CN103483220B - Compound, preparation method thereof and applications of compound in prevention of ulcerative colitis - Google Patents
Compound, preparation method thereof and applications of compound in prevention of ulcerative colitis Download PDFInfo
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Abstract
The invention discloses a compound, a preparation method thereof and applications of the compound in prevention of ulcerative colitis, and more specifically discloses bis{2-(butyroxy)-1-[(butyroxy)methyl]ethyl} 3, 3'-azo bis(6-hydroxybenzoic acid), and the preparation method and the applications thereof. The preparation method comprises following steps: 1, 3-dihydroxyacetone is taken as a synthesis raw material; a symmetrical compound 1, 3-dibutyryloxy-2-propanecarbonyl is synthesized; and dibutyrate 1, 3-dibutyryloxy-2-hydroxypropane is obtained by reduction of 1, 3-dibutyryloxy-2-propanecarbonyl; and then EDCI is taken as a dehydrating agent, DMAP is taken as a catalyst, and the target compound bis{2-(butyroxy)-1-[(butyroxy)methyl]ethyl} 3, 3'-azo bis(6-hydroxybenzoic acid) is obtained by ester condensation reaction of 1, 3-dibutyryloxy-2-propanecarbonyl and 3, 3'-azo bis(6-hydroxybenzoic acid). When bis{2-(butyroxy)-1-[(butyroxy)methyl]ethyl} 3, 3'-azo bis(6-hydroxybenzoic acid) is delivered into colon, 5-aminosalicylic acid and butyric acid are released by decomposition of bis{2-(butyroxy)-1-[(butyroxy)methyl]ethyl} 3, 3'-azo bis(6-hydroxybenzoic acid), and curing effect is realized by combined action of 5-aminosalicylic acid and butyric acid on colon focuses.
Description
Technical field
The invention belongs to medicament for resisting ulcerative colitis technical field, relate to two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3,3 '-azo two (6-hydroxy-benzoic acid) and its preparation method and application.
Background technology
Ulcerative colitis (ulcerative colitis, UC) was first described as by Wilks the independently colonic inflammation disease being different from bacillary dysentery in 1859.It is a kind of chronic nonspecific inflammation of mainly involving rectum, mucous membrane of colon, and clinical manifestation is mainly diarrhoea, stomachache, mucosanguineous feces etc.; Its pathological change is the tissue reaction of filling the air, and comprises the non-opposite sex of holding such as ulceration, crypt abscess, thin vessels inflammation, goblet cell minimizing and all kinds cell infiltration and shows.Can occur, as erythema nodosum, sacroiliitis, ankylosing spondylitis, sclerosing cholangitis, autoimmune hemolytic anemia etc. with various autoimmune disease.Course of disease protracted course of disease reaches the more than ten years, even decades have the possibility of canceration.Its sickness rate is about 3 ~ 14.3/10 ten thousand in American-European countries, China over nearly 10 years case more than 20,000.Epidemiologic data is pointed out, and the sickness rate of UC has the trend increased year by year at home and abroad.This sick cause and onset of disease mechanism is still not very clear; Be difficult to cure, very easily recur; The course of disease is tediously long, has a strong impact on patient body Health and Living quality, is classified as one of modern difficult treatment by the World Health Organization.
Summary of the invention
The problem that the present invention solves is to provide a kind of two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3,3 '-azo two (6-hydroxy-benzoic acid) and preparation method thereof and the pharmaceutical use for the preparation for the treatment of ulcerative colitis.
The present invention is achieved through the following technical solutions:
A kind of compound, this compound is two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3,3 '-azo two (6-hydroxy-benzoic acid), and its structural formula is:
Compound two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyl } 3, the preparation method of 3 '-azo two (6-hydroxy-benzoic acid), comprises following operation:
By 1,3-bis-butyryl acyloxy-2-hydroxy propane and 3, there is ester condensation reaction in 3 '-azo two (6-hydroxy-benzoic acid), generate two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3,3 '-azo two (6-hydroxy-benzoic acid).
Further, comprise the following steps:
1) 1,3-Dihydroxyacetone is become Lipase absobed compound 1,3-bis-butyryl acyloxy-2-carbonyl propane with butyryl oxide, then reduced and generate dibutyrate 1,3-bis-butyryl acyloxy-2-hydroxy propane;
2) adopt EDCI as dewatering agent, DMAP is as catalyzer, by 1,3-bis-butyryl acyloxy-2-hydroxy propane and 3, there is ester condensation reaction in 3 '-azo two (6-hydroxy-benzoic acid), synthesising target compound two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyl } 3,3 '-azo two (6-hydroxy-benzoic acid).
Described preparation specifically comprises the following steps:
1) 1,3-Dihydroxyacetone is dissolved in anhydrous propanone, stirred under nitrogen atmosphere, adds the butyryl oxide being dissolved in anhydrous propanone; drip pyridine, stirring at room temperature, fully concentrated solvent after reaction; with water and extraction into ethyl acetate residue, collected organic layer, uses HCl, NaHCO respectively
3washing, regulates pH to neutral, concentrates to obtain residue with anhydrous sodium sulfate drying; Residue dissolved and is splined on silicagel column, carrying out wash-out using the mixed solution of sherwood oil and ethyl acetate as elutriant, collecting product, steaming after desolventizing, obtain 1,3-bis-butyryl acyloxy-2-carbonyl propane;
1,3-bis-butyryl acyloxy-2-carbonyl propane is dissolved in the mixture of organic solvent and water, is cooled to 5 DEG C, adds NaBH in batches
4, fully add Glacial acetic acid after reaction, then with chloroform dilution, use water, sodium bicarbonate aqueous solution, water washing respectively, anhydrous magnesium sulfate drying, vacuum concentration obtains 1,3-bis-butyryl acyloxy-2-hydroxy propane;
2) by 1,3-bis-butyryl acyloxy-2-hydroxy propane dissolves in organic solvent, 3 are added under stirring, 3 '-azo two (6-hydroxy-benzoic acid) and DMAP, ice bath cools, and then adds EDCI, stirring at room temperature is fully reacted, wash with water and saturated NaCl respectively, anhydrous sodium sulfate drying, vacuum concentration solvent; Residue is dissolved and is splined on silicagel column, wash-out is carried out as elutriant using the mixed solution of sherwood oil and ethyl acetate, collect product, after steaming desolventizes, obtain two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3,3 '-azo two (6-hydroxy-benzoic acid).
Described sherwood oil and ethyl acetate are mixed to get elutriant according to the volume ratio of 10:1 ~ 5.
Two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3,3 '-azo two (6-hydroxy-benzoic acid) is preparing the application in medicament for resisting ulcerative colitis.
Described medicament for resisting ulcerative colitis is the medicine lowering mucosa injury exponential sum disease activity index.
Described medicament for resisting ulcerative colitis is the medicine reducing intestinal tissue MPO level.
Compared with prior art, the present invention has following useful technique effect:
Two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3 provided by the invention, 3 '-azo two (6-hydroxy-benzoic acid) (OLZ-G-B), that the collaborative prodrug of 5-aminosalicylic acid and butyric acid is to treat UC, when it is oral enter colon after, be decomposed into 5-aminosalicylic acid and butyric acid, then the two acting in conjunction plays therapeutic action in colon lesions position.
Two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3 provided by the invention, the preparation method of 3 '-azo two (6-hydroxy-benzoic acid), by 1,3-otan becomes Lipase absobed compound 1 with butyryl oxide, 3-bis-butyryl acyloxy-2-carbonyl propane, reduced generation 1,3-bis-butyryl acyloxy-2-hydroxy propane again; Adopt EDCI as dewatering agent, DMAP as catalyzer, by dibutyrate 1,3-bis-butyryl acyloxy-2-hydroxy propane and 1,3-bis-butyryl acyloxy-2-hydroxy propane generation ester condensation reaction, synthesising target compound.Its synthetic route is simple, and productive rate is high.
Two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3 provided by the invention, 3 '-azo two (6-hydroxy-benzoic acid), should in the preparation of medicament for resisting ulcerative colitis, effectively can lower mouse mucosa injury exponential sum and improve DAI degree, reduce intestinal tissue MPO level, and to showing as the mucous membrane of colon micropathological damage tool improvement result to a certain degree of serious acute chemical damage.Compare with Sodium propanecarboxylate mixture control group with use 5-aminosalicylic acid, be all better than 5-aminosalicylic acid and Sodium propanecarboxylate mixture control group at multiple Testing index and DAI index.
Accompanying drawing explanation
Fig. 1 is two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3, the effect diagram that 3 '-azo two (6-hydroxy-benzoic acid) (OLZ-G-B) changes Mouse Weight;
Fig. 2 is two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3, and 3 '-azo two (6-hydroxy-benzoic acid) (OLZ-G-B) is to the effect diagram of mouse DAI index variation;
Fig. 3 is two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3, and 3 '-azo two (6-hydroxy-benzoic acid) (OLZ-G-B) is to the effect diagram of mouse weight index of the colon;
Fig. 4 is two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3, and 3 '-azo two (6-hydroxy-benzoic acid) (OLZ-G-B) is to the effect diagram of mouse Colon mucosa injury index;
Fig. 5 is two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3, and 3 '-azo two (6-hydroxy-benzoic acid) (OLZ-G-B) organizes the effect diagram of MPO activity to mouse Colon.
Embodiment
The invention provides 5-ASA and butyric acid works in coordination with prodrug two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyl } 3, the effect of 3 '-azo two (6-hydroxy-benzoic acid) (OLZ-G-B) and preparation and resistive connection enteritis.Below in conjunction with specific embodiment, the present invention is described in further detail, and the explanation of the invention is not limited.
Adopt pyridine as catalyzer, two molecule butyryl oxides and 1,3-Dihydroxyacetone are reacted and generates ester; Again it is become alcohol with sodium borohydride reduction, with 3,3 '-azo two (6-hydroxy-benzoic acid) generates OLZ-G-B under EDCI/DMAP catalysis.By Fourier's infrared analysis, nucleus magnetic hydrogen spectrum analysis and mass spectroscopy determination target compound structure after synthesising target compound, and measure the Determination of oil-water partition coefficient (n-Octanol/water) of OLZ-G-B; Investigate the effect of OLZ-G-B anti-mouse experimental colitis on this basis.
1,5-ASA and segmented intestine targeted collaborative prodrug two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyl } 3 of butyric acid, the synthesis of 3 '-azo two (6-hydroxy-benzoic acid) (OLZ-G-B)
Two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3, two (6-hydroxy-benzoic acid) synthetic route of 3 '-azo:
Selection 1,3-Dihydroxyacetone is synthesis material, first synthesizes symmetrical compound 1,3-bis-butyryl acyloxy-2-carbonyl propane, then reduce generation dibutyrate 1,3-bis-butyryl acyloxy-2-hydroxy propane.Select to adopt EDCI as dewatering agent, DMAP is as catalyzer, make itself and 1,3-bis-butyryl acyloxy-2-hydroxy propane generation ester condensation reaction, generate target compound two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyl } 3,3 '-azo two (6-hydroxy-benzoic acid).
The synthesis of 1.21,3-bis-butyryl acyloxy-2-carbonyl propane
1.5g(16.7mmol) 1,3-Dihydroxyacetone is dissolved in 170mL anhydrous propanone, and stirred under nitrogen atmosphere adds the 10.91mL(67mmol being dissolved in 50mL anhydrous propanone in this heterogeneous solution) butyryl oxide, be added dropwise to 10mL pyridine, stirring at room temperature.Detection reaction progress, until otan reacts completely, vacuum concentration solvent.With water and extraction into ethyl acetate residue, get organic layer, use 1mol/LHCl, 1mol/LNaHCO respectively
3washing, regulates pH to neutral, concentrates, obtain colorless oil with anhydrous sodium sulfate drying.Residue is dissolved and is splined on silicagel column, sherwood oil: ethyl acetate (20: 1) is as eluent, and ethyl acetate ratio increases progressively.With 2,4 dinitrophenyl hydrazine as developer, collect product, solvent evaporated under reduced pressure, obtaining 1,3-bis-butyryl acyloxy-2-carbonyl propane 2.0g, is white crystals; Productive rate is 65.8%.Its fusing point is 47-48 DEG C.
Qualitative identification: at chloroform: methyl alcohol=5: under the development system of 1, silica gel G 254 thin layer plate does not show under 253.7nm ultraviolet wavelength, and after spraying 2,4 dinitrophenyl hydrazine oven dry, show a spot, Rf value is 0.70.
The structure elucidation of 1,3-bis-butyryl acyloxy-2-carbonyl propane:
1) 1,3-bis-butyryl acyloxy-2-carbonyl propane hydrogen spectrum
Proton nmr spectra (CDCl
3) ownership as follows:
δ 0.9848 (t, 6H) is three hydrogen on butyryl radicals α C, because there being two butyryl radicalies, so plant hydrogen totally six; δ 1.7153 (m, 4H) is two hydrogen on butyryl radicals β C, because there being two butyryl radicalies, so plant hydrogen totally four; δ 2.4151 (t, 4H) is two hydrogen on butyryl radicals γ C, because there being two butyryl radicalies, so plant hydrogen totally four; Four hydrogen on the C atom that δ 4.7609 (s, 4H) connects for otan two hydroxyls.
2) 1,3-bis-butyryl acyloxy-2-carbonyl propane mass spectrum
MS(ESI)m/z231[M+H]
+。
In sum, gained compound is 1,3-bis-butyryl acyloxy-2-carbonyl propane.
The synthesis of 1.31,3-bis-butyryl acyloxy-2-hydroxy propane
1.0g(5.5mmol) 1,3-bis-butyryl acyloxy-2-carbonyl propane is dissolved in 15mL tetrahydrofuran (THF) and 1mL water, and this heterogeneous solution is cooled to 5 DEG C, and a point short run adds 0.24gNaBH
4, be added dropwise to 1mL Glacial acetic acid after 30min to destroy excessive NaBH
4, then with chloroform dilution, use water, sodium bicarbonate aqueous solution, water washing respectively.Anhydrous magnesium sulfate drying, vacuum concentration obtains 1,3-bis-butyryl acyloxy-2-hydroxy propane 1.2g, productive rate 94.5%, colorless oil.
Qualitative identification: at chloroform: methyl alcohol=5: under the development system of 1, silica GF254 fluorescent plate under 253.7nm ultraviolet wavelength on display a spot, and spray 2,4 dinitrophenyl hydrazine dry after, immaculate, Rf value is 0.58.
1.4 2 { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3, the synthesis of 3 '-azo two (6-hydroxy-benzoic acid)
1.32g(5.66mmol) 1,3-bis-butyryl acyloxy-2-hydroxy propane is dissolved in 30mLCH
2cl
2in, add 0.425g(1.415mmol under stirring) 3,3 '-azo two (6-hydroxy-benzoic acid) and 130mg(1.06mmol) DMAP, ice bath cooling 20min.20mL CH will be dissolved in
2cl
21.6g(8.35mmol) EDCI instills in above-mentioned solution, stirring at room temperature 16h.Wash with water and saturated NaCl respectively, anhydrous sodium sulfate drying, vacuum concentration.Residue is dissolved and is splined on silicagel column, sherwood oil: ethyl acetate (10: 1) is as eluent, and ethyl acetate ratio increases progressively.Collect product, solvent evaporated obtains two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3, two (6-hydroxy-benzoic acid) 0.51g of 3 '-azo, yellow powder; Productive rate is 49.5%.Its fusing point is 234.0 DEG C.
Qualitative identification: at sherwood oil: ethyl acetate: under the development system of acetic acid=2mL: 1mL: 1 droplet, silica GF254 fluorescent plate under 253.7nm ultraviolet wavelength on a display spot, Rf value is 0.60.
Two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3, two (6-hydroxy-benzoic acid) structure elucidation of 3 '-azo:
1) two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3, two (6-hydroxy-benzoic acid) FTIR spectrum of 3 '-azo
At 3453.92cm
-1neighbouring broad peak is the absorption peak of the hydroxyl on phenyl ring, at 2970.17cm
-1, 2930.31cm
-1, 2877.60cm
-1, be ν CH3-, ν-CH2-on butyryl radicals, the absorption peak of ν-CH2-; At 1735.81cm
-1for the stretching vibration peak of ester bond C=O key; At 1180.35cm
-1the asymmetrical stretching vibration peak of ester bond C-O-C, therefore prove have ester bond to generate; At 840.81cm
-1, 860.70cm
-1for the charateristic avsorption band of phenyl ring.
2) two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3, two (6-hydroxy-benzoic acid) proton nmr spectra of 3 '-azo
Proton nmr spectra (DMSO) ownership is as follows:
δ 0.8865 (t, 12H) is three hydrogen on butyryl radicals α C, because there being 4 butyryl radicalies, so plant hydrogen totally 12; δ 1.5620 (m, 8H) is two hydrogen on butyryl radicals β C, because there being 4 butyryl radicalies, so plant hydrogen totally 8; δ 2.2682 (t, 8H) is two hydrogen on butyryl radicals γ C, because there being 4 butyryl radicalies, so plant hydrogen totally 8; δ 3.5145 (d, 2H) is a rear connected hydrogen for otan carbonyl C reduces, because there being 2 otans, so plant hydrogen totally 2; Four hydrogen on the C atom that δ 4.0072 (s, 8H) connects for otan two hydroxyls, because there being 2 otans, so plant hydrogen totally 8; δ 7.1996 (d, 2H), δ 8.0507 (d, 2H), δ 8.2372 (s, 2H) is benzene ring hydrogen; δ 10.8528 (s, 1H), δ 10.8813 (s, 1H) is the hydrogen of the hydroxyl on phenyl ring.
3) two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3, two (6-hydroxy-benzoic acid) mass spectrum of 3 '-azo
m/z729.3
In sum, this compound is two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3,3 '-azo two (6-hydroxy-benzoic acid).
Said synthesis route is simple, and productive rate is high.
Further, control is as follows in above process:
In the building-up process of 1,3-bis-butyryl acyloxy-2-carbonyl propane, reaction solvent should select anhydrous propanone; Two hydroxyls that reaction is 1,3-Dihydroxyacetone all become ester with butyryl oxide, should ensure that butyryl oxide is appropriate, about general selection 4 times of equivalents.In the building-up process of 1,3-bis-butyryl acyloxy-2-hydroxy propane, temperature of reaction need be strict controlled in less than 5 DEG C, ensures the generation without other by products.Two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3, in the building-up process of 3 '-azo two (6-hydroxy-benzoic acid), reaction system is strictly anhydrous.
Below, select the mouse TNBS colitis model similar to acute human and active ulcerativ e colitis, further illustrate two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3,3 '-azo two (6-hydroxy-benzoic acid) is for the therapeutic action of ulcerative colitis.
2, two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3,3 '-azo two (6-hydroxy-benzoic acid) (OLZ-G-B) is to the therapeutic action of mouse TNBS colitis
The foundation of 2.1 mouse TNBS colitis model
Mouse experiment room routine is raised, and 48 mouse are divided into 4 groups immediately, often organizes 12.With picric acid by each group of mouse label respectively, weigh and record successively.Before modeling, fasting 12h, freely drinks water.By the slight micro-anesthesia of ether of the mouse of fasting 12h, insert in mouse Colon with blunt nosed gastric perfusion needle per anum, head end is about about 4cm apart from anus, slow injected slurry volume mark is the 50% ethanolic soln 0.1ml of the TNBS of 2.5%, after keeping mouse to be inverted the downward posture 30s of head, put back to normal in cage raising; Normal group mouse gives normal saline enema.Administration is started after modeling 24h.
2.2 experiment grouping and observation index
Normal group, model control group, 5-aminosalicylic acid and Sodium propanecarboxylate mixture (5-ASA+BA) control group and two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3 are established in experiment, two (6-hydroxy-benzoic acid) (OLZ-G-B) group of 3 '-azo, gastric infusion, every day is administered once, successive administration 5 days.Normal and model control group all gives the CMC-Na aqueous solution of 0.5% of equivalent.5-aminosalicylic acid and Sodium propanecarboxylate mixture (5-ASA+BA) control group dosage are [100mg (5-ASA)+40mg (Sodium propanecarboxylate)] kg
-1; Two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3, two (6-hydroxy-benzoic acid) (OLZ-G-B) dosage 240mgkg of 3 '-azo
-1.After modeling the 6th day, put to death by the mouse anesthesia of fasting 12h, back of the body position is fixed.Mouse cuts belly open, takes out total colectomy.Longitudinally cut off colon along mesentery, rinse out intestinal contents with cold saline, the situation of visual inspection colonic mucosal injury.
Colonic mucosal injury index score standard: 0 point: not damaged; 1 point: without ulcer, contrafluxion; 2 points: have ulcer; 3 points: only a position exists ulcer and inflammation; There is ulcer and inflammation in 4 points: two multiple positions; 5 points: ulcer is more than 2cm.
Measure total colectomy length and weigh, calculating weight index of the colon (total colectomy weight/total colectomy length).Get colonic pathological change site tissue one piece ,-20 DEG C of preservations, measure MPO active.
Other observation index: 1) add up each group of dead mouse situation; 2) Mouse Weight change is observed; 3) observe mouse disease activity index (Disease activity index, DAI), DAI standards of grading are in table 1; Wherein stool in mice is occulted blood to measure and is adopted o tolidine, and criterion is in table 2.
Table 1 mouse DAI standards of grading
Table 2 o tolidine measures fecal occult blood result of determination
| Result | Phenomenon |
| Normally | Still do not develop the color after adding reagent 2min |
| Occult blood+ | After adding reagent 10s, just aobvious light blue, become blue gradually |
| Occult blood ++ | After adding reagent, just aobvious light blue, turn blue brown gradually |
| Occult blood +++ | Blue brown is shown immediately after adding reagent |
MPO determination of activity: adopt dianisidine method.Get mouse Colon tissue to weigh, add the pH7.0PBS of 9 times of volumes, the centrifugal 30min of 4 DEG C of homogenate 30s, 12000rpm, abandons supernatant liquor, takes off the HTAB solution that layer tissue 50mg adds 0.5%, 4 DEG C of homogenate 30s, 12000rpm pelleted by centrifugation 15min.Get supernatant liquor 0.1m, add ODD solution 2.9ml, mix fast, in 460nm METHOD FOR CONTINUOUS DETERMINATION 3min, calculate per minute absorbancy changing value.Being defined in the amount that per minute under 25 DEG C of conditions decomposes 1 micromole's superoxide is a unit of enzyme activity.
The enzymic activity of 100 μ l sample tissue units is:
Note: 1molL
-1h
2o
2absorbancy change be 1.13 × 10
4, so according to definition, 1 unit MPO is 1 μm of olL
-1h
2o
2absorbance, be 1.13 × 10
-2.
2.3 statistical analysis
Experimental data is with means standard deviation
represent, statistical method adopts t check analysis.P<0.05 is that difference has statistical significance.
2.4 experimental result
1) on the impact of mouse survival situation: Normal group 12, TNBS group 7,5-aminosalicylic acid and Sodium propanecarboxylate mixture (5-ASA+BA) control group 6, two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3,3 '-azo two (6-hydroxy-benzoic acid) (OLZ-G-B) organizes 6.
2) on the impact of Mouse Weight change: each group Mouse Weight result of variations as described in Figure 1.After modeling, Mouse Weight declines, and after pharmacological agent, body weight is gone up gradually.Two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3,3 '-azo two (6-hydroxy-benzoic acid) (OLZ-G-B) is organized Mouse Weight and is recovered the most obvious.
3) on the impact of mouse DAI index: after TNBS modeling, mouse all there will be bloody stool, just rare, movable, feed minimizing after 24h, the situations such as weight loss.The DAI index variation of each group of mouse as shown in Figure 2, TNBS group mouse DAI index obviously raises, modeling is basicly stable after 3 days, after drug treatment, DAI index significantly reduces, two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3, two (6-hydroxy-benzoic acid) (OLZ-G-B) impact on DAI index of 3 '-azo is better than 5-aminosalicylic acid and Sodium propanecarboxylate mixture (5-ASA+BA) control group.
4) on the impact of mouse weight index of the colon and colonic mucosal injury index
Mucous hyperemia oedema is there is in mouse Colon after the process of TNBS bowel lavage, intestines wall thickening, there are necrosis and ulceration in surface, and compared with normal group, the mucosa injury index of TNBS group significantly increases (P < 0.01) and shows that Mouse Ulcerative Colitis Model is set up good; Compared with TNBS group, two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3, the mucosa injury index that 3 '-azo two (6-hydroxy-benzoic acid) (OLZ-G-B) is organized significantly declines (P < 0.01).Some animals occurs that the damage of colon transmural is downright bad, and enteron aisle exists hardening, distortion or narrow in various degree.Colon lengths shortens, and its weight index of the colon significantly increases (P < 0.01), and compared with TNBS group, the mouse weight index of the colon of administration group declines (P < 0.01) all to some extent.Experimental result as shown in Figure 3, Figure 4.
5) on the impact of MPO activity in mouse Colon tissue
Mouse Colon, after the process of TNBS bowel lavage, obviously raises because MPO in the colons such as inflammation, oedema, ulceration is active.Compared with normal group, in TNBS group mouse Colon tissue, MPO is active significantly increases (P < 0.01); Compared with TNBS group, two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3,3 '-azo two (6-hydroxy-benzoic acid) (OLZ-G-B) organizes the active significantly decline (P < 0.01) of MPO in mouse Colon tissue.Experimental result as shown in Figure 5.
To sum up, experimental result shows, two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3 provided, 3 '-azo two (6-hydroxy-benzoic acid) (OLZ-G-B) effectively can lower Rat mucosal damage index and improve DAI degree, reduce colon MPO level, and to showing as the mucous membrane of colon micropathological damage tool improvement result to a certain degree of serious acute chemical damage.
Claims (8)
1. compound two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyl } 3, the preparation method of 3 '-azo two (6-hydroxy-benzoic acid), is characterized in that, comprise following operation:
By 1,3-bis-butyryl acyloxy-2-hydroxy propane and 3, there is ester condensation reaction in 3 '-azo two (6-hydroxy-benzoic acid), generate two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3,3 '-azo two (6-hydroxy-benzoic acid).
2. compound two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyl } 3 as claimed in claim 1, the preparation method of 3 '-azo two (6-hydroxy-benzoic acid), is characterized in that, comprise the following steps:
1) 1,3-Dihydroxyacetone is become Lipase absobed compound 1,3-bis-butyryl acyloxy-2-carbonyl propane with butyryl oxide, then reduced and generate dibutyrate 1,3-bis-butyryl acyloxy-2-hydroxy propane;
2) adopt EDCI as dewatering agent, DMAP is as catalyzer, by 1,3-bis-butyryl acyloxy-2-hydroxy propane and 3, there is ester condensation reaction in 3 '-azo two (6-hydroxy-benzoic acid), synthesising target compound two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyl } 3,3 '-azo two (6-hydroxy-benzoic acid).
3. compound two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyl } 3 as claimed in claim 2, the preparation method of 3 '-azo two (6-hydroxy-benzoic acid), it is characterized in that, specifically comprise the following steps:
1) 1,3-Dihydroxyacetone is dissolved in anhydrous propanone, stirred under nitrogen atmosphere, adds the butyryl oxide being dissolved in anhydrous propanone; drip pyridine, stirring at room temperature, fully concentrated solvent after reaction; with water and extraction into ethyl acetate residue, collected organic layer, uses HCl, NaHCO respectively
3washing, regulates pH to neutral, concentrates to obtain residue with anhydrous sodium sulfate drying; Residue dissolved and is splined on silicagel column, carrying out wash-out using the mixed solution of sherwood oil and ethyl acetate as elutriant, collecting product, steaming after desolventizing, obtain 1,3-bis-butyryl acyloxy-2-carbonyl propane;
1,3-bis-butyryl acyloxy-2-carbonyl propane is dissolved in the mixture of organic solvent and water, is cooled to 5 DEG C, adds NaBH in batches
4, fully add Glacial acetic acid after reaction, then with chloroform dilution, use water, sodium bicarbonate aqueous solution, water washing respectively, anhydrous magnesium sulfate drying, vacuum concentration obtains 1,3-bis-butyryl acyloxy-2-hydroxy propane;
2) by 1,3-bis-butyryl acyloxy-2-hydroxy propane dissolves in organic solvent, 3 are added under stirring, 3 '-azo two (6-hydroxy-benzoic acid) and DMAP, ice bath cools, and then adds EDCI, stirring at room temperature is fully reacted, wash with water and saturated NaCl respectively, anhydrous sodium sulfate drying, vacuum concentration solvent; Residue is dissolved and is splined on silicagel column, wash-out is carried out as elutriant using the mixed solution of sherwood oil and ethyl acetate, collect product, after steaming desolventizes, obtain two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3,3 '-azo two (6-hydroxy-benzoic acid).
4. compound two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyl } 3 as claimed in claim 3, the preparation method of 3 '-azo two (6-hydroxy-benzoic acid), it is characterized in that, described sherwood oil and ethyl acetate are mixed to get elutriant according to the volume ratio of 10:1 ~ 5.
5. adopt the compound that the method in Claims 1 to 4 described in any one is obtained, it is characterized in that, this compound is two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyls } 3,3 '-azo two (6-hydroxy-benzoic acid), its structural formula is:
6. compound two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyl } 3 according to claim 5,3 '-azo two (6-hydroxy-benzoic acid) is preparing the application in medicament for resisting ulcerative colitis.
7. apply as claimed in claim 6, it is characterized in that, described medicament for resisting ulcerative colitis is the medicine lowering mucosa injury exponential sum disease activity index.
8. apply as claimed in claim 6, it is characterized in that, described medicament for resisting ulcerative colitis is the medicine reducing intestinal tissue MPO level.
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