progressive and irreversible destruction of lung structures caused by scar formation, ultimately leading to organ dysfunction, resulting in destruction of lung structures and loss of respiratory function. Pulmonary fibrosis can be caused by viral infection, exposure to radiation therapy, chemotherapeutic drugs, and aerosolized environmental toxins. Also, pulmonary fibrosis occurs in some bone marrow transplant recipients with chronic transplant resistant host disease and in some individuals with chronic inflammatory diseases including scleroderma and rheumatoid arthritis. The causes of most patients with pulmonary fibrosis are unknown, and the most common disease type with pulmonary fibrosis as the main manifestation is idiopathic pulmonary fibrosis. Idiopathic pulmonary fibrosis refers to a pulmonary interstitial disease that presents with lung injury in the absence of a clear etiology and progressively worsens, ultimately leading to respiratory failure and death. The incidence and mortality of idiopathic pulmonary fibrosis increases year by year, with an average survival period of only 2.8 years after diagnosis, with mortality rates higher than that of most tumors, known as a "neoplastic-like disease".
The traditional drug therapy for pulmonary fibrosis adopts an anti-inflammatory strategy, but the single use of anti-inflammatory drugs such as glucocorticoid and immunosuppressant cannot completely improve the prognosis and the fatality rate of patients, and cannot fundamentally reverse the inflammatory process. At present, the main marketed drugs for pulmonary fibrosis are pirfenidone and nintedanib. Pirfenidone is a multi-target small molecule pyridone compound, plays anti-fibrosis, anti-inflammation and anti-oxidation effects by down-regulating the levels of a growth factor and an inflammatory factor for promoting fibrosis, and clinical experiments prove that pirfenidone can slow down the progress of idiopathic pulmonary fibrosis and prolong the life of patients with pulmonary fibrosis, and is approved to be marketed in Japan in 2008. Nintedanib is a tyrosine kinase inhibitor and can treat pulmonary fibrosis by inhibiting the signaling pathways of VEGFR, PDGFR and FGFR. In the clinical application process, both the two drugs have the defects of short half-life period, large side effect and unsuitability for long-term administration.
In recent years, research shows that the NO/sGC/cGMP pathway plays an important role in fibrotic diseases, and the sGC agonist drug Riociguat blocks a TGF-beta/ERK signal pathway by activating the sGC-cGMP pathway, thereby playing an anti-fibrotic role. Therefore, the search for novel sGC agonists, which exert the anti-fibrosis and anti-tissue remodeling effects by up-regulating the cGMP level, will become a new strategy for drug treatment of pulmonary fibrosis diseases.
The patent application No. 201810520915.3 discloses compounds of the general structural formula (I) wherein R4Selected from-CN, -CH2NH2，-COOH、COOR5、-CH2-NR6R7Wherein R is5Selected from C1-C6 fatty chain, C3-C6 fatty ring, and mixtures thereofSubstituted or unsubstituted aromatic hydrocarbon, substituted or unsubstituted aromatic heterocycle; r6、R7Are each independently selected from-CH3、-C2H5(ii) a or-NR6R7Is a substituted or unsubstituted aliphatic heterocycle.
Figure BDA0002087451850000021
The patent also discloses the application of the compound with the general formula (I) in preparing the anti-lung cancer medicament. However, the compounds of the general formula (I) do not have the function of activating sGC.
Disclosure of Invention
The invention aims to provide a 5-pyrimidine-6-oxygen-pyrazolopyridine derivative and pharmaceutically usable salts thereof, and also provides a preparation method and application thereof in preparing medicaments for treating pulmonary fibrosis.
In order to solve the technical problems, the technical scheme of the invention is as follows:
the invention provides a 5-pyrimidine-6-oxygen-pyrazolopyridine derivative and a pharmaceutically acceptable salt thereof, wherein the structural general formula is shown as formula (II):
Figure BDA0002087451850000022
wherein R is1Or R2Selected from hydrogen, methyl, trifluoromethyl, aromatic rings or substituted aromatic rings;
R3is selected from C1-C6Fatty chain, C3-C6Alicyclic or substituted C3-C6Alicyclic rings, aromatic rings or substituted aromatic rings, aromatic heterocycles or substituted aromatic heterocycles;
x and Y are independently selected from hydrogen, hydroxyl, amino, halogen;
R4selected from hydrogen, the following structural fragments and pharmaceutically acceptable salts thereof:
Figure BDA0002087451850000031
wherein R is5Is selected from C1-C6Fatty chain, C3-C6Alicyclic or substituted C3-C6Alicyclic rings, aromatic rings or substituted aromatic rings, aromatic heterocycles or substituted aromatic heterocycles; z is selected from CH2、O、N。
Preferably, said substituent C3-C6The substituent in the alicyclic structure being C1-C6Fatty chains or C3-C6A fat ring. C1-C6The aliphatic chain is aliphatic hydrocarbon with 1-6 carbon atoms constituting straight chain or branched chain, C3-C6The alicyclic ring refers to a hydrocarbon having 3 to 6 carbon atoms constituting the alicyclic ring.
Preferably, the aromatic ring is selected from benzene, naphthalene, anthracene or phenanthrene, and the aromatic heterocycle is selected from pyrrole, furan, thiophene, imidazole, thiazole, oxazole, pyrazole, isoxazole, thiadiazole, oxadiazole, tetrazole, pyridine, pyrimidine, pyrazine, pyridazine, purine, quinoline, isoquinoline, indole, acridine, carbazole, and bioisosteres of the above ring systems.
Preferably, the substituent in the substituted aromatic ring or substituted aromatic heterocyclic structure is selected from hydroxyl, methyl, amino, trifluoromethyl or halogen.
Preferably, X and Y are selected from hydroxyl, amino.
Preferably, the pharmaceutically acceptable salt is selected from: hydrochloride, sulfate, phosphate, perchlorate, methanesulfonate, trifluoromethanesulfonate, formate, acetate, propionate, butyrate, maleate, succinate, trifluoroacetate, succinate, salicylate, DL-aspartate, D-aspartate, L-aspartate, DL-glutamate, D-glutamate, L-glutamate, glycerate, stearate, DL-tartrate, D-tartrate, L-tartrate, (±) mandelate, (R) - (-) mandelate, (S) - (+) mandelate, citrate, mucate, malonate, benzoate, DL-malate, (±) lactate, L- (+) -lactate, D- (+) -lactate, succinate, maleate, citrate, maleate, lactate, maleate, citrate, maleate, lactate, citrate, lactate, and acetate, Pamoate, D-alpha-galacturonate, glycerate, DL-cysteine, D-cysteine, L-cysteine, (4S) -hydroxy-L-prolinate, cyclopropane-1, 1-dicarboxylate, 2-methylmalonate, caseinate, prolinate, fumarate, 1-hydroxy-2-naphthoate, phosphonoacetate, carbonate, bicarbonate, 3-phosphonopropionate, DL-pyroglutamate, D-pyroglutamate, L-pyroglutamate, p-toluenesulfonate, benzenesulfonate, ethanesulfonate, (+/-) camphorsulfonate, naphthalenesulfonate, 1R- (-) -camphorsulfonate, 1S- (+) -camphorsulfonate, glycerate, DL-cysteine, D-L-cysteine, L-hydroxy-2-naphthoate, phosphonoacetate, carbonate, bicarbonate, 3-phosphonopropionate, DL-pyroglutamate, D-pyroglutamate, L-pyroglutamate, p-toluenesulfonate, benzenesulfonate, ethanesulfonate, camphorsulfonate, camphor sulfonate, camphor sulfonate, camphor, or alcohol, camphor, and water, camphor, or alcohol, camphor, water, 1, 5-naphthalenedisulfonate, 1, 2-ethanedisulfonate, 1, 3-propanedisulfonate, 3- (N-morpholino) propanesulfonate, biphenylsulfonate, isethionate, 1-hydroxy-2-naphthalenesulfonate, dihydrogenphosphate, potassium hydrogenphosphate, dipotassium phosphate, potassium phosphate, sodium hydrogenphosphate, disodium phosphate, sodium dihydrogenphosphate, calcium phosphate, calcium triphosphate, hexafluorophosphate, vinylphosphate, 2-hydroxyethyl phosphate, and phenylphosphate.
Preferably, the derivative preferably has the following structure:
2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diol (compound 1);
2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) -5-morpholinopyrimidine-4, 6-diol (compound 2);
2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) -5-piperidinylpyrimidine-4, 6-diol (compound 3);
2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) -5-pyrrolidinylpyrimidine-4, 6-diol (compound 4);
5- (dimethylamino) -2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diol (compound 5);
5- (diethylamino) -2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diol (compound 6);
2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diamine (compound 7);
2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) -5-morpholinopyrimidine-4, 6-diamine (compound 8);
2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) -5-piperidinylpyrimidine-4, 6-diamine (compound 9);
2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) -5-pyrrolidinylpyrimidine-4, 6-diamine (compound 10);
2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) -N5, N5-dimethylpyrimidine-4, 5, 6-triamine (compound 11);
2- (6- ((3-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diamine (compound 12);
2- (6- ((4-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diamine (compound 13);
2- (3-methyl-1-phenyl-6- ((2-methyl) benzyloxy) -1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diamine (compound 14);
2- (3-methyl-1-phenyl-6- ((3-methyl) benzyloxy) -1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diamine (compound 15);
2- (3-methyl-1-phenyl-6- ((4-methyl) benzyloxy) -1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diamine (compound 16);
2- (3-methyl-1-phenyl-6- ((4-trifluoromethyl) benzyloxy) -1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diamine (compound 17);
2- (6-benzyloxy-3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diamine (compound 18);
2- (3-methyl-1-phenyl-6- (thiazole-2-methoxy) -1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diamine (compound 19);
2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) -5-nitrosopyrimidine-4, 6-diamine (compound 20);
2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 5, 6-triamine (compound 21);
n- (4, 6-diamino-2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) acetamide (compound 22);
methyl (4, 6-diamino-2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidin-5-yl) carbamate (compound 23);
n-butyl (4, 6-diamino-2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidin-5-yl) carbamate (compound 24);
cyclopentyl (4, 6-diamino-2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidin-5-yl) carbamate (compound 25).
The invention also provides a preparation method of the 5-pyrimidine-6-oxygen-pyrazolopyridine derivative, which comprises the following specific steps:
when R is4Is composed of
Figure BDA0002087451850000051
The method comprises the following specific steps:
s1, aminolysis of cyano in the structure into hydroxyamidine by the compound I under the action of hydroxylamine hydrochloride to obtain a compound III;
s2, reducing the compound III by zinc powder to obtain a compound IV;
s3, Compound IV and R4Reacting substituted diethyl malonate to obtain a compound V;
scheme 1 is shown below:
Figure BDA0002087451850000052
preferably, one skilled in the art can further substitute two OH groups on the basis of compound V by conventional techniques to other groups, such as H, NH2And a halogen.
When R is4Is composed of
Figure BDA0002087451850000053
The specific steps thereofThe method comprises the following steps:
s1, aminolysis of cyano in the structure into hydroxyamidine by the compound I under the action of hydroxylamine hydrochloride to obtain a compound III;
s2, reducing the compound III by zinc powder to obtain a compound IV;
s3, Compound IV and R4Reacting substituted malononitrile to obtain a compound VI;
scheme 2 is shown below:
Figure BDA0002087451850000054
preferably, one skilled in the art can further combine two NH groups on the basis of compound VI2Substitution to other groups such as H, OH, halogen, respectively, is by conventional techniques.
When X and Y are amino groups, the amino group,
Figure BDA0002087451850000061
the method comprises the following specific steps:
s1, aminolysis of cyano in the structure into hydroxyamidine by the compound I under the action of hydroxylamine hydrochloride to obtain a compound III;
s2, reducing the compound III by zinc powder to obtain a compound IV;
s3, reacting the compound IV with hydrogen chloride gas at room temperature to obtain a compound VII;
s4, reacting the compound VII with nitrosomalononitrile silver salt to obtain a compound VIII;
s5, reducing the compound VIII with zinc powder to obtain a compound IX;
s6, reacting the compound IX with different substituted acyl chloride compounds under the alkaline condition to obtain a compound X;
the reaction scheme is as follows:
Figure BDA0002087451850000062
preferably, the skilled person is also able to substitute groups in the compounds XFurther reacting two NH groups2Substitution to other groups such as H, OH, halogen, respectively, is by conventional techniques.
Wherein R is1、R2、R3As defined above.
The invention also provides application of the 5-pyrimidine-6-oxygen-pyrazolopyridine derivative and pharmaceutically acceptable salts thereof as an sGC agonist, and further overcomes the defects of the compound disclosed by the patent and shown in the general formula (I).
The 5-pyrimidine-6-oxy-pyrazolopyridine derivative and pharmaceutically-usable salts thereof are applied to preparation of drugs for treating pulmonary fibrosis.
The invention is further illustrated: on one hand, the structural mother nucleus of the 5-pyrimidine-6-oxygen-pyrazolopyridine derivative and pharmaceutically usable salts thereof is pyrazolopyridine, and the structure can directly inhibit a TGF-beta/Smad classical pathway to play a role in resisting fibrosis; on the other hand, after a pyrimidine ring is introduced into the 5-position of pyrazolopyridine, the compound has the function of activating sGC, and can play an anti-fibrosis role by inhibiting TGF-beta/ERK non-classical pathway.
Compared with the prior art, the invention has the following beneficial effects:
1. the compound synthesized by the invention has novel structure;
2. the compound synthesized by the invention blocks TGF-beta pathway after activating sGC, plays a role in resisting pulmonary fibrosis, and can be applied to related medicines.
Drawings
FIG. 1 shows the effect of the derivatives of the invention on cGMP levels in HPASMCs.
Detailed Description
The present invention will be described in detail with reference to examples. It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
Example 1
2- (6- ((2-Fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diol (Compound 1)
Figure BDA0002087451850000071
0.22g of sodium methoxide (4mmol) was weighed into a 100mL eggplant-shaped bottle, 40mL of methanol was weighed into the reaction bottle, and the mixture was stirred at room temperature to obtain 0.5g of the compound 6- (2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3, 4-b)]Pyridine-5-formamidine (1.33mmol) is added into a reaction bottle, 0.18g dimethyl malonate (1.33mmol) is weighed by a rubber head dropper by a decrement method and slowly dripped into the reaction bottle, and finally 10mL methanol is added to rinse the bottle mouth. After the reaction apparatus was transferred to 70 ℃ and subjected to a reflux reaction for 12 hours, the starting material, 2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3, 4-b), was monitored by TLC]Pyridine-5-pyridyl) formamidine was not completely reacted. The reaction was continued for 8H and the starting material 2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3, 4-b) was monitored by TLC]Pyridine-5-pyridyl) formamidine is still not reacted completely and is not reacted to a degree that is substantially greater than before, and the reaction is stopped. The reaction solution was neutralized with glacial acetic acid, concentrated under reduced pressure and spin-dried to obtain 0.95g of crude product. Dissolving the crude solid with 50mL of methanol, mixing with 80-100 mesh silica gel, loading the mixture into a column with 200-300 mesh silica gel, purifying by column chromatography, separating and purifying an elution system with dichloromethane and methanol at a ratio of 30:1 to obtain 0.32g of white solid. Yield: 54%, melting point: 266.9-269.1 ℃, HPLC: 98.64 percent.1H NMR(500MHz,DMSO-d6):δ12.07(br s,1H,-OH),11.54(br s,1H,-OH),8.66(s,1H),8.14(d,J＝7.7Hz,2H,Ar-H),7.61(t,J＝7.0Hz,1H,Ar-H),7.55(t,J＝8.0Hz,2H,Ar-H),7.45～7.37(m,1H,Ar-H),7.37～7.26(m,2H,Ar-H),7.21(t,J＝7.5Hz,1H,Ar-H),5.66(s,2H,-CH2-),5.30(s,1H),2.58(s,3H,-CH3).13C NMR(126MHz,DMSO-d6):δ160.48(d,1JC,F＝243.75Hz),160.10,155.88(2C),148.70,144.82,139.11,135.28,130.59,130.53,130.39,129.67(2C),126.18,124.96,123.97(d,2JC,F＝15.0Hz),120.17(2C),115.81(d,2JC,F＝21.25Hz),112.51,112.28,89.07,63.16,12.64.HRMS(ESI)m/z calcd for[C24H18FN5O3+H]+:444.1394；found:444.1454[M+H]+,466.1272[M+Na]+.
Example 2
2- (6- ((2-Fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) -5-morpholinopyrimidine-4, 6-diol (Compound 2)
Figure BDA0002087451850000081
According to the synthesis method of the compound 1, the compound 6- (2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b]Pyridine-5-carboxamidine and diethyl morpholinylmalonate were the starting materials to give a yellow solid. Yield: 34%, melting point: 221.7-221.9 ℃, HPLC: 96.98 percent.1H NMR(400MHz,DMSO-d6):δ12.03(br s,1H,-OH),8.63(s,1H),8.12(d,J＝7.7Hz,2H,Ar-H),7.65～7.57(m,1H,Ar-H),7.54(t,J＝8.0Hz,2H,Ar-H),7.41(m,1H,Ar-H),7.32(m,1H,Ar-H),7.21(t,J＝7.4Hz,1H,Ar-H),5.66(s,2H,-CH2-),3.71～3.65(m,4H,-CH2-),3.09～3.02(m,4H,-CH2-),2.58(s,3H,-CH3).13C NMR(101MHz,DMSO-d6):δ160.44(d,1JC,F＝244.0Hz),160.06,151.65(2C),148.57,144.80,139.12,135.22,130.59,130.51,130.40,129.68(2C),126.16,124.97,124.07(d,2JC,F＝14.0Hz),120.14(2C),115.83(d,2JC,F＝21.0Hz),113.65,112.48,112.35,66.91(2C),63.18,49.94(2C),12.65.HRMS(ESI)m/z calcd for[C28H25FN6O4+H]+:529.1921；found:529.2004[M+H]+.
Example 3
2- (6- ((2-Fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) -5-piperidinylpyrimidine-4, 6-diol (Compound 3)
Figure BDA0002087451850000082
According to the synthesis method of the compound 1, the compound 6- (2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b]Pyridine-5-carboxamidine and piperidinylmalonic acidDiethyl ester was the starting material to give a white solid. Yield: 41%, melting point: 230.0-231.5 ℃, HPLC: 97.34 percent.1H NMR(400MHz,DMSO-d6):δ11.38(br s,1H,-OH),8.62(s,1H),8.17～8.07(d,J＝7.8Hz,2H,Ar-H),7.66～7.58(m,1H,Ar-H),7.53(t,J＝7.9Hz,2H,Ar-H),7.44～7.35(m,1H,Ar-H),7.34～7.26(dd,J＝16.0,8.1Hz,2H,Ar-H),7.20(t,J＝7.4Hz,1H,Ar-H),5.66(s,2H,-CH2-),3.34(m,4H,-CH2-),2.58(s,3H,-CH3),1.76(m,4H,-CH2-),1.50(m,2H,-CH2-).13C NMR(101MHz,DMSO-d6):δ160.44(d,1JC,F＝244.0Hz),160.04,152.76(2C),148.51,144.85,139.05,135.14,130.58,130.50,130.32,129.64(2C),126.15,124.94,124.03(d,2JC,F＝14.0Hz),120.11(2C),115.79(d,2JC,F＝20.0Hz),112.84,112.41,106.94,63.14,51.97(2C),23.98(2C),21.97,12.55.HRMS(ESI)m/z calcd for[C29H27FN6O3+H]+:527.2129；found:527.2176[M+H]+.
Example 4
2- (6- ((2-Fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) -5-pyrrolidinylpyrimidine-4, 6-diol (Compound 4)
Figure BDA0002087451850000091
According to the synthesis method of the compound 1, the compound 6- (2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b]Pyridine-5-carboxamidine and diethyl pyrrolidinylmalonate were the starting materials to give a white solid. Yield: 39%, melting point: 260.2-262.2 ℃, HPLC: 98.33 percent.1H NMR(500MHz,DMSO-d6):δ11.17(br s,1H,-OH),8.59(s,1H),8.12(d,J＝7.7Hz,2H,Ar-H),7.66～7.58(m,1H,Ar-H),7.53(t,J＝8.0Hz,2H,Ar-H),7.39((m,1H,Ar-H),7.31(dd,J＝16.0,8.3Hz,2H,Ar-H),7.19(t,J＝7.2Hz,1H,Ar-H),5.66(s,2H,-CH2-),3.49(t,J＝6.6Hz,4H,-CH2-),2.57(s,3H,-CH3),2.04(t,J＝6.7Hz,4H,-CH2-).13C NMR(126MHz,DMSO-d6):δ160.49(d,1JC,F＝243.8Hz),160.12,152.51(2C),148.52,144.67,139.23,134.97,130.51,130.44,130.38,129.63(2C),126.04,124.94,124.20(d,2JC,F＝13.8Hz),120.09(2C),115.80(d,2JC,F＝21.3Hz),113.60,112.41,104.21,63.07,53.36(2C),25.05(2C),12.62.HRMS(ESI)m/z calcd for[C28H25FN6O3+H]+:513.1972；found:513.2129[M+H]+,514.2103[M+2H]+.
Example 5
5- (dimethylamino) -2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diol (Compound 5)
Figure BDA0002087451850000101
According to the synthesis method of the compound 1, the compound 6- (2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b]Pyridine-5-formamidine and diethyl dimethylaminomethomalonate are used as raw materials to obtain a reddish brown solid. Yield: 32%, melting point: 239.3-242.1 ℃, HPLC: 98.56 percent.1H NMR(500MHz,DMSO-d6):δ11.21(br s,1H,-OH),8.60(s,1H),8.12(d,J＝8.1Hz,2H,Ar-H),7.62(t,J＝7.3Hz,1H,Ar-H),7.53(t,J＝7.7Hz,2H,Ar-H),7.40(dd,J＝13.4,7.3Hz,1H,Ar-H),7.35～7.25(m,2H,Ar-H),7.20(t,J＝7.4Hz,1H,Ar-H),5.64(s,2H,-CH2-),3.00(s,6H,-CH3),2.55(s,3H,-CH3).13C NMR(126MHz,DMSO-d6):δ160.45(d,1JC,F＝243.8Hz),160.02,152.75(2C),148.48,144.68,139.17,135.09,130.48,130.42,130.31,129.58(2C),125.99,124.93,124.14(d,2JC,F＝13.8Hz),119.99(2C),115.78(d,2JC,F＝21.3Hz),113.09,112.43,107.54,63.10(2C),43.66,12.58.HRMS(ESI)m/z calcd for[C26H23FN6O3+H]+:487.1816；found:487.1925[M+H]+.
Example 6
5- (diethylamino) -2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diol (Compound 6)
Figure BDA0002087451850000102
According to the synthesis method of the compound 1, the compound 6- (2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b]Pyridine-5-carboxamidine and diethyl diethylaminomalonate were the starting materials, giving a white solid. Yield: 41%, melting point: 266.5-269.1 ℃, HPLC: 97.68 percent.1H NMR(500MHz,DMSO-d6):δ11.02(br s,1H,-OH),8.66(s,1H),8.15(dd,J＝8.6,1.0Hz,2H,Ar-H),7.65～7.58(m,1H,Ar-H),7.56～7.52(m,2H,Ar-H),7.43～7.37(m,1H,Ar-H),7.34～7.28(m,2H,Ar-H),7.17(t,J＝7.4Hz,1H,Ar-H),5.68(s,2H,-CH2-),3.42(m,4H,-CH2-),2.59(s,3H,-CH3),1.07(t,J＝7.1Hz,6H,-CH3).13C NMR(126MHz,DMSO-d6):δ160.42(d,1JC,F＝242.5Hz),160.17,159.97,159.17,148.54,144.74,139.26,135.49,134.91,130.44,130.01,129.67(2C),126.06,124.87,124.37(d,2JC,F＝13.8Hz),124.30,120.11(2C),115.78(d,2JC,F＝21.3Hz),113.82,112.43,63.03,49.39(2C),12.64,10.86(2C).HRMS(ESI)m/z calcd for[C28H27FN6O3+H]+:515.2129；found:515.2225[M+H]+.
Example 7
2- (6- ((2-Fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diamine (Compound 7)
Figure BDA0002087451850000111
0.6g of the compound 6- (2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] is weighed]Pyridine-5-carboxamidine (1.6mmol) was added to a 25mL microwave tube and then in a volume ratio VMeOH:V 1,4-Dioxane5mL of methanol and 10mL of 1, 4-dioxane were measured in a ratio of 1:2Adding into a microwave tube, adding stirring bar, performing ultrasonic treatment for 5min to dissolve the raw material, and dropping 0.11g malononitrile (1.6mmol) slowly into the microwave tube by using a rubber head dropper. After the reaction apparatus was transferred to a microwave reaction at 130 ℃ for 2 hours, the starting material 2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3, 4-b) was monitored by TLC]Pyridine-5-pyridyl) formamidine reaction is completed, and the reaction is stopped. The reaction solution was transferred to 100mL, and then concentrated under reduced pressure and spin-dried to obtain 0.8g of a crude product. Dissolving the crude solid with 50mL of methanol, mixing with 80-100 mesh silica gel, loading the mixture into a column with 200-300 mesh silica gel, purifying by column chromatography, separating and purifying an elution system with dichloromethane and methanol at a ratio of 30:1 to obtain 0.36g of white solid. Yield: 51%, melting point: 278.0-280.7 ℃, HPLC: 96.67 percent.1H NMR(500MHz,DMSO-d6):δ8.30(s,1H),8.15(d,J＝7.9Hz,2H,Ar-H),7.68(t,J＝7.2Hz,1H,Ar-H),7.52(t,J＝7.9Hz,2H,Ar-H),7.35(m,1H,Ar-H),7.31～7.25(m,2H,Ar-H),7.19(t,J＝7.4Hz,1H,Ar-H),6.17(br s,4H,-NH2),5.62(s,2H,-CH2-),5.42(s,1H),2.55(s,3H,-CH3).13C NMR(101MHz,DMSO-d6):δ164.42,163.52,160.55,160.10(d,1JC,F＝242.0Hz),147.88,144.01,139.55,133.43,129.84,129.76,129.65,129.54(2C),125.65,125.00,124.98(d,2JC,F＝13.0Hz),121.04,119.82(2C),115.48(d,2JC,F＝21.0Hz),111.88,81.53,61.75,12.67.HRMS(ESI)m/z calcd for[C24H20FN7O+H]+:442.1713；found:442.1769[M+H]+.
Example 8
2- (6- ((2-Fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) -5-morpholinopyrimidine-4, 6-diamine (Compound 8)
Figure BDA0002087451850000121
According to the synthesis method of the compound 7, the compound raw material 6- (2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b]Pyridine-5-formamidine and morpholinyl malononitrile as starting materials gave a white solid. Yield: 49%, melting point: 221.9～224.6℃，HPLC：99.53％。1H NMR(500MHz,DMSO-d6):δ8.31(s,1H),8.11(d,J＝7.8Hz,2H,Ar-H),7.67(m,1H,Ar-H),7.52(t,J＝7.9Hz,2H,Ar-H),7.36～7.31(m,1H,Ar-H),7.28(dd,J＝16.6,9.0Hz,2H,Ar-H),7.18(t,J＝7.4Hz,1H),6.04(br s,4H,-NH2),5.62(s,2H,-CH2-),3.83～3.67(m,4H,-CH2-),3.01～2.91(m,4H,-CH2-),2.53(m,3H,-CH3).13C NMR(126MHz,DMSO-d6):δ161.03,160.50,160.05,159.97(d,1JC,F＝245.0Hz),147.88,143.98,139.55,133.55,129.81,129.74,129.64,129.52(2C),125.65,125.03(d,2JC,F＝13.8Hz),125.01,120.69,119.82(2C),115.47(d,2JC,F＝20.0Hz),111.89,106.81,67.62(2C),61.74,48.39(2C),12.63.HRMS(ESI)m/z calcd for[C28H27FN8O2+H]+:527.2241；found:527.2285[M+H]+.
Example 9
2- (6- ((2-Fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) -5-piperidinylpyrimidine-4, 6-diamine (Compound 9)
Figure BDA0002087451850000122
According to the synthesis method of the compound 7, the compound raw material 6- (2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b]Pyridine-5-formamidine and piperidinyl malononitrile as starting materials to give a white solid. Yield: 54%, melting point: 222.9-224.5 ℃, HPLC: 98.40 percent.1H NMR(500MHz,DMSO-d6):δ8.32(s,1H),8.13(d,J＝8.4Hz,2H,Ar-H),7.68(t,J＝7.6Hz,1H,Ar-H),7.51(t,J＝8.0Hz,2H,Ar-H),7.36～7.22(m,3H,Ar-H),7.18(t,J＝7.4Hz,1H,Ar-H),5.94(br s,4H,-NH2),5.63(s,2H,-CH2-),2.93(m,4H,-CH2-),2.54(s,3H,-CH3),1.65(m,4H,-CH2-),1.53(m,2H,-CH2-).13C NMR(126MHz,DMSO-d6):δ160.87,160.50,160.09(d,1JC,F＝242.5Hz),159.67,147.85,143.98,139.55,133.59,129.80,129.73,129.59(2C),129.53,125.65,125.04(d,2JC,F＝13.8Hz),125.01,120.73,119.80(2C),115.48(d,2JC,F＝21.3Hz),111.90,108.21,61.74,49.10(2C),27.31(2C),23.77,12.63.HRMS(ESI)m/z calcd for[C29H29FN8O+H]+:525.2448；found:525.2511[M+H]+.
Example 10
2- (6- ((2-Fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) -5-pyrrolidinylpyrimidine-4, 6-diamine ((Compound 10)
Figure BDA0002087451850000131
According to the synthesis method of the compound 7, the compound raw material 6- (2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b]Pyridine-5-formamidine and pyrrolidinyl malononitrile are used as raw materials to obtain a white solid. Yield: 46%, melting point: 216.9-219.2 ℃, HPLC: 97.45 percent.1H NMR(500MHz,DMSO-d6):δ8.32(s,1H),8.12(d,J＝7.9Hz,2H,Ar-H),7.67(t,J＝7.4Hz,1H,Ar-H),7.51(t,J＝7.7Hz,2H,Ar-H),7.37～7.25(m,3H,Ar-H),7.18(t,J＝7.3Hz,1H,Ar-H),5.95(br s,4H,-NH2),5.63(s,2H,-CH2-),3.02(s,4H,-CH2-),2.54(s,3H,-CH3),1.94(s,4H,-CH2-).13C NMR(126MHz,DMSO-d6):δ161.38,160.44,160.10(d,1JC,F＝242.5Hz),159.58,147.84,143.98,139.52,133.56,129.82,129.76,129.65,129.53(2C),125.66,125.02(d,2JC,F＝13.8Hz),124.99,120.63,119.79(2C),115.50(d,2JC,F＝21.3Hz),111.90,103.35,61.79,47.58(2C),25.69(2C),12.63.HRMS(ESI)m/z calcd for[C28H27FN8O+H]+:511.2292；found:511.2339[M+H]+.
Example 11
2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3, 4-b)]Pyridin-5-yl) -N5,N5-dimethylpyrimidine-4, 5, 6-triamine (Compound 11)
Figure BDA0002087451850000132
According to the synthesis method of the compound 7, the compound raw material 6- (2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b]Pyridine-5-formamidine and dimethylamino malononitrile as raw materials to obtain a white solid. Yield: 44%, melting point: 224.3-226.5 ℃, HPLC: 98.16 percent.1H NMR(500MHz,DMSO-d6):δ8.31(s,1H),8.13(d,J＝8.1Hz,2H,Ar-H),7.67(t,J＝7.0Hz,1H,Ar-H),7.51(t,J＝7.7Hz,2H,Ar-H),7.33(dd,J＝10.3,4.6Hz,1H,Ar-H),7.30～7.25(m,2H,Ar-H),7.18(m,1H t,J＝7.4Hz,1H,Ar-H),5.99(br s,4H,-NH2),5.63(s,2H,-CH2-),2.69(s,6H,-CH3),2.54(s,3H,-CH3).13C NMR(126MHz,DMSO-d6):δ160.81,160.46,160.08(d,1JC,F＝242.5Hz),159.63,147.83,143.97,139.54,133.54,129.79,129.72,129.62,129.52(2C),125.63,125.05(d,2JC,F＝13.8Hz),125.02,120.75,119.78(2C),115.47(d,2JC,F＝20.0Hz),111.89,107.87,61.71,41.19(2C),12.64.HRMS(ESI)m/z calcd for[C26H25FN8O+H]+:485.2135；found:485.2186[M+H]+.
Example 12
2- (6- ((3-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diamine compound 12)
Figure BDA0002087451850000141
According to the synthesis method of the compound 7, the compound raw material 6- (3-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b]Pyridine-5-formamidine and malononitrile were used as starting materials to give a white solid. Yield: 43%, melting point: 251.4-253.5 ℃, HPLC: 99.10 percent.1H NMR(400MHz,DMSO-d6):δ8.33(s,1H),8.17(dd,J＝8.7,1.0Hz,2H,Ar-H),7.57～7.51(m,2H,Ar-H),7.45～7.38(m,3H,Ar-H),7.29(m,1H,Ar-H),7.15～7.08(m,1H,Ar-H),6.21(br s,4H,-NH2),5.56(s,2H,-CH2-),5.44(s,1H),2.55(s,3H,-CH3).13C NMR(101MHz,DMSO-d6):δ164.33(2C),163.34,162.47(d,1JC,F＝242.0Hz),160.67,147.99,144.03,141.04,139.55,133.45,130.76,129.59(2C),125.61,123.14,120.75,119.88(2C),114.46(d,2JC,F＝21.0Hz),113.99(d,2JC,F＝22.0Hz),111.87,81.48,67.21,12.67.HRMS(ESI)m/z calcd for[C24H20FN7O+H]+:442.1713；found:442.1789[M+H]+.
Example 13
2- (6- ((4-Fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diamine (Compound 13)
Figure BDA0002087451850000151
According to the synthesis method of the compound 7, the compound raw material 6- (4-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b]Pyridine-5-formamidine and malononitrile were used as starting materials to give a white solid. Yield: 47%, melting point: 254.6-255.6 ℃, HPLC: 98.47 percent.1H NMR(500MHz,DMSO-d6):δ8.29(s,1H),8.19(d,J＝7.7Hz,2H,Ar-H),7.63(dd,J＝8.6,5.6Hz,2H,Ar-H),7.55(t,J＝8.0Hz,2H),7.30(t,J＝7.4Hz,1H,Ar-H),7.23～7.17(m,2H,Ar-H),6.15(br s,4H,-NH2),5.53(s,2H,-CH2-),5.42(s,1H),2.55(s,3H,-CH3).13C NMR(126MHz,DMSO-d6):δ164.43(2C),163.58,161.93(d,1JC,F＝241.3Hz),160.86,148.05,143.97,139.61,134.18,133.29,129.58(2C),129.57(d,2JC,F＝11.3Hz,2C),125.59,121.06,119.90(2C),115.49(d,2JC,F＝21.3Hz,2C),111.78,81.55,67.27,12.66.HRMS(ESI)m/z calcd for[C24H20FN7O+H]+:442.1713；found:442.1789[M+H]+.
Example 14
2- (3-methyl-1-phenyl-6- ((2-methyl) benzyloxy) -1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diamine (Compound 14)
Figure BDA0002087451850000152
According to the synthesis method of the compound 7, the compound raw material 3-methyl-6- ((2-methyl) benzyloxy) -1-phenyl-1H-pyrazolo [3,4-b]Pyridine-5-formamidine and malononitrile were used as starting materials to give a white solid. Yield: 52%, melting point: 227.2-228.0 ℃, HPLC: 98.36 percent.1H NMR(500MHz,DMSO-d6):δ8.26(s,1H),8.19(d,J＝8.1Hz,2H,Ar-H),7.53(m,3H,Ar-H),7.29(t,J＝7.4Hz,1H,Ar-H),7.18(m,3H,Ar-H),6.12(br s,4H,-NH2),5.53(s,2H,-CH2-),5.41(s,1H),2.54(s,3H,-CH3),2.40(s,3H,-CH3).13C NMR(126MHz,DMSO-d6):δ164.40(2C),163.69,160.95,148.06,143.95,139.65,136.60,135.67,133.12,130.33,129.53(2C),128.22,127.98,126.12,125.61,121.22,119.93(2C),111.66,81.58,66.58,19.12,12.68.HRMS(ESI)m/z calcd for[C25H23N7O+H]+:438.1964；found:438.2040[M+H]+.
Example 15
2- (3-methyl-1-phenyl-6- ((3-methyl) benzyloxy) -1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diamine (Compound 15)
Figure BDA0002087451850000161
According to the synthesis method of the compound 7, the compound raw material 3-methyl-6- ((3-methyl) benzyloxy) -1-phenyl-1H-pyrazolo [3,4-b]Pyridine-5-formamidine and malononitrile were used as starting materials to give a white solid. Yield: 54%, melting point: 237.2-239.8 ℃, HPLC: 98.23 percent.1H NMR(500MHz,DMSO-d6):δ8.27(s,1H),8.21(d,J＝7.8Hz,2H,Ar-H),7.54(t,J＝8.0Hz,2H,Ar-H),7.39(s,1H,Ar-H),7.35～7.27(m,2H,Ar-H),7.24(t,J＝7.6Hz,1H,Ar-H),7.08(d,J＝7.5Hz,1H,Ar-H),6.18(br s,4H,-NH2),5.49(s,2H,-CH2-),5.42(s,1H),2.55(s,3H,-CH3),2.30(s,3H,-CH3).13C NMR(126MHz,DMSO-d6):δ164.42(2C),163.58,160.98,148.09,143.96,139.64,137.81,137.77,133.18,129.58(2C),128.60,128.41,128.12,125.56,124.50,121.07,119.90(2C),111.70,81.52,67.98,21.51,12.67.HRMS(ESI)m/z calcd for[C25H23N7O+H]+:438.1964；found:438.2081[M+H]+.
Example 16
2- (3-methyl-1-phenyl-6- ((4-methyl) benzyloxy) -1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diamine (compound 16)
Figure BDA0002087451850000162
According to the synthesis method of the compound 7, the compound raw material 3-methyl-6- ((4-methyl) benzyloxy) -1-phenyl-1H-pyrazolo [3,4-b]Pyridine-5-formamidine and malononitrile were used as starting materials to give a white solid. Yield: 52%, melting point: 227.9-230.2 ℃, HPLC: 98.31 percent.1H NMR(500MHz,DMSO-d6):δ8.26(s,1H),8.20(d,J＝7.7Hz,2H,Ar-H),7.54(t,J＝8.0Hz,2H,Ar-H),7.43(d,J＝7.9Hz,2H,Ar-H),7.29(t,J＝7.4Hz,1H,Ar-H),7.17(d,J＝7.9Hz,2H,Ar-H),6.15(br s,4H,-NH2),5.49(s,2H,-CH2-),5.41(s,1H),2.54(s,3H,-CH3),2.28(s,3H,-CH3).13C NMR(126MHz,DMSO-d6):δ164.37(2C),163.57,160.99,148.08,143.94,139.63,136.93,134.85,133.14,129.59(2C),129.27(2C),127.57(2C),125.57,121.09,119.89(2C),111.66,81.52,67.92,21.20,12.66.HRMS(ESI)m/z calcd for[C25H23N7O+H]+:438.1964；found:438.2041[M+H]+.
Example 17
2- (3-methyl-1-phenyl-6- ((4-trifluoromethyl) benzyloxy) -1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diamine (Compound 17)
Figure BDA0002087451850000171
According to the synthesis method of the compound 7, the compound raw material 3-methyl-1-phenyl-6- ((4-trifluoromethyl) benzyloxy) -1H-pyrazolo [3,4-b]Pyridine-5-formamidine and malononitrile were used as starting materials to give a white solid. Yield: 47%, melting point: 254.6-255.6 ℃, HPLC: 98.47 percent.1H NMR(500MHz,DMSO-d6):δ8.29(s,1H),8.19(d,J＝7.7Hz,2H,Ar-H),7.63(dd,J＝8.6,5.6Hz,2H,Ar-H),7.55(t,J＝8.0Hz,2H),7.30(t,J＝7.4Hz,1H,Ar-H),7.23～7.17(m,2H,Ar-H),6.15(br s,4H,-NH2),5.53(s,2H,-CH2-),5.42(s,1H),2.55(s,3H,-CH3).13C NMR(126MHz,DMSO-d6):δ164.43(2C),163.58,161.93(d,1JC,F＝241.3Hz),160.86,148.05,143.97,139.61,134.18,133.29,129.58(2C),129.57(d,2JC,F＝11.3Hz,2C),125.59,121.06,119.90(2C),115.49(d,2JC,F＝21.3Hz,2C),111.78,81.55,67.27,12.66.HRMS(ESI)m/z calcd for[C24H20FN7O+H]+:442.1713；found:442.1789[M+H]+.
Example 18
2- (6-benzyloxy-3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diamine (compound 18)
Figure BDA0002087451850000172
According to the synthesis method of the compound 7, the compound raw material 6-benzyloxy-3-methyl-1-phenyl-1H-pyrazolo [3,4-b]Pyridine-5-formamidine and malononitrile were used as starting materials to give a white solid. Yield: 49%, melting point: 264.7-267.9 ℃, HPLC: 96.97 percent.1H NMR(500MHz,DMSO-d6):δ8.28(s,1H),8.17(d,J＝8.0Hz,2H,Ar-H),7.54(m,4H,Ar-H),7.37(t,J＝7.5Hz,2H,Ar-H),7.28(m,2H,Ar-H),6.19(br s,4H,-NH2),5.54(s,2H,-CH2-),5.42(s,1H),2.55(s,3H,-CH3).13C NMR(126MHz,DMSO-d6):δ164.34(2C),163.49,160.90,148.04,143.97,139.58,137.96,133.24,129.60(2C),128.72(2C),127.78,127.39(2C),125.59,120.99,119.88(2C),111.71,81.50,67.96,12.67.HRMS(ESI)m/z calcd for[C24H21N7O+H]+:424.1808；found:424.1886[M+H]+.
Example 19
2- (3-methyl-1-phenyl-6- (thiazole-2-methoxy) -1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diamine (Compound 19)
Figure BDA0002087451850000181
According to the synthesis method of the compound 7, the compound raw material 3-methyl-1-phenyl-6- (thiazole-2-methoxyl) -1H-pyrazolo [3,4-b]Pyridin-5-yl) formamidine and malononitrile were used as starting materials to give a white solid. Yield: 48%, melting point: 227.5-230.0 ℃, HPLC: 98.06 percent.1H NMR(500MHz,DMSO-d6):δ9.02(s,1H),8.30(d,J＝7.8Hz,2H,Ar-H),8.28(s,1H,Ar-H),8.04(s,1H,Ar-H),7.56(t,J＝8.0Hz,2H,Ar-H),7.31(t,J＝7.4Hz,1H,Ar-H),6.15(br s,4H,-NH2),5.79(s,2H,-CH2-),5.39(s,1H),2.56(s,3H,-CH3).13C NMR(126MHz,DMSO-d6):δ164.34(2C),163.31,160.36,155.87,147.78,144.05,143.74,139.57,134.32,133.59,129.66(2C),125.74,120.95,120.11(2C),112.04,81.52,60.69,12.67.HRMS(ESI)m/z calcd for[C21H18N8OS+H]+:431.1324；found:431.1418[M+H]+.
Example 20
2- (6- ((2-Fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) -5-nitrosopyrimidine-4, 6-diamine (Compound 20)
Figure BDA0002087451850000182
16.3g of the compound 6- (2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] was weighed]Putting pyridine-5-formamidine hydrochloride (0.039mol) into a 250mL eggplant-shaped bottle, measuring 120mL methanol, adding into a reaction bottle, and performing ultrasonic treatment for 3min to fully dissolve the raw materials, wherein the bottle is white after dissolutionThe color solution was stirred at room temperature. Weighing 8g of compound nitrosomalononitrile silver salt (0.039mol), slowly adding into a reaction bottle, changing the reaction bottle into a yellow solution in the feeding process, generating white precipitate in the solution after the feeding is finished, and finally replenishing 30mL of methanol to rinse the bottle mouth. After stirring the mixture at room temperature for reaction for 2 hours, no white precipitate is generated, and the reaction is stopped. The reaction solution was subjected to suction filtration to remove a white precipitate, and the filtrate was concentrated under reduced pressure and dried by rotary evaporation to obtain 18g of a yellow solid. Transferring 18g of the yellow solid to a 100mL eggplant type bottle, weighing 50mL of 2-methylpyridine, adding the 2-methylpyridine into the reaction bottle, completely dissolving the yellow solid, carrying out reflux reaction in an oil bath kettle at 130 ℃ for 0.5H, and then monitoring the reaction progress by TLC to find that the 2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazole [3,4-b ] is]Pyridine-5-pyridyl) formamidine has reacted completely and the reaction is stopped. The reaction solution was concentrated under reduced pressure at 70 ℃ and spin-dried to obtain 18.3g of a brownish black solid. Dissolving the crude solid with 200mL of ethyl acetate, mixing the crude solid with 80-100 meshes of silica gel, loading the mixture into a column with 200-300 meshes of silica gel, purifying by column chromatography, separating and purifying an elution system with petroleum ether and ethyl acetate of 2:1 to obtain 10.3g of dark green solid. Yield: 55%, HPLC: 98.69 percent.1H NMR(500MHz,CDCl3):δ10.18(s,1H,-NH2),9.21(s,1H,-NH2),8.56(s,1H,-NH2),8.53(s,1H),8.18(s,1H,-NH2),8.13(d,J＝7.9Hz,2H,Ar-H),7.70(t,J＝6.9Hz,1H,Ar-H),7.54(t,J＝7.9Hz,2H,Ar-H),7.31(m,3H,Ar-H),7.20(t,J＝7.5Hz,1H,Ar-H),5.65(s,2H,-CH2-),2.57(s,3H,-CH3).13C NMR(101MHz,DMSO-d6):δ164.42,163.52,160.55,160.10(d,1JC,F＝242.0Hz),147.88,144.01,139.55,133.43,129.84,129.76,129.65,129.54(2C),125.65,125.00,124.98(d,2JC,F＝13.0Hz),121.04,119.82(2C),115.48(d,2JC,F＝21.0Hz),111.88,81.53,61.75,12.67.HRMS(ESI)m/z calcd for[C24H19FN8O2+H]+:471.1615；found:471.1690[M+H]+.
Example 21
2- (6- ((2-Fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 5, 6-triamine (Compound 21)
Figure BDA0002087451850000191
10g of the compound 2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3, 4-b) was weighed]Putting pyridine-5-yl) -5-nitrosopyrimidine-4, 6-diamine (0.021mol) into a 250mL eggplant-shaped bottle, then weighing 90mL ethyl acetate, adding into a reaction bottle, carrying out ultrasonic treatment for 3min to obtain a dark green turbid solution in the bottle, stirring at room temperature, weighing 14g reduced zinc powder (0.21mol), slowly adding into the reaction bottle, and finally adding 30mL glacial acetic acid to rinse the bottle mouth. After stirring the reaction at room temperature for 12 hours, the progress of the reaction was monitored by TLC, and the starting material was found to have reacted completely, and the reaction was stopped. And (3) filtering, removing residual zinc powder in the reaction liquid and zinc acetate solids generated in the reaction process, and concentrating and spin-drying the filtrate at 60 ℃ under reduced pressure to obtain 11.2g of gray solids. Dissolving the crude solid with 140mL of methanol, mixing with 80-100 mesh silica gel, loading the mixture into a column with 200-300 mesh silica gel, purifying by column chromatography, separating and purifying an elution system with dichloromethane and methanol at a ratio of 30:1 to obtain 6.5g of yellow solid. Yield: 67%, melting point: 242.8-245.2 ℃, HPLC: 98.31 percent.1H NMR(500MHz,DMSO-d6):δ8.25(s,1H),8.16(d,J＝7.8Hz,2H,Ar-H),7.70(t,J＝7.2Hz,1H,Ar-H),7.51(t,J＝8.0Hz,2H,Ar-H),7.36～7.32(m,1H,Ar-H),7.30～7.25(m,2H,Ar-H),7.19(t,J＝7.4Hz,1H,Ar-H),5.76(br s,4H,-NH2),5.61(s,2H,-CH2-),3.97(br s,2H,-NH2),2.53(s,3H,-CH3).13C NMR(126MHz,DMSO-d6):δ160.68,160.10(d,1JC,F＝242.5Hz),152.57,151.95(2C),147.69,143.84,139.62,133.14,129.80,129.73,129.53(2C),125.57,125.05(d,2JC,F＝13.8Hz),125.02,121.04,119.75(2C),115.45(d,2JC,F＝20.0Hz),111.95,106.02,61.71,12.69.HRMS(ESI)m/z calcd for[C24H21FN8O+H]+:457.1822；found:457.1907[M+H]+.
Example 22
N- (4, 6-diamino-2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) acetamide (compound 22)
Figure BDA0002087451850000201
0.5g of the compound 2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3, 4-b) is weighed]Putting pyridine-5-yl) pyrimidine-4, 5, 6-triamine (1.1mmol) into a 100mL eggplant-shaped bottle, then measuring 40mL acetonitrile, adding into a reaction bottle, carrying out ultrasonic treatment for 3min, stirring at room temperature, weighing 0.5g anhydrous potassium carbonate (3.6mmol), slowly adding into the reaction bottle, transferring a reaction device into a cold trap, controlling the temperature to be 0-5 ℃, weighing 0.14g acetyl chloride (1.7mmol) by a decrement method, slowly adding into the reaction bottle, and finally replenishing 10mL acetonitrile to rinse the bottle mouth. After the reaction is stirred in a cold trap for 5 hours, the reaction progress is monitored by TLC, a product with lower polarity is found to be generated, but the raw materials are not completely reacted, then the reaction device is transferred to room temperature to continue stirring for reaction for 4 hours, the reaction progress is monitored by TLC, the reaction is found to be complete, and the reaction is stopped. Suction filtration was carried out to remove potassium carbonate, and the filtrate was concentrated under reduced pressure at 40 ℃ and spin-dried to give 0.6g of a tan solid. Dissolving the crude solid with 50mL of methanol, mixing with 80-100 mesh silica gel, loading the mixture into a column with 200-300 mesh silica gel, purifying by column chromatography, separating and purifying an elution system with dichloromethane and methanol at a ratio of 30:1 to obtain 0.35g of white solid. Yield: 64%, melting point: 256.2-259.5 ℃, HPLC: 98.62 percent.1H NMR(500MHz,DMSO-d6):δ8.61(s,1H,-NH-),8.33(s,1H),8.15(d,J＝7.8Hz,2H,Ar-H),7.68(t,J＝7.2Hz,1H,Ar-H),7.55～7.47(t,J＝7.9Hz,2H,Ar-H),7.36～7.25(m,3H,Ar-H),7.18(t,J＝7.4Hz,1H,Ar-H),6.05(br s,4H,-NH2),5.63(s,2H,-CH2-),2.56(s,3H,-CH3),2.04(s,3H,-CH3).13C NMR(126MHz,DMSO-d6):δ169.95,160.99,160.49,160.40,160.08(d,1JC,F＝242.5Hz),147.88,143.98,139.53,133.41,129.77,129.70,129.63,129.53(2C),125.67,125.04,125.02(d,2JC,F＝13.8Hz),120.80,119.82(2C),115.46(d,2JC,F＝20.0Hz),111.87,94.75,61.76,23.64,12.65.HRMS(ESI)m/z calcd for[C26H23FN8O2+H]+:499.1928；found:499.2011[M+H]+.
Example 23
Methyl (4, 6-diamino-2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidin-5-yl) carbamate (compound 23)
Figure BDA0002087451850000211
1g of the compound 2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3, 4-b) is weighed]Pyridine-5-yl) pyrimidine-4, 5, 6-triamine (2.2mmol) is put into a 100mL eggplant-shaped bottle, then 40mL pyridine is weighed and added into a reaction bottle, ultrasonic treatment is carried out for 3min, the bottle is a reddish brown solution, and stirring is carried out for 1h at room temperature. Then the reaction device is transferred to a cold trap, the temperature is controlled to be 0 to-5 ℃, 0.4mL (3.3mmol) of methyl chloroformate is measured by a 0.5mL pipette and slowly added into a reaction bottle, white smoke is generated in the feeding process, and finally 10mL of pyridine is added to rinse the bottle mouth. After stirring the reaction in a cold trap for 4 hours, monitoring the reaction process by TLC, finding that a product with lower polarity is generated, finding that the reaction is complete, and stopping the reaction. The reaction solution was concentrated under reduced pressure at 60 ℃ and spin-dried to obtain 1.2g of a reddish brown solid. Dissolving the crude solid with 60mL of methanol, mixing with 80-100 mesh silica gel, loading the mixture into a column with 200-300 mesh silica gel, purifying by column chromatography, separating and purifying an elution system with dichloromethane and methanol at a ratio of 30:1 to obtain 0.8g of light yellow solid. Yield: 71%, melting point: 235.4-238.6 ℃, HPLC: 99.64 percent.1H NMR(500MHz,DMSO-d6):δ8.31(s,1H),8.14(d,J＝7.9Hz,2H,Ar-H),7.99(br s,1H,-NH-),7.68(t,J＝7.3Hz,1H,Ar-H),7.51(t,J＝8.0Hz,2H,Ar-H),7.38～7.24(m,3H,Ar-H),7.19(t,J＝7.4Hz,1H,Ar-H),6.06(br s,4H–NH2),5.63(s,2H,-CH2-),3.64(s,3H,-OCH3),2.54(s,3H,-CH3).13C NMR(126MHz,DMSO-d6):δ161.02,160.74,160.48,160.11(d,1JC,F＝242.5Hz),155.66,147.90,143.99,139.54,133.41,129.80,129.74,129.70,129.53(2C),125.67,125.03,125.00(d,2JC,F＝13.8Hz),120.74,119.83(2C),115.47(d,2JC,F＝21.3Hz),111.86,94.31,61.73,52.22,12.65.HRMS(ESI)m/z calcd for[C26H23FN8O3+H]+:515.1877；found:515.1963[M+H]+.
Example 24
N-butyl (4, 6-diamino-2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidin-5-yl) carbamate (compound 24)
Figure BDA0002087451850000221
According to the synthesis method of compound 23, 2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3, 4-b)]Pyridin-5-yl) pyrimidine-4, 5, 6-triamine and n-butyl chloroformate were used as starting materials to give a pale yellow solid. Yield: 70%, melting point: 224.6-227.1 ℃, HPLC: 98.23 percent.1H NMR(500MHz,DMSO-d6):δ8.32(s,1H),8.14(d,J＝7.9Hz,2H,Ar-H),7.97(br s,1H,-NH-),7.68(t,J＝7.3Hz,1H,Ar-H),7.51(t,J＝7.9Hz,2H,Ar-H),7.37～7.25(m,3H,Ar-H),7.18(t,J＝7.4Hz,1H,Ar-H),6.02(br s,4H–NH2),5.63(s,2H,-CH2-),4.04(m,3H,-OCH2-),2.54(s,3H,-CH3),1.58(m,2H,-CH2-),1.46～1.36(m,2H,-CH2-),1.27～1.22(m,3H,-CH3).13C NMR(101MHz,DMSO-d6):δ160.40,160.26,160.12(d,1JC,F＝242.0Hz),159.98,155.33,148.00,144.10,139.47,133.72,129.85,129.76,129.72,129.51(2C),125.70,125.03,125.00,124.90(d,2JC,F＝14.0Hz),119.82(2C),115.48(d,2JC,F＝20.0Hz),111.95,94.27,64.64,61.88,31.12,19.14,14.18,12.66.HRMS(ESI)m/z calcd for[C29H29FN8O3+H]+:557.2347；found:557.2439[M+H]+.
Example 25
Cyclopentyl (4, 6-diamino-2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidin-5-yl) carbamate (compound 25)
Figure BDA0002087451850000222
According to the synthesis method of compound 23, 2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3, 4-b)]Pyridine-5-yl) pyrimidine-4, 5, 6-triamine and cyclopentyl chloroformate are used as raw materials to obtain a light yellow solid. Yield: 52%, melting point: 207.5-210.2 ℃, HPLC: 98.86 percent.1H NMR(400MHz,DMSO-d6):δ8.31(s,1H),8.13(d,J＝7.9Hz,2H),7.91(br s,1H,-NH-),7.67(t,J＝7.3Hz,1H,Ar-H),7.51(t,J＝8.0Hz,2H,Ar-H),7.36～7.25(m,3H,Ar-H),7.21～7.16(m,1H,Ar-H),6.01(br s,4H–NH2),5.63(s,2H,-CH2-),5.05(m,1H,-CH-),2.54(s,3H,-CH3),1.90～1.50(m,8H,-CH2-).13C NMR(126MHz,DMSO-d6):δ160.87,160.62,160.47,160.11(d,1JC,F＝243.8Hz),155.06,147.89,143.98,139.54,133.42,129.79,129.73,129.68,129.52(2C),125.66,125.02,124.99(d,2JC,F＝12.5Hz),120.70,119.81(2C),115.46(d,2JC,F＝20.0Hz),111.86,94.64,77.23,61.77,32.82(2C),23.91(2C),12.65.HRMS(ESI)m/z calcd for[C30H29FN8O3+H]+:569.2347；found:569.2434[M+H]+.
Example 26
Inhibition experiment of compound on proliferation of human pulmonary artery smooth muscle cells and human lung fibroblasts
The inhibition rate of the synthesized compound on human pulmonary artery smooth muscle cell lines (HPASMCs) and human pulmonary fibroblast cell lines (HLF1) is determined by CCK-8 method with Riociguat as positive control, and IC of each compound is calculated50The value is obtained.
(1) Experimental materials:
a. cell lines: human lung fibroblast cell line and human pulmonary artery smooth muscle cell line (purchased from Nanjing Enjinjing Biotechnology Ltd., cultured in RPMI1640 medium containing 10% fetal bovine serum).
b. Drugs and reagents: 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfonated benzene) -2H-tetrazole monosodium salt (CCK-8) and dimethyl sulfoxide (DMSO) were purchased from Sigma; 96-well cell culture plates were purchased from Costar corporation; pancreatin, newborn bovine serum, fetal bovine serum, RPMI-1640, DMEM and penicillin-streptomycin were purchased from Gibco; EnoGeneCellTMA Counting Kit-8(CCK-8) cell viability detection Kit (E1CK-000208-10) is purchased from Nanjing Enjing Biotech, Inc.; paclitaxel injection, tai chi group sichuan tai chi pharmaceutical co ltd, 5mL:30mg, batch No.: 14121201, respectively; other reagents were purchased from Sigma, unless otherwise specified.
c. Instruments and consumables: a ChemBase CBS-CJ-1FD superclean workbench; MCO-15AC carbon dioxide incubator (SANYO, Sanyo, Japan); XD-202 fluorescence inverted biomicroscopy (Yongxin optics, Inc. of south Jing Jiangnan); thermo MK3 microplate reader (us thermoelectric).
(2) The method comprises the following operation steps:
a. according to CCK-8 staining method, cell with living cell proportion of more than 90% is taken for experiment, and EnoGeneCell is adopted for cell proliferation inhibition experimentTMCounting Kit-8(CCK-8) cell viability detection Kit.
b. Digesting and counting cells to obtain a concentration of 1 × 105Cell suspension/mL, 100. mu.L of cell suspension per well in 96-well plates (1X 10 per well)4Individual cells).
c.96 well plates at 37 ℃ 5% CO2Culturing in an incubator for 24 hours; adding 100 μ L of corresponding culture medium containing medicine into each well, and simultaneously setting up a negative control group, a solvent control group, a positive control group, and 5 more wells per group.
d.96 well plates were placed at 37 ℃ in 5% CO2Culturing for 72 hours in an incubator; add 10. mu.L of CCK-8 solution to each well, incubate the plate in the incubator for 4 hours, carefully aspirate the medium from the wells.
e. Measuring OD value at 450nm with enzyme labeling instrument, and respectively calculating inhibition rate and IC of the compound on human lung fibroblast and human pulmonary artery smooth muscle cell50The value is obtained.
f. Dose setting: dose 1 group: 100 mu M; dose 2 groups: 50 mu M; dose 3 groups: 25 mu M; dose 4 groups: 12.5 mu M; dose 5 groups: 6.25 mu M; dose 6 groups: 3.125 μ M; dose 7 groups: 1.5625 μ M; dose 8 groups: 0.78125 μ M; dose 9 groups: 0.390625 μ M; dose 10 groups: 0.1953125 μ M.
(3) Data processing:
data are expressed as mean and SD using GraphPad Prism 5.0 statistical software and comparison between groups using t-test. The IC of the compound was calculated by fitting a curve with the concentration as abscissa and the inhibition as ordinate50The value is obtained. Measuring the absorbance OD value of each hole at 450nm on a microplate reader, and calculating the cell growth inhibition rate according to the following formula:
Figure BDA0002087451850000241
TABLE 1 inhibition of HPASMCs and HLF1 cell proliferation by Compounds
Figure BDA0002087451850000242
Figure BDA0002087451850000251
Figure BDA0002087451850000261
The experimental results show that: after a pyrimidine ring is introduced on the basis of the compound I-9 (patent 201810520915.3), the proliferation effect of the compound on HPASMCs and HLF1 cell strains can be obviously improved. The compound protected by the patent has obvious anti-fibrosis effect and can be better applied to treating pulmonary fibrosis diseases.
Example 27
Agonism of Compounds on sGC enzyme Activity
A compound (compound 14) with good anti-fibrosis activity is selected to carry out an sGC enzyme agonistic activity experiment, and the effect of the target compound on sGC is examined by calculating the relative fold of increase of the cGMP concentration level.
The experimental principle is as follows:
the effect of compounds on cGMP enzyme levels in Human pulmonary artery smooth muscle cells (hpasmic) was tested using HTRF technology using HTRF cGMP kit drug discovery kit (Cat No.62GM2PEG, Cisbio) with Riociguat and Sildenafil as positive control drugs. HPASMC cells were cultured in 15% FBS-containing high-sugar DMEM medium at 37 ℃ for 48 hours, after which the cells were collected and the cell concentration was diluted to about 500 cells/5. mu.L with the medium for use. Each compound was initially dissolved in DMSO at 10mM, and then diluted to 10 μ M with the dilution in the kit. mu.L of cells (. about.500) and 5. mu.L of compound (10. mu.M) were added to a 384-well plate, and after incubation at 37 ℃ for 0.5h, 5. mu.L of a cGMP donor labeled with d2 and 5. mu.L of a cGMP antibody coated with a cryptate of europium were added to the reaction system, the mixture was incubated at room temperature for 1h, and data at excitation wavelength 620nm and emission wavelength 665nm were collected on an HTRF multifunctional microplate reader.
The Ratio (Ratio) at two wavelengths was calculated as (Signal 665nm)/(Signal 665nm) 100%,
further by calculating Delta F (100%) (Ratio Standard sample-Ratio reactive Control)/(Ratio reactive Control) 100
To evaluate the effect of the compounds in increasing intracellular cGMP levels, two duplicate wells were set for each experimental group, and the average Delta F value was calculated in three independent replicates.
The specific data are shown in fig. 1, and the test results show that: compound 14 increased cGMP at lower levels with and without significant differences from Riociguat when no PDE-5 inhibitor IBMX was added, regardless of whether sGC inhibitor ODQ was added (fig. 1-a), mainly due to increased cGMP in pulmonary artery smooth muscle cells being degraded by intracellular phosphodiesterase-5 (PDE-5); after adding PDE-5 inhibitor IBMX, the compound 14 and the Ricociguat significantly increase the cGMP level in pulmonary artery smooth muscle cells, and the ODQ experiment group added with the sGC inhibitor has more significant increase of the cGMP level compared with the ODQ experiment group not added (figure 1-B), which shows that the compound 14 and the Ricociguat have the same action mechanism, increase of the intracellular cGMP generation by activating sGC, and the capability of increasing the cGMP of the Ricociguat is obviously stronger than that of the compound 14.
The experimental results also show that: the former patent, compound I-9 (patent 201810520915.3), was unable to increase cGMP levels in pulmonary artery smooth muscle cells, but compound 14 significantly increased cGMP levels in pulmonary artery smooth muscle cells when a pyrimidine ring was introduced based on the pyrazolo [3,4-b ] pyridine nucleus. Further illustrating the claimed structure of the present application is an essential group for activating sGC, and thus promoting cGMP production.
The foregoing examples are set forth to illustrate the present invention more clearly and are not to be construed as limiting the scope of the invention, which is defined in the appended claims to which the invention pertains, as modified in all equivalent forms, by those skilled in the art after reading the present invention.

Claims (8)

1. A5-pyrimidine-6-oxygen-pyrazolopyridine derivative and a pharmaceutically acceptable salt thereof are characterized in that the structural general formula is shown as formula (II):
Figure FDA0003128059970000011
wherein R is1Or R2Selected from hydrogen, methyl, trifluoromethyl or an aromatic ring;
R3selected from aromatic rings, substituted aromatic rings or aromatic heterocycles;
the aromatic ring is benzene; the aromatic heterocyclic ring is thiazole;
the substituted aromatic ring is a substituted benzene ring; the substituent is selected from methyl, trifluoromethyl or halogen;
x and Y are selected from hydroxyl and amino;
R4selected from hydrogen, the following structural fragment:
-NO-NH2
Figure FDA0003128059970000012
wherein R is5Is selected from C1-C6Fatty chain, C3-C6An alicyclic ring; z is selected from CH2、O。
2. The 5-pyrimidine-6-oxo-pyrazolopyridine derivative according to claim 1, wherein the pharmaceutically acceptable salt is selected from the group consisting of: hydrochloride, sulfate, phosphate, perchlorate, methanesulfonate, trifluoromethanesulfonate, formate, acetate, propionate, butyrate, maleate, succinate, trifluoroacetate, succinate, salicylate, DL-aspartate, D-aspartate, L-aspartate, DL-glutamate, D-glutamate, L-glutamate, glycerate, stearate, DL-tartrate, D-tartrate, L-tartrate, (±) mandelate, (R) - (-) mandelate, (S) - (+) mandelate, citrate, mucate, malonate, benzoate, DL-malate, (±) lactate, L- (+) -lactate, D- (+) -lactate, succinate, maleate, citrate, maleate, lactate, maleate, citrate, maleate, lactate, citrate, lactate, and acetate, Pamoate, D-alpha-galacturonate, glycerate, DL-cysteine, D-cysteine, L-cysteine, (4S) -hydroxy-L-prolinate, cyclopropane-1, 1-dicarboxylate, 2-methylmalonate, caseinate, prolinate, fumarate, 1-hydroxy-2-naphthoate, phosphonoacetate, carbonate, bicarbonate, 3-phosphonopropionate, DL-pyroglutamate, D-pyroglutamate, L-pyroglutamate, p-toluenesulfonate, benzenesulfonate, ethanesulfonate, (+/-) camphorsulfonate, naphthalenesulfonate, 1R- (-) -camphorsulfonate, 1S- (+) -camphorsulfonate, glycerate, DL-cysteine, D-L-cysteine, L-hydroxy-2-naphthoate, phosphonoacetate, carbonate, bicarbonate, 3-phosphonopropionate, DL-pyroglutamate, D-pyroglutamate, L-pyroglutamate, p-toluenesulfonate, benzenesulfonate, ethanesulfonate, camphorsulfonate, camphor sulfonate, camphor sulfonate, camphor, or alcohol, camphor, and water, camphor, or alcohol, camphor, water, 1, 5-naphthalenedisulfonate, 1, 2-ethanedisulfonate, 1, 3-propanedisulfonate, 3- (N-morpholino) propanesulfonate, biphenylsulfonate, isethionate, 1-hydroxy-2-naphthalenesulfonate, dihydrogenphosphate, potassium hydrogenphosphate, dipotassium phosphate, potassium phosphate, sodium hydrogenphosphate, disodium phosphate, sodium phosphate, calcium triphosphate, hexafluorophosphate, vinylphosphate, 2-hydroxyethyl phosphate and phenylphosphate.
3. The 5-pyrimidine-6-oxo-pyrazolopyridine derivative according to claim 1 or 2, wherein the derivative has the following structure:
2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diol
2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) -5-morpholinopyrimidine-4, 6-diol
2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) -5-piperidinylpyrimidine-4, 6-diol
2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) -5-pyrrolidinylpyrimidine-4, 6-diol
5- (dimethylamino) -2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diol
5- (diethylamino) -2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diol
2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diamine
2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) -5-morpholinopyrimidine-4, 6-diamine
2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) -5-piperidinylpyrimidine-4, 6-diamine
2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) -5-pyrrolidinylpyrimidine-4, 6-diamine
2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) -N5, N5-dimethylpyrimidine-4, 5, 6-triamine
2- (6- ((3-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diamine
2- (6- ((4-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diamine
2- (3-methyl-1-phenyl-6- ((2-methyl) benzyloxy) -1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diamine
2- (3-methyl-1-phenyl-6- ((3-methyl) benzyloxy) -1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diamine
2- (3-methyl-1-phenyl-6- ((4-methyl) benzyloxy) -1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diamine
2- (3-methyl-1-phenyl-6- ((4-trifluoromethyl) benzyloxy) -1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diamine
2- (6-benzyloxy-3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diamine
2- (3-methyl-1-phenyl-6- (thiazole-2-methoxy) -1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 6-diamine
2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) -5-nitrosopyrimidine-4, 6-diamine
2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidine-4, 5, 6-triamine
N- (4, 6-diamino-2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) acetamide
Methyl (4, 6-diamino-2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidin-5-yl) carbamate
N-butyl (4, 6-diamino-2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidin-5-yl) carbamate
Cyclopentyl (4, 6-diamino-2- (6- ((2-fluorobenzyloxy) -3-methyl-1-phenyl-1H-pyrazolo [3,4-b ] pyridin-5-yl) pyrimidin-5-yl) carbamate.
4. A preparation method of 5-pyrimidine-6-oxygen-pyrazolopyridine derivatives is characterized in that R4Is composed of
Figure FDA0003128059970000031
The method comprises the following specific steps:
s1, aminolysis of cyano in the structure into hydroxyamidine by the compound I under the action of hydroxylamine hydrochloride to obtain a compound III;
s2, reducing the compound III by zinc powder to obtain a compound IV;
s3, Compound IV and R4Reacting substituted diethyl malonate to obtain a compound V;
the reaction scheme is as follows:
Figure FDA0003128059970000032
wherein R is1、R2、R3As defined in claim 1.
5. A preparation method of 5-pyrimidine-6-oxygen-pyrazolopyridine derivatives is characterized in that R4Is composed of
Figure FDA0003128059970000033
The method comprises the following specific steps:
s1, aminolysis of cyano in the structure into hydroxyamidine by the compound I under the action of hydroxylamine hydrochloride to obtain a compound III;
s2, reducing the compound III by zinc powder to obtain a compound IV;
s3, Compound IV and R4Reacting substituted malononitrile to obtain a compound VI;
the reaction scheme is as follows:
Figure FDA0003128059970000034
wherein R is1、R2、R3As defined in claim 1.
6. A preparation method of 5-pyrimidine-6-oxygen-pyrazolopyridine derivatives is characterized in that R4is-NO-NH2
Figure FDA0003128059970000041
The method comprises the following specific steps:
s1, aminolysis of cyano in the structure into hydroxyamidine by the compound I under the action of hydroxylamine hydrochloride to obtain a compound III;
s2, reducing the compound III by zinc powder to obtain a compound IV;
s3, reacting the compound IV with hydrogen chloride gas at room temperature to obtain a compound VII;
s4, reacting the compound VII with nitrosomalononitrile silver salt to obtain a compound VIII;
s5, reducing the compound VIII with zinc powder to obtain a compound IX;
s6, reacting the compound IX with different substituted acyl chloride compounds under the alkaline condition to obtain a compound X;
the reaction scheme is as follows:
Figure FDA0003128059970000042
wherein R is1、R2、R3As defined in claim 1.
7. Use of 5-pyrimidine-6-oxo-pyrazolopyridine derivatives according to any of claims 1-3 and pharmaceutically acceptable salts thereof for the preparation of sGC agonists.
8. Use of 5-pyrimidine-6-oxo-pyrazolopyridine derivatives according to any of claims 1-3 and pharmaceutically acceptable salts thereof for the manufacture of a medicament for the treatment of pulmonary fibrosis.
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