CN103483220A - 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 the medicament for resisting ulcerative colitis technical field, relate to two 2-(butyryl acyloxy)-1-[(butyryl acyloxy) and methyl] ethyl } 3,3 '-azo two (6-hydroxy-benzoic acid) and its preparation method and application.
Background technology
Ulcerative colitis (ulcerative colitis, UC) at first was described as being different from the independently colonic inflammation disease of bacillary dysentery by Wilks 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 that the non-opposite sex of holding such as ulceration, crypt abscess, polyangitis disease, goblet cell minimizing and all kinds cell infiltration show.Can occur with the various autoimmune disease, as erythema nodosum, sacroiliitis, ankylosing spondylitis, sclerosing cholangitis, autoimmune hemolytic anemia etc.Course of disease protracted course of disease reaches the more than ten years, even decades the possibility of canceration is arranged.Its sickness rate is about 3～14.3/10 ten thousand in American-European countries, China over nearly 10 years case surpass 20,000.The epidemiologic data prompting, the sickness rate of UC has the trend increased year by year at home and abroad.This disease 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, by the World Health Organization, is classified as one of modern difficult treatment.
Summary of the invention
That the problem that the present invention solves is to provide is a kind of two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyl } 3,3 '-azo two (6-hydroxy-benzoic acid) and preparation method thereof and for the preparation of the pharmaceutical use 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] ethyl } 3,3 '-azo two (6-hydroxy-benzoic acid), its structural formula is:
Compound two 2-(butyryl acyloxy)-1-[(butyryl acyloxy) and methyl] ethyl } 3, the preparation method of 3 '-azo two (6-hydroxy-benzoic acid) comprises following operation:
By 1,3-bis-butyryl acyloxies-2-hydroxy propane and 3, ester condensation reaction occurs in 3 '-azo two (6-hydroxy-benzoic acids), generate two 2-(butyryl acyloxy)-1-[(butyryl acyloxy) and methyl] ethyl } 3,3 '-azo two (6-hydroxy-benzoic acid).
Further, comprise the following steps:
1) 1,3-Dihydroxyacetone is become to ester synthetic compound 1 with butyryl oxide, 3-bis-butyryl acyloxies-2-carbonyl propane, then its reduction is generated to dibutyrate 1,3-bis-butyryl acyloxies-2-hydroxy propane;
2) adopt EDCI as dewatering agent, DMAP is as catalyzer, by 1,3-bis-butyryl acyloxies-2-hydroxy propane and 3, ester condensation reaction occurs in 3 '-azo two (6-hydroxy-benzoic acid), synthesising target compound two 2-(butyryl acyloxy)-1-[(butyryl acyloxy) and 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, stirs under nitrogen protection, add the butyryl oxide that is dissolved in anhydrous propanone; drip pyridine, stirring at room, fully concentrated solvent after reaction; water and ethyl acetate extraction leftover, collected organic layer, use respectively HCl, NaHCO
3washing, regulate pH to neutral, with anhydrous sodium sulfate drying, concentrates to obtain residue; Residue is dissolved and be splined on silicagel column, the mixed solution of sherwood oil and ethyl acetate of usining carries out wash-out as elutriant, collects product, after steaming and desolventizing, obtains 1,3-, bis-butyryl acyloxies-2-carbonyl propane;
1,3-, bis-butyryl acyloxies-2-carbonyl propane is dissolved in the mixture of organic solvent and water, is cooled to 5 ℃, add in batches NaBH
4, fully after reaction, add Glacial acetic acid, then with the chloroform dilution, water, sodium bicarbonate aqueous solution, water washing respectively, anhydrous magnesium sulfate drying, vacuum concentration obtains 1,3-, bis-butyryl acyloxies-2-hydroxy propane;
2) by 1,3-bis-butyryl acyloxies-2-hydroxy propane is dissolved in organic solvent, add 3 under stirring, 3 '-azo two (6-hydroxy-benzoic acid) and DMAP, ice bath is cooling, then adds EDCI, stirring at room is fully reacted, water and saturated NaCl washing respectively, anhydrous sodium sulfate drying, vacuum concentration solvent; Residue is dissolved and be splined on silicagel column, the mixed solution of sherwood oil and ethyl acetate of usining carries out wash-out as elutriant, collects product, after steaming and desolventizing, two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyl } 3,3 '-azo two (6-hydroxy-benzoic acid).
Described sherwood oil and ethyl acetate are that the volume ratio according to 10:1～5 is mixed to get elutriant.
Two 2-(butyryl acyloxy)-1-[(butyryl acyloxy) and methyl] ethyl } 3, two (6-hydroxy-benzoic acid) application in preparing medicament for resisting ulcerative colitis of 3 '-azo.
Described medicament for resisting ulcerative colitis is for lowering the medicine of mucosa injury exponential sum disease activity index.
Described medicament for resisting ulcerative colitis is for reducing the medicine of intestinal tissue MPO level.
Compared with prior art, the present invention has following useful technique effect:
Provided by the invention two 2-(butyryl acyloxy)-1-[(butyryl acyloxy) and methyl] ethyl } 3,3 '-azo two (6-hydroxy-benzoic acid) (OLZ-G-B), that the collaborative prodrug of 5-aminosalicylic acid and butyric acid is with treatment UC, when it is oral enter colon after, be decomposed into 5-aminosalicylic acid and butyric acid, then the two acting in conjunction is in colon lesions position performance therapeutic action.
Provided by the invention two 2-(butyryl acyloxy)-1-[(butyryl acyloxy) and methyl] ethyl } 3, the preparation method of 3 '-azo two (6-hydroxy-benzoic acid), by 1, the 3-otan becomes ester synthetic compound 1 with butyryl oxide, 3-bis-butyryl acyloxies-2-carbonyl propane, again its reduction is generated to 1,3-, bis-butyryl acyloxies-2-hydroxy propane; Adopt EDCI as dewatering agent, DMAP is as catalyzer, by dibutyrate 1, and 3-bis-butyryl acyloxies-2-hydroxy propane and 1,3-, bis-butyryl acyloxies-2-hydroxy propane generation ester condensation reaction, synthesising target compound.Its synthetic route is simple, and productive rate is high.
Provided by the invention two 2-(butyryl acyloxy)-1-[(butyryl acyloxy) and methyl] ethyl } 3,3 '-azo two (6-hydroxy-benzoic acid), should be in the preparation of medicament for resisting ulcerative colitis, can effectively lower mouse mucosa injury exponential sum and improve the DAI degree, reduce intestinal tissue MPO level, and to showing as the mucous membrane of colon micropathological damage tool improvement effect to a certain degree of serious acute chemical damage.Compare with Sodium propanecarboxylate mixture control group with using 5-aminosalicylic acid, in a plurality of detection indexs and DAI index, all be better than 5-aminosalicylic acid and Sodium propanecarboxylate mixture control group.
The accompanying drawing explanation
Fig. 1 is two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyl } 3,3 '-azo two (6-hydroxy-benzoic acid) (OLZ-G-B) changes Mouse Weight affects figure;
Fig. 2 is two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyl } 3,3 '-azo two (6-hydroxy-benzoic acid) is (OLZ-G-B) on the figure that affects of mouse DAI index variation;
Fig. 3 is two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyl } 3,3 '-azo two (6-hydroxy-benzoic acid) is (OLZ-G-B) on the figure that affects of mouse weight index of the colon;
Fig. 4 is two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyl } 3,3 '-azo two (6-hydroxy-benzoic acid) is (OLZ-G-B) on the figure that affects of mouse colonic mucosal injury index;
Fig. 5 is two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyl } 3,3 '-azo two (6-hydroxy-benzoic acid) is (OLZ-G-B) on the figure that affects of the MPO of mouse colon activity.
Embodiment
The invention provides the collaborative prodrug two of 5-ASA and butyric acid { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyl } 3,3 '-azo two (6-hydroxy-benzoic acid) (OLZ-G-B) and the effect of 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 are reacted with 1,3-Dihydroxyacetone and generate ester; Again it is become to alcohol with sodium borohydride reduction, with 3,3 '-azo two (6-hydroxy-benzoic acid) generates OLZ-G-B under EDCI/DMAP catalysis.Determine the target compound structure by Fourier's infrared analysis, nucleus magnetic hydrogen spectrum analysis and mass spectroscopy after synthesising target compound, and measure the Determination of oil-water partition coefficient (n-Octanol/water) of OLZ-G-B; Investigate on this basis the effect of the anti-mouse experiment colitis of OLZ-G-B.
1, the segmented intestine targeted collaborative prodrug two of 5-ASA and butyric acid { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyl } 3, (OLZ-G-B) synthetic of 3 '-azo two (6-hydroxy-benzoic acid)
Two 2-(butyryl acyloxy)-1-[(butyryl acyloxy) and methyl] ethyl } 3, two (6-hydroxy-benzoic acid) synthetic routes of 3 '-azo:
The selection 1,3-Dihydroxyacetone is synthesis material, first synthesizes symmetrical compound 1,3-bis-butyryl acyloxies-2-carbonyl propane, then its reduction is generated to dibutyrate 1,3-bis-butyryl acyloxies-2-hydroxy propane.Select to adopt EDCI as dewatering agent, DMAP is as catalyzer, make itself and 1,3-bis-butyryl acyloxies-2-hydroxy propane generation ester condensation reaction, generation target compound two 2-(butyryl acyloxy)-1-[(butyryl acyloxy) and methyl] ethyl } 3,3 '-azo two (6-hydroxy-benzoic acid).
1.21,3-bis-butyryl acyloxies-2-carbonyl propane synthetic
1.5g(16.7mmol) 1,3-Dihydroxyacetone is dissolved in the 170mL anhydrous propanone, under nitrogen protection, stirs, and adds the 10.91mL(67mmol that is dissolved in the 50mL anhydrous propanone in this heterogeneous solution) butyryl oxide, be added dropwise to the 10mL pyridine, stirring at room.The detection reaction progress, until otan reacts completely, the vacuum concentration solvent.Water and ethyl acetate extraction leftover, get organic layer, uses respectively 1mol/LHCl, 1mol/LNaHCO
3washing, regulate pH to neutral, concentrated with anhydrous sodium sulfate drying, obtains colorless oil.Residue is dissolved and be splined on silicagel column, sherwood oil: ethyl acetate (20: 1) is as eluent, and the ethyl acetate ratio increases progressively.As developer, collect product with 2,4 dinitrophenyl hydrazine, solvent evaporated under reduced pressure, obtain 1,3-, bis-butyryl acyloxies-2-carbonyl propane 2.0g, is white crystals; Productive rate is 65.8%.Its fusing point is 47-48 ℃.
Qualitative identification: at chloroform: under the development system of methyl alcohol=5: 1, silica gel G 254 thin layer plates do not show under the 253.7nm ultraviolet wavelength, after spraying 2,4 dinitrophenyl hydrazine and drying, show a spot, and the Rf value is 0.70.
The structure elucidation of 1,3-, bis-butyryl acyloxies-2-carbonyl propane:
1) 1,3-bis-butyryl acyloxies-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 two butyryl radicalies are arranged, so plant totally six of hydrogen; δ 1.7153 (m, 4H) is two hydrogen on butyryl radicals β C, because two butyryl radicalies are arranged, so plant totally four of hydrogen; δ 2.4151 (t, 4H) is two hydrogen on butyryl radicals γ C, because two butyryl radicalies are arranged, so plant totally four of hydrogen; Four hydrogen on the C atom that δ 4.7609 (s, 4H) connects for two hydroxyls of otan.
2) 1,3-bis-butyryl acyloxies-2-carbonyl propane mass spectrum
MS(ESI)m/z231[M+H]
+。
In sum, the gained compound is 1,3-, bis-butyryl acyloxies-2-carbonyl propane.
1.31,3-bis-butyryl acyloxies-2-hydroxy propane synthetic
1.0g(5.5mmol) 1,3-bis-butyryl acyloxies-2-carbonyl propane is dissolved in 15mL tetrahydrofuran (THF) and 1mL water, and this heterogeneous solution is cooled to 5 ℃, and a minute short run adds 0.24gNaBH
4, be added dropwise to the 1mL Glacial acetic acid after 30min to destroy excessive NaBH
4, then with chloroform dilution, water, sodium bicarbonate aqueous solution, water washing respectively.Anhydrous magnesium sulfate drying, vacuum concentration obtains 1,3-, bis-butyryl acyloxies-2-hydroxy propane 1.2g, productive rate 94.5%, colorless oil.
Qualitative identification: at chloroform: under the development system of methyl alcohol=5: 1, the silica GF254 fluorescent plate shows a spot under the 253.7nm ultraviolet wavelength, and after spraying 2,4 dinitrophenyl hydrazine and drying, immaculate, the Rf value is 0.58.
Two 1.4 { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyl } 3,3 '-azo two (6-hydroxy-benzoic acid) synthetic
1.32g(5.66mmol) 1,3-bis-butyryl acyloxies-2-hydroxy propane is dissolved in 30mLCH
2cl
2in, add 0.425g(1.415mmol under stirring) 3,3 '-azo two (6-hydroxy-benzoic acids) and 130mg(1.06mmol) DMAP, the cooling 20min of ice bath.To be dissolved in 20mL CH
2cl
21.6g(8.35mmol) EDCI splashes in above-mentioned solution, stirring at room 16h.Water and saturated NaCl washing respectively, anhydrous sodium sulfate drying, vacuum concentration.Residue is dissolved and be splined on silicagel column, sherwood oil: ethyl acetate (10: 1) is as eluent, and the ethyl acetate ratio increases progressively.Collect product, solvent evaporated obtains two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyl } 3, two (6-hydroxy-benzoic acid) 0.51g of 3 '-azo, yellow powder; Productive rate is 49.5%.Its fusing point is 234.0 ℃.
Qualitative identification: at sherwood oil: ethyl acetate: acetic acid=2mL: 1mL: under the development system of 1, the silica GF254 fluorescent plate shows a spot under the 253.7nm ultraviolet wavelength, and the Rf value is 0.60.
Two 2-(butyryl acyloxy)-1-[(butyryl acyloxy) and methyl] ethyl } 3, two (6-hydroxy-benzoic acid) structure elucidations of 3 '-azo:
1) two 2-(butyryl acyloxy)-1-[(butyryl acyloxy) and methyl] ethyl } 3, two (6-hydroxy-benzoic acid) the Fourier's infrared spectras of 3 '-azo
At 3453.92cm
-1the absorption peak that near broad peak is the hydroxyl on phenyl ring, at 2970.17cm
-1, 2930.31cm
-1, 2877.60cm
-1, be ν CH3-on butyryl radicals, ν-CH2-, the absorption peak of ν-CH2-; At 1735.81cm
-1stretching vibration peak for ester bond C=O key; At 1180.35cm
-1the asymmetrical stretching vibration peak of ester bond C-O-C, therefore proof has ester bond to generate; At 840.81cm
-1, 860.70cm
-1charateristic avsorption band for phenyl ring.
2) two 2-(butyryl acyloxy)-1-[(butyryl acyloxy) and methyl] ethyl } 3, two (6-hydroxy-benzoic acid) proton nmr spectras 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 4 butyryl radicalies are arranged, so plant totally 12 of hydrogen; δ 1.5620 (m, 8H) is two hydrogen on butyryl radicals β C, because 4 butyryl radicalies are arranged, so plant totally 8 of hydrogen; δ 2.2682 (t, 8H) is two hydrogen on butyryl radicals γ C, because 4 butyryl radicalies are arranged, so plant totally 8 of hydrogen; δ 3.5145 (d, 2H) is hydrogen of connected after otan carbonyl C reduction, because 2 otans are arranged, so plant totally 2 of hydrogen; Four hydrogen on the C atom that δ 4.0072 (s, 8H) connects for two hydroxyls of otan, because 2 otans are arranged, so plant totally 8 of hydrogen; δ 7.1996 (d, 2H), δ 8.0507 (d, 2H), δ 8.2372 (s, 2H) is the benzene ring hydrogen; δ 10.8528 (s, 1H), the hydrogen that δ 10.8813 (s, 1H) is the hydroxyl on phenyl ring.
3) two 2-(butyryl acyloxy)-1-[(butyryl acyloxy) and methyl] ethyl } 3, two (6-hydroxy-benzoic acid) mass spectrums of 3 '-azo
m/z729.3
In sum, this compound be two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyl } 3,3 '-azo two (6-hydroxy-benzoic acid).
Said synthesis route is simple, and productive rate is high.
Further, in said process, control is as follows:
In the building-up process of 1,3-, bis-butyryl acyloxies-2-carbonyl propane, reaction solvent should be selected anhydrous propanone; Reaction all becomes ester for two hydroxyls of 1,3-Dihydroxyacetone with butyryl oxide, should guarantee that butyryl oxide is appropriate, generally selects 4 times of equivalent left and right to get final product.In the building-up process of 1,3-, bis-butyryl acyloxies-2-hydroxy propane, temperature of reaction need be strict controlled in below 5 ℃, guarantees the generation without other by products.Two 2-(butyryl acyloxy)-1-[(butyryl acyloxy) and methyl] ethyl } 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) and methyl] ethyl } 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] ethyl } 3,3 '-azo two (6-hydroxy-benzoic acid) is (OLZ-G-B) to the therapeutic action of mouse TNBS colitis
2.1 the foundation of mouse TNBS colitis model
The mouse experiment chamber is conventional raises, and 48 mouse are divided into 4 groups immediately, 12 every group.With picric acid, by each group mouse label respectively, weigh and record successively.Fasting 12h before modeling, freely drink water.By the slight micro-anesthesia of ether for the mouse of fasting 12h, insert the mouse colonic with blunt nosed gavage pin per anum, head end is apart from the about 4cm of anus left and right, slowly inject the 50% ethanolic soln 0.1ml of the TNBS that volume fraction is 2.5%, after keeping mouse to be inverted a downward posture 30s, put back to normal raising the in cage; The Normal group mouse gives normal saline enema.Start administration 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] ethyl } 3 established in experiment, 3 '-azo two (6-hydroxy-benzoic acid) is group (OLZ-G-B), gastric infusion, be administered once every day, successive administration 5 days.Normal and model control group all gives 0.5% the CMC-Na aqueous solution 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) and methyl] ethyl } 3,3 '-azo two (6-hydroxy-benzoic acid) is dosage 240mgkg (OLZ-G-B)
-1.After modeling the 6th day, the mouse anesthesia of fasting 12h to be put to death, back of the body position is fixing.Mouse is cut belly open, takes out total colectomy.Vertically cut off colon along mesentery, with cold saline, rinse out intestinal contents, the situation of visual inspection colonic mucosal injury.
Colonic mucosal injury index standards of grading: 0 minute: not damaged; 1 minute: without ulcer, contrafluxion; 2 minutes: ulcer is arranged; 3 minutes: only a position existed ulcer and inflammation; 4 minutes: two a plurality of positions existed ulcer and inflammation; 5 minutes: ulcer surpassed 2cm.
Measure total colectomy length and weigh, calculating weight index of the colon (total colectomy weight/total colectomy length).Get one, colonic pathological change position tissue ,-20 ℃ of preservations, measure the MPO activity.
Other observation index: 1) statistics is respectively organized the dead mouse situation; 2) observing Mouse Weight changes; 3) observe mouse disease activity index (Disease activity index, DAI), the DAI standards of grading are in Table 1; Wherein stool in mice is occulted blood to measure and is adopted adjacent first benzidine method, and criterion is in Table 2.
Table 1 mouse DAI standards of grading
The adjacent first benzidine method of table 2 is measured the fecal occult blood result of determination
| Result | Phenomenon |
| Normally | After adding reagent 2min, still do not develop the color |
| Occult blood+ | After adding reagent 10s, just aobvious light blue, become gradually blue |
| Occult blood ++ | After adding reagent, just aobvious light blue, turn gradually blue brown |
| Occult blood +++ | Show immediately blue brown after adding reagent |
MPO determination of activity: adopt the dianisidine method.Get mouse colon and weigh, add the pH7.0PBS of 9 times of volumes, 4 ℃ of homogenate 30s, the centrifugal 30min of 12000rpm, abandon supernatant liquor, takes off layer tissue 50mg and add 0.5% HTAB solution, 4 ℃ of homogenate 30s, the centrifugal 15min of 12000rpm condition.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 ℃ of conditions decomposes 1 micromole's superoxide is a unit of enzyme activity.
The enzymic activity of 100 μ l sample tissue units is:
Annotate: 1molL
-1h
2o
2absorbancy to change be 1.13 * 10
4so, according to definition, 1 MPO of unit is 1 μ molL
-1h
2o
2absorbance, be 1.13 * 10
-2.
2.3 statistical analysis
Experimental data is with means standard deviation
mean, statistical method adopts the t check analysis.There is statistical significance P<0.05 for difference.
2.4 experimental result
1) on the impact of mouse survival situation: 12 of Normal groups, 7 of TNBS groups, 6 of 5-aminosalicylic acid and Sodium propanecarboxylate mixture (5-ASA+BA) control groups, two 2-(butyryl acyloxy)-1-[(butyryl acyloxy) and methyl] ethyl } 3,3 '-azo two (6-hydroxy-benzoic acid) (OLZ-G-B) is organized 6.
2) impact Mouse Weight changed: each organizes the Mouse Weight result of variations as described in Figure 1.After modeling, Mouse Weight descends, and after pharmacological agent, body weight is gone up gradually.Two 2-(butyryl acyloxy)-1-[(butyryl acyloxy) and methyl] ethyl } 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 the TNBS modeling, mouse all there will be bloody stool, just rare after 24h, and movable, feed reduces, the situations such as weight loss.Each DAI index variation of organizing mouse as shown in Figure 2, TNBS group mouse DAI index obviously raises, modeling is basicly stable after 3 days, after drug treatment, the DAI index significantly reduces, two 2-(butyryl acyloxy)-1-[(butyryl acyloxy) and methyl] ethyl } 3,3 '-azo two (6-hydroxy-benzoic acid) (OLZ-G-B) is better than 5-aminosalicylic acid and Sodium propanecarboxylate mixture (5-ASA+BA) control group to the impact of DAI index.
4) on the impact of mouse weight index of the colon and colonic mucosal injury index
The mouse colon mucous hyperemia oedema occurs after the TNBS bowel lavage is processed, the intestines wall thickening, and there are necrosis and ulceration in surface, with normal group, compares, and the mucosa injury index of TNBS group significantly increases (P<0.01) and shows that Mouse Ulcerative Colitis Model foundation is good; Compare two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyl } 3, the mucosa injury index that 3 '-azo two (6-hydroxy-benzoic acid) (OLZ-G-B) is organized significantly descend (P<0.01) with the TNBS group.The saturating wall damage of colon necrosis appears in the part mouse, and there be hardening, distortion or narrow in various degree in enteron aisle.The colon contraction in length, its weight index of the colon significantly increases (P<0.01), with the TNBS group, compares, and the mouse weight index of the colon of administration group is decline (P<0.01) to some extent all.Experimental result as shown in Figure 3, Figure 4.
5) on the impact of MPO activity in mouse colon
The mouse colon, after the TNBS bowel lavage is processed, obviously raises because MPO in the colons such as inflammation, oedema, ulceration is active.Compare active significantly increase (P<0.01) of MPO in TNBS group mouse colon with normal group; With the TNBS group, compare, two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyl } 3,3 '-azo two (6-hydroxy-benzoic acid) (OLZ-G-B) organize the active significantly decline (P<0.01) of MPO in mouse colon.Experimental result as shown in Figure 5.
To sum up, experimental result shows, two { 2-(the butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyl } 3 provided, 3 '-azo two (6-hydroxy-benzoic acid) (OLZ-G-B) can effectively lower rat mucosa injury exponential sum and improve the DAI degree, reduce the MPO of colon level, and to showing as the mucous membrane of colon micropathological damage tool improvement effect to a certain degree of serious acute chemical damage.
Claims (8)
1. a compound, is characterized in that, this compound is two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyl } 3, and 3 '-azo two (6-hydroxy-benzoic acid), its structural formula is:
2. 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, comprises following operation:
By 1,3-bis-butyryl acyloxies-2-hydroxy propane and 3, ester condensation reaction occurs in 3 '-azo two (6-hydroxy-benzoic acids), generate two 2-(butyryl acyloxy)-1-[(butyryl acyloxy) and methyl] ethyl } 3,3 '-azo two (6-hydroxy-benzoic acid).
3. compound two as claimed in claim 2 { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyl } 3, the preparation method of 3 '-azo two (6-hydroxy-benzoic acid), is characterized in that, comprises the following steps:
1) 1,3-Dihydroxyacetone is become to ester synthetic compound 1 with butyryl oxide, 3-bis-butyryl acyloxies-2-carbonyl propane, then its reduction is generated to dibutyrate 1,3-bis-butyryl acyloxies-2-hydroxy propane;
2) adopt EDCI as dewatering agent, DMAP is as catalyzer, by 1,3-bis-butyryl acyloxies-2-hydroxy propane and 3, ester condensation reaction occurs in 3 '-azo two (6-hydroxy-benzoic acid), synthesising target compound two 2-(butyryl acyloxy)-1-[(butyryl acyloxy) and methyl] ethyl } 3,3 '-azo two (6-hydroxy-benzoic acid).
4. compound two as claimed in claim 3 { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyl } 3, the preparation method of 3 '-azo two (6-hydroxy-benzoic acid), is characterized in that, specifically comprises the following steps:
1) 1,3-Dihydroxyacetone is dissolved in anhydrous propanone, stirs under nitrogen protection, add the butyryl oxide that is dissolved in anhydrous propanone; drip pyridine, stirring at room, fully concentrated solvent after reaction; water and ethyl acetate extraction leftover, collected organic layer, use respectively HCl, NaHCO
3washing, regulate pH to neutral, with anhydrous sodium sulfate drying, concentrates to obtain residue; Residue is dissolved and be splined on silicagel column, the mixed solution of sherwood oil and ethyl acetate of usining carries out wash-out as elutriant, collects product, after steaming and desolventizing, obtains 1,3-, bis-butyryl acyloxies-2-carbonyl propane;
1,3-, bis-butyryl acyloxies-2-carbonyl propane is dissolved in the mixture of organic solvent and water, is cooled to 5 ℃, add in batches NaBH
4, fully after reaction, add Glacial acetic acid, then with the chloroform dilution, water, sodium bicarbonate aqueous solution, water washing respectively, anhydrous magnesium sulfate drying, vacuum concentration obtains 1,3-, bis-butyryl acyloxies-2-hydroxy propane;
2) by 1,3-bis-butyryl acyloxies-2-hydroxy propane is dissolved in organic solvent, add 3 under stirring, 3 '-azo two (6-hydroxy-benzoic acid) and DMAP, ice bath is cooling, then adds EDCI, stirring at room is fully reacted, water and saturated NaCl washing respectively, anhydrous sodium sulfate drying, vacuum concentration solvent; Residue is dissolved and be splined on silicagel column, the mixed solution of sherwood oil and ethyl acetate of usining carries out wash-out as elutriant, collects product, after steaming and desolventizing, two { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyl } 3,3 '-azo two (6-hydroxy-benzoic acid).
5. compound two as claimed in claim 4 { 2-(butyryl acyloxy)-1-[(butyryl acyloxy) methyl] ethyl } 3, the preparation method of 3 '-azo two (6-hydroxy-benzoic acid), it is characterized in that, described sherwood oil and ethyl acetate are that the volume ratio according to 10:1～5 is mixed to get elutriant.
6. two 2-(butyryl acyloxy)-1-[(butyryl acyloxy) and methyl] ethyl } 3, two (6-hydroxy-benzoic acid) application in preparing medicament for resisting ulcerative colitis of 3 '-azo.
7. application as claimed in claim 6, is characterized in that, described medicament for resisting ulcerative colitis is for lowering the medicine of mucosa injury exponential sum disease activity index.
8. application as claimed in claim 6, is characterized in that, described medicament for resisting ulcerative colitis is for reducing the medicine of intestinal tissue MPO level.
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| CN105566153A (en) * | 2014-10-14 | 2016-05-11 | 中国医学科学院药物研究所 | Azobenzene derivatives and their preparation method, pharmaceutical composition and use |
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| CN101610670A (en) * | 2006-11-03 | 2009-12-23 | 萨利克斯药品公司 | The preparation and the purposes of 2-hydroxyl-5-phenylazobenzoic acid derivatives |
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| CN101610670A (en) * | 2006-11-03 | 2009-12-23 | 萨利克斯药品公司 | The preparation and the purposes of 2-hydroxyl-5-phenylazobenzoic acid derivatives |
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| 赖俊英: "《中国优秀博硕士学位论文》", 31 December 2006 * |
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| CN105566153A (en) * | 2014-10-14 | 2016-05-11 | 中国医学科学院药物研究所 | Azobenzene derivatives and their preparation method, pharmaceutical composition and use |
| CN105566153B (en) * | 2014-10-14 | 2019-05-31 | 中国医学科学院药物研究所 | Azobenzene derivatives and its preparation method and pharmaceutical composition and purposes |
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