CN110511222A - Pyrazolo-pyridines and its purposes in treatment diabetes - Google Patents

Pyrazolo-pyridines and its purposes in treatment diabetes Download PDF

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Publication number
CN110511222A
CN110511222A CN201910877861.0A CN201910877861A CN110511222A CN 110511222 A CN110511222 A CN 110511222A CN 201910877861 A CN201910877861 A CN 201910877861A CN 110511222 A CN110511222 A CN 110511222A
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compound
pyrazolo
pyridines
pharmaceutically acceptable
acceptable salt
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王广忠
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2013—Organic compounds, e.g. phospholipids, fats
    • A61K9/2018—Sugars, or sugar alcohols, e.g. lactose, mannitol; Derivatives thereof, e.g. polysorbates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2022—Organic macromolecular compounds
    • A61K9/205—Polysaccharides, e.g. alginate, gums; Cyclodextrin
    • A61K9/2054—Cellulose; Cellulose derivatives, e.g. hydroxypropyl methylcellulose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems

Abstract

The invention discloses the pyrazolo-pyridines as shown in compound 1 or its pharmaceutically acceptable salt,

Description

Pyrazolo-pyridines and its purposes in treatment diabetes
Technical field
The present invention relates to field of medicaments, and in particular to pyrazolo-pyridines and its use in treatment diabetes On the way.
Background technique
Diabetes are a kind of metabolic diseases of multi-pathogenesis, since the absolute or relative deficiency of insulin causes blood glucose rise So as to cause organism metabolic disorder.Diabetes can be divided into two kinds of insulin-dependent (I type) and non-insulin-depending type (II type) Type, wherein type-2 diabetes mellitus is most commonly seen, accounts for 90% of diabetic or more.
According to the difference of function and effect, oral hypoglycemic agents can be divided into the mainly medicine to promote insulin secretion as main function Object (sulfonylureas, meglitinide, DPP-IV inhibitor) and drug (biguanides, thiazolidine two that blood glucose is reduced by other mechanism Ketone, alpha-glucosidase inhibitor).Sulfonylureas and meglitinide directly stimulate islet β cell insulin；DPP-4 inhibitor Decomposition by reducing internal GLP-1 increases GLP-1 concentration and and then promotes islet β cell insulin；Biguanides Main pharmacological is to reduce the output of liver glucose；The main pharmacological of thiazolidinediones is to improve insulin to support It is anti-；The main pharmacological of alpha-glucosidase inhibitor is the digestion and absorption for delaying carbohydrate in enteron aisle.
DPP-IV is that one kind is distributed widely in the intracorporal glycoprotein of people, serine protease is functionally similar to, by right The shearing of polypeptide makes its inactivation, to have the function that regulation of physiological functions.DPP-IV is constant to the shearing position of substrate, is The proline or alanine of its N-terminal penultimate.GLP-1 is a kind of endogenic hormone, with the raising of postprandial blood sugar, by L- cell in small intestine secretes the secretion for generating, and then stimulating insulin.Therefore, the secretion of GLP-1 and the intake of blood glucose are close Cut phase is closed.Therapeutic scheme based on GLP-1 can efficiently control blood glucose without putting on weight, and it is bad will not to generate hypoglycemia etc. Reaction.Substrate of the GLP-1 as DPP-IV, half-life period is very short, is just sheared rapidly after secretion in 1-2min, inactivates by DPP-IV, Even become antagonist.Therefore, the small-molecule chemical antidiabetic for inhibiting DPP-IV can be researched and developed based on the mechanism of action.
China is the most country of global diabetic's number, and the DPP-IV inhibitor listed at home at present has western lattice Arrange spit of fland, saxagliptin, vildagliptin, Li Gelieting and Egelieting.Past 30 Years come, and diabetes mellitus in China illness rate sharply increases Add: 1980 were 5.5% less than 1%, 2001 year, and 2008 are 9.7%, and 2013 are 10.9%.The elderly, male, city Resident, developed area resident, overweight and overweight people diabetes prevalence are higher.Therefore, new treatment diabetes are found Drug has important scientific meaning and economic value, and is inhibiting about the pyrazolo-pyridines that the present invention mentions Research is not yet reported that so far in terms of the anti-diabetic of DPP-IV.
Summary of the invention
The present invention provides a kind of pyrazolo-pyridines, structure is shown in formula I:
Wherein R1、R2It is independently selected from hydrogen or methyl.
Further, the pyrazolo-pyridines Formulas I pharmaceutically acceptable salt.
Further, the pyrazolo-pyridines Formulas I is selected from compound 1, compound 2, compound 3, and structure is such as Shown in lower:
On the other hand, the change similar the present invention provides a kind of structure of pyrazolo-pyridines shown in formula I Object 4 is closed, 4 structure of compound is as follows:
The compound 1, compound 2, compound 3 and compound 4 structured data and its synthetic method referring to embodiment Part.
The present invention also provides the screening of the external activity of the compound, the oral glucose tolerance of mouse (OGTT) are real It tests, the research of pharmacokinetic profile and hERG Inhibition test.
The composition of pyrazolo-pyridines Formulas I tablet formulation of the present invention are as follows: the pyrazolopyridines chemical combination Object Formulas I or its pharmaceutically acceptable salt 0.1-10 parts, 3-30 parts of disintegrating agent, 20-90 parts of filler, 2-70 parts of stabilizer, 0.5-5 parts of lubricant.Suitable amount of adhesive can also be added except above-mentioned prescription.The auxiliary material for wherein playing disintegrating agent is low takes For hydroxypropyl cellulose (L-HPC), sodium carboxymethyl starch (CMS-Na), sodium carboxymethylcellulose (CMC-Na), crospovidone (PVPP) one kind, two or more composition formed with certain proportion.Play the auxiliary material of filler wherein as cream Sugar, mannitol, xylitol, maltose alcohol, dextrin, pregelatinized starch, one kind of microcrystalline cellulose (MCC), two kinds or two kinds The composition formed above with certain proportion.The auxiliary material for wherein playing stabilizer function is starch, povidone (PVP), copolyvidone Deng or their various combinations.Wherein the auxiliary material of super fatting agent effect is magnesium stearate, zinc stearate, polyethylene glycol (PEG), sugarcane Sugar fatty acid ester, sodium stearyl fumarate, stearic acid, talcum powder, silica one kind, two or more to be centainly to compare The composition of example composition.The auxiliary material for wherein playing adhesive is water, ethyl alcohol, hydroxypropyl methyl cellulose (HPMC), ethyl Cellulose (EC), one kind of povidone (PVP) or starch, two or more mixture.Other, which also may be selected, has filling The filler of effect, other disintegrating agents with calving disaggregation, other with the stabilizer of stabilization and other there is lubrication The lubricant of effect is applied in the present invention.
Preparation method of the invention includes the steps that following sequence:
(1) the pyrazolo-pyridines Formulas I is uniformly mixed with stabilizer.
(2) above-mentioned mixed-powder is mediated with adhesive, is pelletized, whole grain after drying；
(3) gained particle is uniformly mixed with rest materials, tabletting.
Or prepared by the step of taking following sequence
(1) that the pyrazolo-pyridines Formulas I is dissolved in adhesive with stabilizer is spare.
(2) it is mediated with disintegrating agent, filler in above-mentioned adhesive and prescription, granulation, whole grain after drying；
(3) lubricant is added, is uniformly mixed, tabletting.
Or prepared by the step of taking following sequence
(1) it is crushed after the pyrazolo-pyridines Formulas I being prepared into solid dispersions with stabilizer.
(2) piece is prepared with conventional wet lay granulating process or direct tablet compressing technique after mixing solid dispersions with rest materials Agent.
Specific embodiment
Embodiment 1: the synthesis of compound 1
Under 0 DEG C, condition of nitrogen gas, the dry THF of 2- benzyl oxygroup -3,3- dihydro-pyrazolo [1,5-a] pyridine-3-carboxylic acid Methylene chloride (3mmoL) solution of the drying of 2mmol/L oxalyl chloride and DMF (1 drop) solution of drying are added dropwise in (20mL) solution. It is added dropwise, reaction solution reacts at room temperature 2h.It is concentrated under reduced pressure, residue is dissolved in dry THF (10mL, this step repetition three It is secondary), obtained acyl chlorides is immediately using being not required to be further purified.Under condition of nitrogen gas, by trimethyl aluminium (2M hexane solution, It 1.5mmoL) is added in dry toluene (15mL) solution of 2,3,5,6- tetra- fluoro- 4- phenylanilines (1.1mmoL).React liquid chamber It is transferred in dry toluene (30mL) solution of acyl chlorides obtained above after temperature stirring 2h.After 90 DEG C of heating stirrings of reaction solution are stayed overnight It is cooled to room temperature, the dilute hydrochloric acid quenching reaction of 1mmoL, liquid separation is added.The extraction of water phase ethyl acetate, the organic phase after merging are used 1mmoL sodium hydroxide solution and salt water washing, it is dry, it is concentrated under reduced pressure.Crude product chromatographs (petrol ether/ethyl acetate=9: 1) through column Purifying, obtains compound 1.Through structural identification, synthesized compound1H-NMR and13C-NMR data and document (Targeting Myeloid Differentiation Using Potent 2-Hydroxypyrazolo[1,5-a]pyridine Scaffold-BasedHuman Dihydroorotate Dehydrogenase Inhibitors(JMed Chem.2018, Jun 25)) it is consistent.
Embodiment 2: the synthesis of compound 2
Under 0 DEG C, condition of nitrogen gas, 2- benzyl oxygroup -7- methyl -3,3- dihydro-pyrazolo [1,5-a] pyridine-3-carboxylic acid is dry Methylene chloride (3mmoL) solution of the drying of 2mmol/L oxalyl chloride and the DMF (1 of drying are added dropwise in dry THF (20mL) solution Drop) solution.It is added dropwise, reaction solution reacts at room temperature 2h.It is concentrated under reduced pressure, residue is dissolved in dry THF (10mL, this step Suddenly in triplicate), obtained acyl chlorides is immediately using being not required to be further purified.Under condition of nitrogen gas, by trimethyl aluminium, (2M hexane is molten Liquid, 1.5mmoL) it is added in dry toluene (15mL) solution of 2,3,5,6- tetra- fluoro- 4- phenylanilines (1.1mmoL).Reaction Liquid is transferred in dry toluene (30mL) solution of acyl chlorides obtained above after 2h is stirred at room temperature.90 DEG C of heating stirring mistakes of reaction solution It is cooled to room temperature after night, the dilute hydrochloric acid quenching reaction of 1mmoL, liquid separation is added.The extraction of water phase ethyl acetate, the organic phase after merging It is dry with 1mmoL sodium hydroxide solution and salt water washing, it is concentrated under reduced pressure.Crude product through column chromatograph (petrol ether/ethyl acetate=9: 1) it purifies, obtains compound 2.Through structural identification, synthesized compound1H-NMR and13C-NMR data and document (Targeting Myeloid Differentiation Using Potent 2-Hydroxypyrazolo[1,5-a] pyridine Scaffold-Based Human Dihydroorotate Dehydrogenase Inhibitors(JMed Chem.2018, Jun 25)) it is consistent.
Embodiment 3: the synthesis of compound 3
Under 0 DEG C, condition of nitrogen gas, 2- benzyl oxygroup -5- methyl -3,3- dihydro-pyrazolo [1,5-a] pyridine-3-carboxylic acid is dry Methylene chloride (3mmoL) solution of the drying of 2mmol/L oxalyl chloride and the DMF (1 of drying are added dropwise in dry THF (20mL) solution Drop) solution.It is added dropwise, reaction solution reacts at room temperature 2h.It is concentrated under reduced pressure, residue is dissolved in dry THF (10mL, this step Suddenly in triplicate), obtained acyl chlorides is immediately using being not required to be further purified.Under condition of nitrogen gas, by trimethyl aluminium, (2M hexane is molten Liquid, 1.5mmoL) it is added in dry toluene (15mL) solution of 2,3,5,6- tetra- fluoro- 4- phenylanilines (1.1mmoL).Reaction Liquid is transferred in dry toluene (30mL) solution of acyl chlorides obtained above after 2h is stirred at room temperature.90 DEG C of heating stirring mistakes of reaction solution It is cooled to room temperature after night, the dilute hydrochloric acid quenching reaction of 1mmoL, liquid separation is added.The extraction of water phase ethyl acetate, the organic phase after merging It is dry with 1mmoL sodium hydroxide solution and salt water washing, it is concentrated under reduced pressure.Crude product through column chromatograph (petrol ether/ethyl acetate=9: 1) it purifies, obtains compound 3.Through structural identification, synthesized compound1H-NMR and13C-NMR data and document (Targeting Myeloid Differentiation Using Potent 2-Hydroxypyrazolo[1,5-a] pyridine Scaffold-Based Human Dihydroorotate Dehydrogenase Inhibitors(JMed Chem.2018, Jun 25)) it is consistent.
Embodiment 4: the synthesis of compound 4
Pd/C (10%), compound 1 (1.0mmoL), dry THF (15mL) and 37% concentrated hydrochloric acid are added in reaction flask (1.0mmoL) reacts 6h under hydrogen atmosphere.After completion of the reaction, mixed liquor diatomite filters, and methanol washs diatomite.Filtrate is dense Contracting, residue column chromatograph (dichloromethane/ethyl acetate=4/1 adds 1% formic acid) purifying, obtain compound 4.Through structural identification, Synthesized compound1H-NMR and13C-NMR data and document (Targeting Myeloid Differentiation Using Potent 2-Hydroxypyrazolo[1,5-a]pyridine Scaffold-Based Human Dihydroorotate Dehydrogenase Inhibitors (JMed Chem.2018, Jun 25)) it is consistent.
Embodiment 5:
One, prescription
1000
Two, processing step
Compound 1, low-substituted hydroxypropyl cellulose (L-HPC), microcrystalline cellulose (MCC), xylitol, starch are mixed equal It after even, with 35% ethyl alcohol softwood of adhesive, is pelletized with 40 meshes, 40 DEG C of dryings, 30 mesh sieves are mixed after magnesium stearate is added It closes uniformly, tabletting to obtain the final product.
Embodiment 6:
One, prescription
1000
Two, processing step
After mixing by compound 1, sodium carboxymethyl starch (CMS-Na), microcrystalline cellulose (MCC), dextrin, povidone, It with 35% ethyl alcohol softwood of adhesive, is pelletized with 40 meshes, 40 DEG C of dryings, 30 mesh sieves mix after polyethylene glycol is added Even, tabletting to obtain the final product.
Embodiment 7:
One, prescription
1000
Two, processing step
After mixing by compound 1, sodium carboxymethylcellulose (CMC-Na), pregelatinized starch, dextrin, starch, with viscous 50% ethyl alcohol softwood of mixture is pelletized with 40 meshes, 40 DEG C of dryings, 30 mesh sieves, is uniformly mixed after magnesium stearate is added, pressure Piece to obtain the final product.
Embodiment 8:
One, prescription
1000
Two, processing step
Compound 1, low-substituted hydroxypropyl cellulose (L-HPC), sodium carboxymethylcellulose (CMC-Na), pregelatinated are formed sediment Powder, starch after mixing, with 50% ethyl alcohol softwood of adhesive, are pelletized, 40 DEG C of dryings, 30 mesh sieves add with 40 meshes It is uniformly mixed after entering magnesium stearate, tabletting to obtain the final product.
Embodiment 9:
One, prescription
1000
Two, processing step
After mixing by compound 1, low-substituted hydroxypropyl cellulose (L-HPC), xylitol, pregelatinized starch, starch, It with 50% ethyl alcohol softwood of adhesive, is pelletized with 40 meshes, 40 DEG C of dryings, 30 mesh sieves mix after magnesium stearate is added Even, tabletting to obtain the final product.
Embodiment 10:
One, prescription
1000
Two, processing step
Compound 2, low-substituted hydroxypropyl cellulose (L-HPC), microcrystalline cellulose (MCC), xylitol, starch are mixed equal It after even, with 35% ethyl alcohol softwood of adhesive, is pelletized with 40 meshes, 40 DEG C of dryings, 30 mesh sieves are mixed after magnesium stearate is added It closes uniformly, tabletting to obtain the final product.
Embodiment 11:
One, prescription
1000
Two, processing step
After mixing by compound 2, sodium carboxymethyl starch (CMS-Na), microcrystalline cellulose (MCC), dextrin, povidone, It with 35% ethyl alcohol softwood of adhesive, is pelletized with 40 meshes, 40 DEG C of dryings, 30 mesh sieves mix after polyethylene glycol is added Even, tabletting to obtain the final product.
Embodiment 12:
One, prescription
1000
Two, processing step
Compound 3, low-substituted hydroxypropyl cellulose (L-HPC), microcrystalline cellulose (MCC), xylitol, starch are mixed equal It after even, with 35% ethyl alcohol softwood of adhesive, is pelletized with 40 meshes, 40 DEG C of dryings, 30 mesh sieves are mixed after magnesium stearate is added It closes uniformly, tabletting to obtain the final product.
Embodiment 13:
One, prescription
1000
Two, processing step
After mixing by compound 3, sodium carboxymethylcellulose (CMC-Na), pregelatinized starch, dextrin, starch, with viscous 50% ethyl alcohol softwood of mixture is pelletized with 40 meshes, 40 DEG C of dryings, 30 mesh sieves, is uniformly mixed after magnesium stearate is added, pressure Piece to obtain the final product.
Test example 1: the screening of external activity
According to DPP-IV vitro detection mechanism, compound can use DPP-IV-Glo to the inhibiting rate of DPP-IVTMAlbumen water Solve the homogeneous luminescent detection system (DPP-IV-Glo of enzymeTMProteaseAssay, Promega cat#G8350) it measures.This is The Laemmli buffer system Laemmli that system is detected containing DPP-IV substrate Gly-Pro- amino luciferin and luciferase activity, DPP-IV-GloTM Luciferase reaction can be activated after being cut by DPP-IV, generate " glow-type " type luminous signal, then use TurnerVeritasTM Microwell plate luminometer detection luminous signal can characterize the activity of DPP-IV.
Experimental method:
Untested compound is dissolved in DMSO to and is configured to the mother solution (≤10M, final concentration) of 8-10 various concentration. The untested compound of above-mentioned various concentration is added into the reaction system of 100 μ L mankind DPP-IV enzymes (0.1nmol/L, final concentration) Solution.After 37 DEG C of incubation 10min, highly sensitive fluorogenic substrate Gly-Pro-AMC (H-glycyl- is added into reaction system prolyl-7-amino-4-methylcoumarin；Sigma-Aldrich, 10 μM, final concentration) start reaction and in 400nm Detection architecture luminous intensity under two wavelength of (exciting light) and 505nm (transmitting light).Pass through enzyme power using standardized mathematical model It learns curve and calculates inhibition constant IC50。
The 1 external IC of compound 1-4 of table50Activity
Xi Gelieting (sun ginseng) Compound 1 Compound 2 Compound 3 Compound 4
IC50(nM) 18.2 15.4 14.8 15.5 209.3
Activity Results show that compound 1,2,3 has good inhibition DPP-IV ability in vitro, and inhibitory activity is better than Positive control drug Xi Gelieting；4 rejection ability of compound is faint.Illustrate compound 1, compound 2 and chemical combination of the present invention Object 3 can be used as exploitation of the DPP-IV inhibitor for diabetes medicament.
Test example 2: oral glucose tolerance (OGTT) experiment
1, experimental animal:
Kunming mouse, 6 week old, weight 18-22g, half male and half female.
2, it is grouped and is administered:
Mouse fasting 12h after adaptive feeding measures fasting blood sugar, is then divided into 3 groups, every group 8.It is dynamic to each group Object stomach-filling gives corresponding test-compound, and blank group gives distilled water.Concrete scheme is as shown in table 2 below:
The OGTT experimental administration scheme of table 2 compound 1 and positive control drug comparison
3, blood sugar detection:
After the last administration, each group mouse fasting 12h, stomach-filling glucose solution 1g/kg, is surveyed respectively when 30min, 60min Determine blood glucose value.
4, test result:
The OGTT experimental result of table 3 compound 1 and positive control drug comparison
Test result shows, compared to the blank group, after glucose load 30min, 60min, 1 group of compound and positive controls Blood glucose reduces obvious.Compared with positive controls, after glucose load 30min, 60min, the ability of 1 group of control blood glucose of compound is better than Positive control drug illustrates that the hypoglycemic effect of compound 1 is relatively preferable.
Test example 3: the research of the pharmacokinetic profile of compound 1
1, experimental animal: SD rat, half male and half female, weight (220-250) g.
2, administration is acquired with sample:
Rat vein administration: SD rat 4, half male and half female, weight 220-250g.Fasting 12h, drinks during test before being administered Water is free.Test-compound is given by the dosage intravenous injection of 10mg/kg.2min, 10min, 30min, 1.0h after administration, 2.0h, 3.0h, 4.0h, 6.0h, 8.0h, 11.0h, 24.0h take blood about 0.2mL through eye socket, set in heparinised tubes, 4000G from Heart 10min, separated plasma, -20 DEG C of preservations are to be measured.
Oral Administration in Rats administration: SD rat 4, half male and half female, weight 220-250g.Fasting 12h before being administered, during test certainly By drinking water.Test-compound is given by the dosage of 50mg/kg is oral.5min, 15min, 30min, 1.0h, 2.0h after administration, 3.0h, 4.0h, 6.0h, 8.0h, 11.0h, 24.0h take blood about 0.2mL to set in heparinised tubes through eye socket, 4000G centrifugation 10min, separated plasma, -20 DEG C of preservations are to be measured.
3, instrument condition and plasma sample measurement:
Instrument: American AB company API3000 type triple quadrupole bar tandem mass spectrometer is ionized equipped with Turbo Ionspray 1.4 data processing system of source and Analyst；Japanese Shimadzu Corporation LC-10ADvp pump, German GerstelAutosampler MPS3C type autosampler, Amada Co., Ltd. Shiseido C18Column (30mm × 2.0mm I.D, 5 μm of partial sizes)；The U.S. Phenomenex company C18Guard column (4 × 3.0mm I.D.).
Blood samples processing: 10 μ L methanol: water (1:1) and 150 μ L inner mark solutions are added into 50 μ L rat plasma samples It (5 μM contain acetonitrile solution), mixes；Vortex mixing 1min is centrifuged 30min (14000G), and 100 μ L is taken to carry out LC/MS/MS analysis.
Configuration standard curve: taking 50 μ L of rat blank plasma, sequentially adds 10 μ L of test-compound standard serial solution, matches System is equivalent to the test-compound blood that plasma concentration is 20,50,100,500,1000,2000,4000,6000,12000ng/mL Slurry samples establish standard curve by operating under " plasma sample processing " item.With testing concentration (x) be abscissa, determinand with The peak area ratio (y) of internal standard compound is ordinate, carries out regressing calculation with weighted least-squares method, acquires linear regression equation, As standard curve.Typical rat plasma sample standard curve are as follows: y=0.0155x+0.000353, r=0.9872.According to mark Directrix curve, the range of linearity that LC/MS/MS method measures test-compound in rat plasma is 20-12000ng/mL.
4, test result:
The pharmacokinetic data of 4 compound 1 of table
Test result shows that pharmacokinetic profile of the compound 1 in SD rat is good, and oral half-life period is greater than 5h, bioavilability are greater than 70%.
Test example 4: the hERG of compound 1 inhibits test:
The drug candidate verified using rubidium ion and the competitive method for entering the channel hERG of compound 1 whether inhibit or Block the channel hERG.
Chinese hamster ovary celI with the density kind of 20,000 cells/wells in 96 orifice plates, in 37 DEG C of 5%CO2Incubator culture.After 48h, use Buffer I (25mmol/LHepes, 150mmol/LNaCl, 1mmol/L MgCl2, 0.8mmol/LNaH2PO4, 2mmol/L CaCl2, pH 7.4 is adjusted to NaOH) and rinsing 3 times, every hole adds 200 μ L buffer II, and (buffer I adds 5.4mmol/L RbCl2 2.5h is incubated for 5mmol/L glucose).The buffer II of half is removed, the buffer II containing compound 1 or DMSO is added. After being incubated for 30min, 3 times are rinsed to remove extracellular rubidium ion with buffer I, is then added and contains compound 1 or DMSO (0.5% Final concentration) buffer III (25mmol/LHepes, 100mmol/LNaCl, 50mmol/LKCl, 1mmol/LMgCl2, 0.8mmol/LNaH2PO4, 2mmol/L CaCl2, be adjusted to pH 7.4 with NaOH) or buffer IV (buffer I adds 5.4mmol/ LKCl).Cell removes supernatant and is transferred to another 96 orifice plate after being incubated at room temperature 10min.200 μ L are added containing 0.1% The buffer III lytic cell of Triton X-100.With ICR8000 Flame Atomic Absorption Spectrometry light instrument (Aurora Biomed Inc., Vancouver, B.C.) measure supernatant and cell pyrolysis liquid rubidium ion.Test result is as follows,
Compound name Highest detection concentration Maximum concentration inhibiting rate (%) IC50(μM)
Compound 1 300μM 79.0±1.2 104.2±7.1
Test result shows compound 1 to the IC in the channel hERG50It is 104.2 ± 7.1 μM, does not inhibit or block hERG Channel illustrates that a possibility that short malicious there are heart is small.

Claims (9)

1. pyrazolo-pyridines or its pharmaceutically acceptable salt, structure are shown in formula I:
Wherein R1、R2It is independently selected from hydrogen or methyl.
2. pyrazolo-pyridines as described in claim 1 or its pharmaceutically acceptable salt, specific structure are as follows:
3. pyrazolo-pyridines as described in claim 1 or its pharmaceutically acceptable salt are as DPP-IV inhibitor Application.
4. pyrazolo-pyridines as described in claim 1 or its pharmaceutically acceptable salt are as antidiabetic medicine Application, the especially treatment of type-2 diabetes mellitus.
5. the tablet containing pyrazolo-pyridines described in claim 1 or its pharmaceutically acceptable salt, feature Be: prescription matches by weight are as follows:
6. tablet as claimed in claim 5, it is characterised in that: the disintegrating agent is low-substituted hydroxypropyl cellulose, starch, carboxylic One of methyl starch sodium, sodium carboxymethylcellulose or crospovidone, two or more composition.
7. tablet as claimed in claim 5, it is characterised in that: the filler is lactose, mannitol, xylitol, malt One of sugar alcohol, dextrin, pregelatinized starch or microcrystalline cellulose, two or more composition.
8. tablet as claimed in claim 5, it is characterised in that: the lubricant is magnesium stearate, zinc stearate, poly- second two Alcohol, sucrose fatty ester, sodium stearyl fumarate, stearic acid, talcum powder, one of silica, two or more Composition.
9. tablet as claimed in claim 5, it is characterised in that: adhesive is water, ethyl alcohol, hydroxypropyl methyl cellulose, ethyl Cellulose, one kind of povidone or starch, two or more mixture.
CN201910877861.0A 2019-09-17 2019-09-17 Pyrazolo-pyridines and its purposes in treatment diabetes Withdrawn CN110511222A (en)

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