CN103622969B - Application of chiral spiro indole-pyran pyrimidine base compound in preparation of anti-inflammatory drugs - Google Patents

Abstract

The invention provides a new medical application of a chiral spiro indole-pyran pyrimidine base compound, particularly application of the compound in preparation of anti-inflammatory drugs. An in vitro cyclooxygenase (COX) activity inhibiting experiment, an experiment of inhibiting generation of an inflammatory molecule (nitric oxide) in primary mice macrophage and inhibition test on an ear swelling and paw edema model in mice, show that the chiral compound has an obvious anti-inflammatory effect and has extremely low gastrointestinal injury effect; therefore, the chiral compound, as an active ingredient, can be prepared into anti-inflammatory drugs in various dosage forms through pharmaceutically acceptable technologies and auxiliary materials, so that the chiral compound has an excellent medical application prospect.

Description

The application of chiral spiro indole-pyran pyridine alkaline compound in preparing anti-inflammatory drug

Technical field

The invention belongs to technological field of biochemistry, relate to a kind of new medical usage of chiral spiro indole-pyran pyridine alkaline compound, relate in particular to the application of chiral spiro indole-pyran pyridine alkaline compound in preparing anti-inflammatory drug.

Background technology

Chiral drug is comprised of the chipal compounds with pharmacologically active, it in human body by with biomacromolecule between mutually chirality mate, identify and bring into play therapeutical effect.Although enantiomer medicine physicochemical properties are in vitro substantially the same, but, the chirality albumen being formed by aminoacid, nucleoside, film etc. due to receptor or the target position of drug molecule effect and nucleic acid molecule etc., they have certain requirement to the space multistory configuration of the drug molecule with its combination, therefore, enantiomer medicine often presents the difference of the aspects such as very large pharmacodynamics, dynamic metabolism in vivo.At twentieth century end, along with people deepen continuously to the research of enantiomer medicine and understanding, FDADeng pharmaceutical control and administration department started enantiomer medicine to be examined as mixture in 1992, required manufacturer that the data of each isomer of enantiomer medicine is provided.This measure has promoted development and the exploitation of chiral drug greatly.The annual sales amount of world's chiral drug was respectively 356,452,614,72,900,000,000 dollars in 1993,1994,1995,1996, increased every year on average with more than 20% amplitude.To world's chiral drug annual sales amount in 2000, increase to 1,233 hundred million dollars, reach medicine year gross sales amount (2000 3900 hundred million dollars) 1/3rd (StinsonS.C, chem & Eng News. 2001, Oct 14, 15).Compare with enantiomer medicine, the chiral drug of one-component also has the advantages such as consumption is few, curative effect is high, untoward reaction is low.Therefore this industry has attracted the drugmaker of countries in the world and relevant unit to be engaged in exploitation chiral drug strongly, makes it to become an emerging high-tech industry.

Chinese patent application 201110138538.5 discloses a kind of volution indole-pyrans pyrimidine base compound and Chiral Synthesis thereof, and it is with 4 -hydroxycoumarin compound or 1 -naphthol compound, dicyano Oxoindole ene compound is reaction substrate, the derivative chirality thiourea of the Colophonium of take is catalyst, the stereoselective New-type spiro indole-pyran pyridine alkaline compound that synthesized.Biological activity test proves, chiral spiro indole-pyrans pyrimidine base compound of the present invention can optionally kill tumor cell, and has stronger effect for dissimilar tumor, illustrates that it has good selectivity anti-tumor activity.Along with further research and the exploration to volution indole-pyrans pyrimidine base compounds property, find that it is inhibited to inflammation, therefore, for a kind of novel anti-inflammatory drug of its anti-inflammatory activity exploitation, there is good biological significance and application prospect.

Summary of the invention

The object of this invention is to provide the medical usage that a kind of chiral spiro indole-pyran pyridine alkaline compound is new---the application in preparing anti-inflammatory drug.

The inhibitory action to inflammation below by cell and laboratory animal explanation chiral spiro indole-pyrans pyrimidine base compound.

1 , cell and laboratory animal source and cultural method

1.1 cell strain

Primary peritoneal macrophage You Zhe research department is from mouse peritoneal fresh extractor, condition of culture: with the MEM culture fluid that adds 10% calf serum at 37 ℃, 5% CO ₂cellar culture under condition.Separation method: get healthy male C57BL/6 mice (body weight 25-31 g), lumbar injection Brewer Thioglycollate meat soup (2 ml/ only), after four days, disconnected neck is put to death mice, gathers macrophage in super-clean bench, rinses mouse peritoneal obtain cell suspension with cell culture medium (6 ml), by its centrifugal (5000 rpm, 5 min), re-suspended cell, counting also carries out follow-up cultivation according to different experiments.

1.2 laboratory animal

C57BL/6 is male mice, body weight 20 ~ 22g, by Lanzhou University's Experimental Animal Center (GLP), provided, the execution of whole experimentation is the guideline (86/609/EEC) of the European laboratory animal of reference Ethics Committee all, and obtains the license of laboratory animal Ethics Committee of Lanzhou University.Animal feeding environment is clean level, 22 ± 2 ℃ of ambient temperatures, humidity 50% ~ 55%, 8:00 ~ 20:00 illumination.6 ~ 8 raisings of the every cage of mice, the drinking-water of can freely ingesting.Male Sprague-Dawley (SD) rat, body weight 140-160g, is provided by Lanzhou University's Experimental Animal Center (GLP).Feeding environment is identical with mice, and 4 raisings of the every cage of rat are tested front fasting but can't help water 12 ~ 16 hours.

1.3 COX kinases body outer suppressioning experiments

The COX that adopts COX detection kit (Cayman Chemical, Ann Arbor, MI) to detect JP-8g suppresses active, and operation is carried out according to its description.Chiral spiro indole-pyran pyridine alkaline compound (JP-8g) (being dissolved in DMSO) is added to reaction buffer (being added with respectively COX-1 and the COX-2 enzyme of fluorogenic substrate).Add arachidonic acid solution, room temperature reaction 2 min, adopt large microplate reader device Varioskan Flash 4.00.53 plate fluorimeter (ThermoScitific, Waltham, MA, USA) detect 590 nm wavelength (absorbing wavelength: absorbance 535 nm), and calculate suppression ratio according to following formula.Experimental result is in Table 1.Table 1 shows certain density JP-8g(50 uM) two kinds of hypotypes that can suppress cyclooxygenase COX (are 24.92% to the suppression ratio of COX-1; To the suppression ratio of COX-2, be 57.35%), illustrate that JP-8g has good anti-inflammatory activity, for good basis is established in follow-up anti-inflammatory drug screening.

1.4 NO concentration detect

The compound JP-8g final concentration that the every hole of primary macrophage adds is respectively 50 μ M, 10 μ M, 2 μ M, 0.4 μ M, 80 μ M, 16 μ M, 3.2 μ M, 0.64 μ M, 0.128 μ M, 0.0256 μ M, adds 1 μ g/ml LPS after 2h.With the culture fluid that contains solvent, as blank (control), each multiple hole of concentration group 6, is placed in CO ₂in incubator, cultivate, respectively after dosing 24,48, tri-time points of 72h carry out respectively follow-up detection.Draw 50 μ l cell conditioned medium liquid to 96 new orifice plates, then add successively 50 μ l Griess Regent with 50 μ l Griess Regent .Lucifuge is surveyed each hole light absorption value at microplate reader 540nm after standing 10 minutes, the results are shown in Table 2.Table 2 shows that JP-8g has obvious inhibitory action to the inflammatory mediator NO in primary macrophage under inflammatory conditions, suppresses respectively the generation of NO at 24 h, 48 h, 72 h concentration dependents, and then performance antiinflammatory action.JP-8g is above-mentioned chiral spiro indole-pyran pyridine alkaline compound, and its structural formula is:

The swollen experiment of 1.5 mouse ear

Get 35 healthy male mices and be divided at random 5 groups, 7 every group.Difference lumbar injection Vehicle, compound JP-8g and positive drug indomethacin, after 20 minutes, 20 μ l dimethylbenzene are evenly spread upon to the wide tow sides of mouse right ear, left ear does not process, after 30 minutes, de-cervical vertebra is put to death mice, along auricle baseline, cut complete left and right ear, use respectively card punch (7 mm diameter) to punch at left and right ear center same position, take off specimen weigh respectively immediately (the results are shown in Table three).Table 3 shows the swollen reaction of the JP-8g ear that obviously inflammation-inhibiting causes on inflammatory animal model (mouse ear swell model), and the swollen rate of ear of JP-8g mice when 10 mg/kg, 25 mg/kg, 50 mg/kg dosage is respectively 47.85%, 38.55%, 31.37%.This shows that JP-8g all has very strong periphery anti-inflammatory activity.

The swollen experiment of 1.6 mice foot

Adopt mice foot swelling inflammatory model, be divided at random 4 groups, 7/group.(1) negative control group: intraperitoneal injection of saline, at the right toes subcutaneous injection 25 μ l pro-inflammatory cytokine carrageenin of mice, left toes subcutaneous injection 25 μ l normal saline in contrast subsequently.(2) anti-inflammatory drug group: lumbar injection (12.5 mg/kg) compound JP-8g, at the right toes subcutaneous injection 25 μ l pro-inflammatory cytokine carrageenin of mice, left toes subcutaneous injection 25 μ l normal saline in contrast subsequently.(3) positive control medicine group: lumbar injection (5 mg/kg) dexamethasone, at the right toes subcutaneous injection 25 μ l pro-inflammatory cytokine carrageenin of mice, left toes subcutaneous injection 25 μ l normal saline in contrast subsequently.The lumbar injection volume of above negative control, positive drug contrast and JP-8g is all 10 ml/kg.From the subcutaneous beginning timing of pro-inflammatory cytokine carrageenin injection toes, respectively 1,2, vola, vernier caliper measurement mice left and right thickness for 3,4,5,6,24,48 h, calculates vola, left and right thickness difference (the results are shown in Table four).It is swollen that table 4 shows that JP-8g starts obviously to reduce mice foot at injection 4 h, until 6 h, close with the strong positive anti-inflammatory drug dexamethasone activity of present clinical use.Illustrate that JP-8g has very strong antiinflammatory inhibitory action to the swollen model of mice foot.

1.7 injury of gastrointestinal tract test experience

Get 9 healthy male rats and be divided at random 3 groups every group 3, oral administration gavage gives solvent control (negative medicine contrast), JP-8g, indomethacin (positive drug contrast) respectively, after 6 hours, de-cervical vertebra is put to death, dissecting rat takes out stomach, along greater gastric curvature, cut off, gastric wall is launched to be placed on ice, with 0.9% ice-cold normal saline, rinse gently, measure ulcer length (mm) and hemorrhage length (mm) on every gastric wall in rats, be added the gastric injury total points (the results are shown in Table five) that is every rat.Table 5 shows that positive control drug indomethacin can cause the obvious gastric bleeding of rat and ulcer, and JP-8g is extremely low to rat pipe film injury.This explanation is compared with conventional medicament, the gastrointestinal damage effect that JP-8g is extremely low.

Above-mentioned experiment shows, chiral spiro indole-pyran pyridine alkaline compound has good inhibitory action to inflammation, compare with traditional anti-inflammatory drug, this compounds is extremely low to gastrointestinal damage at the same time, therefore take it as active component, adopt acceptable technique and adjuvant on pharmaceutics, be prepared into the anti-inflammatory drug of various dosage forms, there is good application prospect.

The specific embodiment

Specific embodiment is to chiral spiro indole-pyran pyridine alkaline compound (JP-8g) to being active component below, and the application in preparing anti-inflammatory drug is described further.

The preparation of embodiment 1, tablet

Formula: JP-8g(purity is greater than 95%) 20.0g

Filler 180.0g

Disintegrating agent 10.0g

Adhesive 6.0g

Lubricant 3.0g

Amount to 200.0g

Technique: make 1000, tablet by the common process of preparing tablet, every containing JP-8g 20mg.

Usage and dosage: one day twice, each 2 ~ 3.

Embodiment 2: the preparation of soft capsule

Formula: JP-8g(purity is greater than 95%) 50.0g

Filler 85.0g

Adhesive 5.0g

Lubricant 10.0g

Amount to 200.0g

Technique: make 1000 by the common process of preparing capsule, every capsules is containing JP-8g 50mg.

Usage and dosage: one day twice, each 2 ~ 3.

The preparation of embodiment 3, injection

Formula: JP-8g(purity is greater than 95%) 100.0g

Citric acid 1.0g

Sodium citrate 0.5g

Sodium chloride 18.0g

Water for injection 2000ml

Technique: by the common process operation of preparing injection, make altogether 1000 of the injections of 2ml, every containing JP-8g 100mg.

Usage and dosage: one day twice, each 1 ~ 2.

The adjuvants such as the filler in the various embodiments described above, disintegrating agent, adhesive, lubricant are adjuvant the most conventional on pharmaceutics.

Claims (2)

1. the application of chiral spiro indole-pyran pyridine alkaline compound in preparing anti-inflammatory drug; The structural formula of described chiral spiro indole-pyran pyridine alkaline compound is:

。

2. the application of chiral spiro indole-pyran pyridine alkaline compound in preparing anti-inflammatory drug as claimed in claim 1, it is characterized in that: take chiral spiro indole-pyran pyridine alkaline compound as active component, adopt acceptable technique and adjuvant on pharmaceutics, be prepared into the anti-inflammatory drug of various dosage forms.