CN101554374B

CN101554374B - Flavonoid compound with inhibit function for dipeptidyl peptidase IV

Info

Publication number: CN101554374B
Application number: CN2008100547501A
Authority: CN
Inventors: 王金戌; 张福军; 肖乔; 陈欣然; 杨汉煜; 刘翠艳; 严艳欣; 刘玉峰
Original assignee: CSPC Zhongqi Pharmaceutical Technology Shijiazhuang Co Ltd
Current assignee: CSPC Zhongqi Pharmaceutical Technology Shijiazhuang Co Ltd
Priority date: 2008-04-08
Filing date: 2008-04-08
Publication date: 2011-05-25
Anticipated expiration: 2028-04-08
Also published as: CN101554374A

Abstract

The invention relates to a flavonoid compound which can be used as an inhibitor for dipeptidyl peptidase IV, and aims at preventing and curing diseases correlative to dipeptidyl peptidase IV, in particular, type II sugar diabetes. The flavonoid compound has a structure shown in the general formula 1, wherein R1 and R7 in the general formula I are aryl parts containing benzene ring, R2, R3, R4, R5, R6, R8, R9 and R10 are H, OH, COOH and alkoxyl parts in the general formula 1.

Description

Has the inhibiting flavone compound of DPP IV

Technical field

The present invention relates to a class and have the inhibiting flavone compound of DPP IV (DDP-IV), can be used for treating the relevant disease of DPP IV, as diabetes, type ii diabetes etc. particularly.

Technical background

Diabetes are to be the chronic metabolic disease of feature with the hyperglycemia, are the big diseases in third place in the world.China has become the high incidence area of onset diabetes, diabetics 3,000 ten thousand is arranged in 2007, and the sickness rate of diabetes is about 5%, and especially the various complication that caused by diabetes are that diabetics is disabled and lethal main cause.The onset diabetes rate is based on type ii diabetes, and the type ii diabetes medicine comprises the insulin of sulfonylurea, biguanides, euglycemic agent, alpha-glucosidase inhibitor, inhibitors of dipeptidyl IV and various dosage forms etc. at present.

DPP IV (E.C.3.4.14.5) is also referred to as CD26, it is a kind of transmembrane serine protease, also there is soluble form, it is a member of prolyl oligopeptidase family, it is that this enzyme is brought into play active main substrate that all N hold the polypeptide that has proline (Pro) or alanine (Ala) on the 2nd, it can hold two amino acid residue Xaa-Pro of hydrolysis or Xaa-Ala (Xaa is an arbitrary amino acid) from peptide chain N, thereby makes active polypeptide partially or completely inactivation occur.

Inhibitors of dipeptidyl IV is the novel oral hypoglycemic of a class, it is responsible for the glucagon-like peptide of degrading by suppressing DDP-IV) activity keep glucagon-like peptide (GLP-1) level in the body, GLP-1 has the promotion insulin secretion, suppress pancreas and rise the plain release of sugar, suppress gastric emptying, increase effects such as β cell quantity.In clinical research, the DDP-IV inhibitor demonstrates excellent curative to diabetes, and this type of medicine can not cause that weight in patients increases, and it causes hypoglycemic risk also very little.

Because DPP IV is a treating diabetes novel targets that just grew up in recent years, there is inhibiting native compound research less to DPP IV both at home and abroad, there is inhibiting natural flavone compounds not appear in the newspapers especially to this enzyme.Flavone compound is a big compounds that exists in the natural plants, has pharmacological action widely.Zhou Xin etc. have summarized the various biologic activity of flavone compound, sum up flavone compound and can suppress the activity of plurality of enzymes perhaps, as [Zhou Xin, Li Hongjie such as hydrolytic enzyme, oxidoreductase, DNA synzyme, RNA polymerase, phosphatase, protein phosphatase kinases, the biological activity of flavone compound and Clinical advances, Chinese Journal of New Drugs, 2007,16 (5), 350], but flavone compound yet there are no report to the inhibitory action of DPP IV.Xu Zhi etc. have reported the application of the bis-flavonoid of discovery in nearly ten years at aspects such as antitumor, antiviral, antioxidation and antiinflammatories, wherein there be not applied research [Xu Zhi, Shu Jianhui, the Tan Guishan of this compounds in the diabetes field, the bis-flavonoid progress, the contemporary Chinese medical journal, 2004,14 (7), 88].Sun Fenglei, burnt Jingjing etc. have been summed up the application of flavonoid natural drug in diabetes and complication field thereof, but specifically be that the flavone of which class formation has also research report [Sun Fenglei of DPP IV inhibitory action and treating diabetes effect, flavonoid natural drug effective ingredient treatment diabetes and complication investigation thereof, Traditional Chinese Medicinal College of Liaoning's journal, 2003,5 (4), 381; Burnt Jingjing, Zhang Ying, flavone compound is in the latest Progress of preventing and treating aspect diabetes and the complication thereof, Chinese Pharmaceutical Journal, 2006,41 (7), 481].

Summary of the invention

The present invention relates to the flavone compound that a class can be used as inhibitors of dipeptidyl IV, it relates to the disease of DPP IV in prevention and treatment, the purposes in the type ii diabetes particularly, the Pharmaceutical composition that comprises this compounds relates to the purposes in the disease, particularly diabetes of DPP IV in prevention and treatment.This flavone compound has following general formula I structure:

General formula I

Or its pharmaceutically acceptable salt and stereoisomer thereof, wherein R1, R7 representative contains the aryl moiety of phenyl ring, and R2, R3, R4, R5, R6, R8, R9, R10 represent H, 0H, COOH, alkoxyl part.This chemical compound suppresses the DPP IV activity, can be used for preventing disease, the particularly diabetes relevant with treating DPP IV.

Particularly, as R1=4 ', 5, when 7-trihydroxyflavone, R2=OH, R3=H, R4=OH, R5=H, R6=H, R7=H, R8=OH, R9=H, R10=H, described chemical compound is an amentoflavone, the concrete structure formula is as follows:

As R1=4 ', 5,7-trihydroxyflavone, R2=CH ₃, when R3=H, R4=OH, R5=H, R6=H, R7=H, R8=OH, R9=H, R10=H, described chemical compound is a sotetsuflavone, the concrete structure formula is as follows:

As R1=4 ', 5-dihydroxy-7-methoxy flavone, R2=CH ₃, R3=H, R4=OH, R5=H, R6=H, R7=H, R8=CH ₃, when R9=H, R10=H, described chemical compound is the rubber tree bisflavone, the concrete structure formula is as follows:

When R1=5-hydroxyl-4 '-when methoxy flavone, R2=OH, R3=H, R4=OH, R5=H, R6=H, R7=H, R8=OH, R9=H, R10=H, described chemical compound is a ginkegetin, the concrete structure formula is as follows:

As R1=H, R2=OH, R3=H, R4=OH, R5=H, R6=H, R7=5 ", 7 ", 4 ' "-when trihydroxyflavone, R8=OH, R9=H, R10=H, described chemical compound is sieve S flavone, the concrete structure formula is as follows:

Terminological interpretation is as follows among the present invention:

Term " flavone " is meant with two phenyl ring and is connected to a series of chemical compounds that basic structure forms by middle three carbochains.

Term " alkoxyl " be meant 10 carbon with interior straight or branched and oxygen atom by singly building the group that is connected, particularly 1-6 carbon straight or branched and oxygen atom are by singly building the group that is connected.

Term " aryl " is meant 6-20 carbon monocycle, dicyclo or the three cyclophane bases that comprise phenyl ring, particularly contains the flavonoid structure of dicyclo.

Term " pharmaceutically acceptable salt " is meant the salt that pharmaceutically acceptable, non-toxic bases or processed with acid are equipped with.Wherein non-toxicity alkali salt comprises aluminum, ammonium, calcium, copper, ferrum, ferrous, lithium, magnesium, manganese, potassium, sodium, zinc salt etc., particularly preferably is potassium, calcium, sodium and ammonium salt.Non-toxicity acid comprises acetic acid, benzenesulfonic acid, benzoic acid, citric acid, fumaric acid, gluconic acid, hydrochloric acid, lactic acid, maleic acid, malic acid, methanesulfonic acid, nitric acid, phosphoric acid, succinic acid, sulphuric acid and tartaric acid etc., particularly preferably is citric acid, hydrochloric acid, maleic acid, malic acid, methanesulfonic acid, succinic acid and tartaric acid.

The chemical compound of general formula I structure of the present invention and pharmaceutically acceptable salt thereof can be with oral methods or the medications of parenteral road.Oral medication can be tablet, granule, capsule, soft capsule; Non-have injection and suppository etc. through the intestinal drug formulation.These preparations are according to the known method preparation of those skilled in the art.For making tablet, granule, capsule, the used adjuvant of soft capsule is the auxiliary agent of conventional usefulness, for example starch, lactose, gelatin, syrup, glycerol, Cera Flava, arabic gum, micropowder silica gel, Pulvis Talci, Polyethylene Glycol.The used solvent of liquid dosage form is water, ethanol, propylene glycol, plant oil such as Semen Maydis oil, Oleum Arachidis hypogaeae semen, Fructus Canarii albi wet goods for example.Containing in the preparation of The compounds of this invention also can have other auxiliary agents, for example surfactant, lubricant, antioxidant, disintegrating agent, antiseptic, correctives, pigment etc.

Therefore the embodiment of back will illustrate the chemical compound of general formula I structure of the present invention, and DPP IV is had stronger inhibitory action, can be used for treating the relevant disease of DPP IV, as diabetes type ii diabetes etc. particularly.

The specific embodiment

Below further set forth the The compounds of this invention pharmacodynamic study by specific embodiment, but this embodiment should not become limitation of the present invention.

Embodiment 1 amentoflavone was tested active inhibition of DPP IV

As R1=4 ', 5, when 7-trihydroxyflavone, R2=OH, R3=H, R4=OH, R5=H, R6=H, R7=H, R8=OH, R9=H, R10=H, compound of Formula I is an amentoflavone, its enzyme inhibition activity experiment to DPP IV is as follows:

Experiment material: amentoflavone is a pale yellow powder, opens up the health bio tech ltd available from Changsha, and specification is the 20mg/ bottle, and HPLC detects purity content greater than 98%; Positive inhibitor Ile-Pro-Ile white powder, available from Sigma company, specification is the 5mg/ bottle, it is greater than 97% that HPLC detects purity; Gly-Pro p-nitroanilide hydrochloride is a pale yellow powder, and available from Sigma company, specification is the 25mg/ bottle, and it is 99% that TLC detects purity; DPP IV is a freeze-dried powder, and available from Sigma company, specification is the 1.1unit/ bottle, and other reagent is homemade analytical pure.

Experimental technique: DPP IV enzyme reaction experiment is carried out in 96 orifice plates, 200 μ l systems, each hole adds 24 μ l 0.02u/ml dipeptides matrix enzyme IV respectively, add 0.5 respectively, 1.25,2.5, positive inhibitor Ile-Pro-Ile and the amentoflavone of 5 and 12.5 μ g/ml, wherein positive inhibitor Ile-Pro-Ile water preparation, amentoflavone is prepared with the Tris buffer of 50% dimethyl sulfoxide and 50%50mM, and set up the dimethyl sulfoxide negative control hole, mend to 160 μ l with the Tris buffer of 50mM, the vibration mixing, hatched 10 minutes in 37 ℃, the Gly-pro-pn substrate that adds 40 μ l 1mM, the vibration mixing, in 37 ℃ of 405nm photometry absorption values, detected once, and detected 60 minutes altogether in per 5 minutes.

When carrying out Ile-Pro-Ile and amentoflavone, take by weighing the fresh Wistar rat kidney of 1g, shred, add the 9ml normal saline, use refiner homogenate with dissecting scissors to the inhibitory action of dipeptidyl peptidase-iv in the Ren Mus homogenate.Homogenate after the homogenate centrifugal 10 minutes in 3000rpm, supernatant in 5000rpm centrifugal 10 minutes are again drawn supernatant, are used for the enzymatic activity experiment after 10 times of the supernatant dilutions.DPP IV enzyme reaction experiment in the Ren Mus homogenate supernatant is carried out in 96 orifice plates, 200 μ l systems, each hole adds 24 μ l 0.02u/ml dipeptides matrix enzyme IV respectively, the Ile-Pro-Ile and the amentoflavone that add variable concentrations are respectively mended to 160 μ l with the Tris buffer of 50mM, the vibration mixing, hatched 10 minutes in 37 ℃, the Gly-pro-pn substrate that adds 40 μ l 1mM, the vibration mixing is in 37 ℃ of 405nm photometry absorption values, detected once, and detected 60 minutes altogether in per 5 minutes.

Experimental result:

Table one, positive inhibitor Ile-Pro-Ile are to the inhibitory action of dipeptidyl peptidase-iv

Table two, amentoflavone are to the inhibitory action of dipeptidyl peptidase-iv

Result by table one can draw, and positive inhibitor Ile-Pro-Ile is to the half-inhibition concentration IC of the inhibition of dipeptidyl peptidase-iv ₅₀Be 2.86 μ g/ml, to the half-inhibition concentration IC of dipeptidyl peptidase-iv inhibition in the Ren Mus homogenate ₅₀Be 4.66 μ g/ml.

Result by table two can draw, and amentoflavone is to the half-inhibition concentration IC of the inhibition of dipeptidyl peptidase-iv ₅₀Be 1.66 μ g/ml, to the half-inhibition concentration IC of dipeptidyl peptidase-iv inhibition in the Ren Mus homogenate ₅₀Be 3.14 μ g/ml.

Conclusion: amentoflavone has very strong inhibitory action to dipeptidyl peptidase-iv, and it is stronger to suppress effect than positive inhibitor Ile-Pro-Ile under the bad situation of self water solublity, also good than positive inhibitor Ile-Pro-Ile to the inhibition effect of dipeptidyl peptidase-iv in the Ren Mus homogenate.

Embodiment 2 sotetsuflavones were tested active inhibition of DPP IV

As R1=4 ', 5,7-trihydroxyflavone, R2=CH ₃, when R3=H, R4=OH, R5=H, R6=H, R7=H, R8=OH, R9=H, R10=H, chemical compound shown in the general formula I is a sotetsuflavone, its enzyme inhibition activity experiment to DPP IV is as follows:

Experiment material: sotetsuflavone is a yellow powder, and purity is greater than 98%, and the experimental technique of reference literature " Xu Yaming, Fang Shengding, what quick; the chemical constituent in the Dacrydium pierrei, Botany Gazette, 1991; 33 (8), 646 " separation and Extraction from Dacrydium pierrei obtains, and all the other reagent are with embodiment 1.

Experimental technique is with embodiment 1.

Experimental result:

Table three, sotetsuflavone are to the inhibitory action of dipeptidyl peptidase-iv

Result by table three can draw, and sotetsuflavone is to the half-inhibition concentration IC of the inhibition of dipeptidyl peptidase-iv ₅₀Be 2.58 μ g/ml, to the half-inhibition concentration IC of dipeptidyl peptidase-iv inhibition in the Ren Mus homogenate ₅₀Be 2.95 μ g/ml.

Conclusion: sotetsuflavone has stronger inhibitory action to dipeptidyl peptidase-iv, and dipeptidyl peptidase-iv in the Ren Mus homogenate is also had significant inhibitory effect, and it is better than positive inhibitor Ile-Pro-Ile to suppress effect.

Embodiment 3 rubber tree bisflavones were tested active inhibition of DPP IV

As R1=4 ', 5-dihydroxy-7-methoxy flavone, R2=CH ₃, R3=H, R4=OH, R5=H, R6=H, R7=H, R8=CH ₃, when R9=H, R10=H, chemical compound shown in the general formula I is the rubber tree bisflavone, its enzyme inhibition activity experiment to DPP IV is as follows:

Experiment material: the rubber tree bisflavone is a pale yellow powder, and purity is greater than 95%, reference literature " Lee, Shwu Woan; Chen, Zeng Tsi; Chen, Ming Tyan; Biflavones of Selaginelladoederlenii Hieron.Zhonghua Yaoxue Zazhi, 1992,44 (6), 537. " experimental technique separation and Extraction from selaginella doederleinii obtains, and all the other reagent are with embodiment 1.

Experimental technique is with embodiment 1.

Experimental result:

Table four, rubber tree bisflavone are to the inhibitory action of dipeptidyl peptidase-iv

Result by table four can draw, and the rubber tree bisflavone is to the half-inhibition concentration IC of the inhibition of dipeptidyl peptidase-iv ₅₀Be 2.50 μ g/ml, to the half-inhibition concentration IC of dipeptidyl peptidase-iv inhibition in the Ren Mus homogenate ₅₀Be 2.67 μ g/ml.

Conclusion: the rubber tree bisflavone has stronger inhibitory action to dipeptidyl peptidase-iv, and dipeptidyl peptidase-iv in the Ren Mus homogenate is also had significant inhibitory effect, and it is better than positive inhibitor Ile-Pro-Ile to suppress effect.

Embodiment 4 ginkegetins were tested active inhibition of DPP IV

When R1=5-hydroxyl-4 '-when methoxy flavone, R2=OH, R3=H, R4=OH, R5=H, R6=H, R7=H, R8=OH, R9=H, R10=H, chemical compound shown in the general formula I is a ginkegetin, and its enzyme inhibition activity experiment to DPP IV is as follows:

Experiment material: ginkegetin is a yellow powder, purity is greater than 98%, and the experimental technique of reference literature " Tang Yuping, Lou Fengchang; Wang Jinghua; Li Yanfang, the research of flavones ingredient in the Folium Ginkgo, Chinese Pharmaceutical Journal; 2001; 36 (4), 231 " separation and Extraction from Folium Ginkgo obtains, and all the other reagent are with embodiment 1.

Experimental technique is with embodiment 1.

Experimental result:

Table five, ginkegetin are to the inhibitory action of dipeptidyl peptidase-iv

Result by table five can draw, and ginkegetin is to the half-inhibition concentration IC of the inhibition of dipeptidyl peptidase-iv ₅₀Be 2.52 μ g/ml, to the half-inhibition concentration IC of dipeptidyl peptidase-iv inhibition in the Ren Mus homogenate ₅₀Be 3.05 μ g/ml.

Conclusion: ginkegetin has stronger inhibitory action to dipeptidyl peptidase-iv, and dipeptidyl peptidase-iv in the Ren Mus homogenate is also had significant inhibitory effect, and it is better than positive inhibitor Ile-Pro-Ile to suppress effect.

Embodiment 5 sieve S flavone were tested active inhibition of DPP IV

As R1=H, R2=OH, R3=H, R4=OH, R5=H, R6=H, R7=5 "; 7 " 4 ' "-and when trihydroxyflavone, R8=OH, R9=H, R10=H, chemical compound shown in the general formula I is sieve S flavone, its enzyme inhibition activity experiment to DPP IV is as follows:

Experiment material: sieve S flavone is a pale yellow powder, purity is greater than 98%, the method of reference literature " David E.Zembower and Heping Zhang; Total Synthesis of Robustaflavone, aPotential Anti-Hepatitis B Agent, J.Org.Chem.; 1998; 63 (25), 9300 " prepares, and all the other reagent are with embodiment 1.

Experimental technique is with embodiment 1.

Experimental result:

Table six, sieve S flavone are to the inhibitory action of dipeptidyl peptidase-iv

Result by table six can draw, and sieve S flavone is to the half-inhibition concentration IC of the inhibition of dipeptidyl peptidase-iv ₅₀Be 2.34 μ g/ml, to the half-inhibition concentration IC of dipeptidyl peptidase-iv inhibition in the Ren Mus homogenate ₅₀Be 3.12 μ g/ml.

Conclusion: sieve S flavone has stronger inhibitory action to dipeptidyl peptidase-iv, and dipeptidyl peptidase-iv in the Ren Mus homogenate is also had significant inhibitory effect, and it is better than positive inhibitor Ile-Pro-Ile to suppress effect.

Claims

1. the application of the rubber tree bisflavone shown in the following formula in preparation DPP IV activity inhibitor

2. the application of rubber tree bisflavone according to claim 1 in preparation prevention and treatment type ii diabetes medicine.

3. a pharmaceutical composition that contains the described rubber tree bisflavone of claim 1 is characterized in that containing rubber tree bisflavone and acceptable accessories.