CN102532137B - 8-pyrazole substituted xanthine A2B adenosine receptor antagonist and synthesis method and application thereof - Google Patents
8-pyrazole substituted xanthine A2B adenosine receptor antagonist and synthesis method and application thereof Download PDFInfo
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Abstract
The invention relates to a 8-pyrazole substituted xanthine A2B adenosine receptor antagonist and a synthesis method and an application thereof. The structural formula of the A2B adenosine receptor antagonist is shown in the specifications, and the antagonist is synthesized by taking 1,3-dialkyl-5,6-diaminouracil and nitrobenzyl pyrazole-4-formic acid as raw materials and improving a Traube reaction. The synthesis method disclosed by the invention has mild conditions, low cost and high yield, is easy to operate, and is suitable for industrial production. As proved by a pharmacological experiment, the affinity and selectivity of an A2B receptor are enhanced due to substitution of the 8-position of xanthine in 8-pyrazole substituted xanthine with nitrobenzyl pyrazole, the secretion of IL-8 can be suppressed, and the release of inflammatory factors is reduced. The receptor antagonist is suitable for treating bronchial asthma.
Description
Technical field
The present invention relates to medicine synthetic field, particularly a kind of 8-pyrazoles substituted xanthine class A
2Badenosine receptor antagonists and synthetic method thereof and application.
Background technology
Bronchial asthma is chronic respiratory tract disease the most common in world wide, its principal feature is that chronic airway inflammation and air flue increase multiple stimulating factor reactivity, clinical Chang Fanfu outbreak, seriously threatening people's health, it is universally acknowledged difficult medical problem, by the World Health Organization, classified as one of four large persistent ailments in disease, according to investigations, the whole world has at least 100,000,000 people to suffer from above asthma.Recent study finds that adenosine is a kind of effective bronchoconstriction factor, by with specific A
2Badenosine Receptors interacts, and collapsible bronchial smooth muscle promotes inflammatory cell to discharge inflammatory factor, participates in the pathophysiological process of bronchial asthma acute and chronic inflammation, in Pathogenesy of Asthma, plays an important role.
A
2Badenosine Receptors is to cause scorching acceptor, and research HML-1 cell, finds non-selective A
2Aadenosine receptor agonist can stimulate interleukin-IL-8 secretion, selectivity A
2Aadenosine receptor agonist does not stimulate IL-8 secretion, and IL-8 of mastocyte secretion and A
2Badenosine Receptors activity is relevant.8-(trifluoro-benzyl-1 of report in 2006
h-pyrazoles)-1,3-dimethyl xanthine is just to A
2Bacceptor has higher avidity and selectivity.A
2Badenosine Receptors is that adenosine promotes mastocyte to produce one of principal recipient of cytokine and inflammatory mediator, optionally blocks A
2Badenosine Receptors is the activity of inflammation-inhibiting cell effectively, can effectively block the release of airway inflammation medium and bronchospasm and reach treatment asthma object.
According to the feature of chemical structure, A
2Breceptor antagonist can be divided into 8-substituted xanthine class and 8-replaces non-xanthine.
US20070219221 and
j. Med. Chem.2008,
51,2267 – 2278 grades are to 8-substituted xanthine class A
2Bsynthetic method and the pharmacological action of adenosine receptor antagonists are studied, and synthetic route is as follows:
R
1, R
2for alkyl, R
3substituted benzene or m-trifluoromethyl benzyl.
With 1,3-, replace-4,5-diaminourea pyrimidine and obtain the substituent purine of different 8-from the carboxyl reaction cyclization of 1-substituted pyrazolecarboxylic-4-formic acid.The method needs more violent reaction conditions, and long reaction time, and particularly m-trifluoromethyl benzyl synthesis condition is complicated, and cost is high, produces toxic substance in reaction process, is difficult for preparation.
Summary of the invention
It is simple that the present invention is that the technical problem that will solve is to provide a kind of synthetic method, easily preparation, and the compound making can suppress the secretion of IL-8, reduces the release of inflammatory factor, treatment asthma is had to the 8-pyrazoles substituted xanthine class A of good curative effect effect
2Badenosine receptor antagonists and synthetic method thereof and application.
Technical solution of the present invention is:
A kind of 8-pyrazoles substituted xanthine class A
2Badenosine receptor antagonists, its structural formula is as follows:
R wherein
1, R
2be respectively a kind of in the alkyl, alkene of hydrogen, alkyl, replacement or two kinds; R
3for nitrobenzyl pyrazoles substituting group.
Above-mentioned 8-pyrazoles substituted xanthine class A
2Badenosine receptor antagonists, is characterized in that: R
1, R
2for hydrogen, methyl, and R
1, R
2identical or different.
Above-mentioned 8-pyrazoles substituted xanthine class A
2Badenosine receptor antagonists, R
1, R
2for identical substituting group.
A kind of 8-pyrazoles substituted xanthine class A
2Bthe synthetic method of adenosine receptor antagonists, its special character is:
Nitrobenzyl pyrazoles-4-formic acid and the carbodiimide hydrochloride of equimolar amount are dissolved in methyl alcohol, divide the formula 2 adding for 5 times with nitrobenzyl pyrazoles-4-formic acid equimolar amount, stir 8～16h, filter, except desolventizing, washing, is dissolved in methyl alcohol and sodium hydroxide solution, makes mixing solutions PH=8～12, in stirring and dissolving postposition 300～400W microwave, react 10～50 min, be cooled to room temperature, with hydrochloric acid, in ice bath, be acidified to pH=2～5, separate out 8-pyrazoles substituted xanthine class A
2Badenosine receptor antagonists.
Above-mentioned 8-pyrazoles substituted xanthine class A
2Bthe synthetic method of adenosine receptor antagonists, with hydrochloric acid pH=3～4 after acidifying in ice bath.
Above-mentioned 8-pyrazoles substituted xanthine class A
2Bthe synthetic method of adenosine receptor antagonists, R in described formula 2
1, R
2for methyl, reaction formula is:
By dimethyl urea 0.18mol, cyanoacetic acid 0.15～0.2mol, at 90～110 ℃, in diacetyl oxide, stir 1～2h, remove acetic acid, diacetyl oxide under reduced pressure, residuum adds water, add activated carbon decolorizing, filter, cooling, with NaOH, adjust pH=8～10, suction filtration, obtains 1,3-dimethyl-6-Urea,amino-pyrimidine; 1,3-dimethyl-6-Urea,amino-pyrimidine is dissolved in acetum, and average mark adds and 1 for 5 times, the Sodium Nitrite of 3-dimethyl-6-Urea,amino-pyrimidine equimolar amount, is cooled to room temperature after adding, and stirs 1～3h, filter, obtain 1,3-dimethyl-5-nitroso-group-6-Urea,amino-pyrimidine; 1,3-dimethyl-5-nitroso-group-6-Urea,amino-pyrimidine is dissolved in ammoniacal liquor, and average mark adds V-Brite B 0.1～0.3 mol for 5 times, be down to room temperature and continue to stir 10～30min, cooling in ice bath, filter collecting precipitation, wash 1,3-dimethyl-5,6-diaminourea pyrimidine; Mp:210～214 ℃.
Above-mentioned 8-pyrazoles substituted xanthine class A
2Bthe synthetic method of adenosine receptor antagonists, temperature when 1,3-dimethyl-6-Urea,amino-pyrimidine dissolves is 50～75 ℃, is then cooled to 50 ℃.
Above-mentioned-pyrazoles substituted xanthine class A
2Bthe synthetic method of adenosine receptor antagonists, removing after acetic acid, diacetyl oxide amount of water when residuum adds water under reduced pressure is 20～100mL.
Above-mentioned 8-pyrazoles substituted xanthine class A
2Bthe synthetic method of adenosine receptor antagonists, the reaction formula of preparing nitrobenzyl pyrazoles-4-formic acid is:
Add 0.1mol ethyl cyanoacetate, 0.1～0.15mol triethyl orthoformate, diacetyl oxide 0.1～1g and zinc chloride catalyst 0.1～0.2g, stir, 100～120 ℃ of 1～2h that reflux, by Claisen ester condensation, continue to be heated to 130～140 ℃ and make it to react completely, and steam from fractional column is suitable for reading the low boilers that when reaction generates; Reaction solution, through cooling crystallization, filters and uses washing with alcohol, obtains oxyethyl group methylene radical ethyl cyanoacetate, Mp:50～51 ℃; 0.1mol oxyethyl group methylene radical ethyl cyanoacetate and 0.1mol hydrazine hydrate stir 10～14h in being dissolved in ethanol, and vacuum rotary steam is removed ethanol, obtain 3-amino-pyrazol-4-ethyl formate, Mp:102 ℃; 3-amino-pyrazol-4-ethyl formate is dissolved in glacial acetic acid, adds rare HCl solution and the 0.1～0.3mol sodium nitrite solution of 0.1～0.2mol under stirring, reaction mixture stirs 10～30min at-5～0 ℃, add ethanol, backflow 0.5～1.5h, vacuum rotary steam is removed ethanol, and residue washes with water, filter, use the dichloromethane extraction aqueous solution, use dried over mgso organic phase, filter, concentrating under reduced pressure obtains pyrazoles-4-ethyl formate, Mp:69 ℃; Pyrazoles-4-ethyl formate is dissolved in acetone soln, add salt of wormwood and 0.1～0.15mol nitrobenzyl bromine, at 40～70 ℃, reaction 12～20h, removes by filter salt of wormwood, adds solid potassium hydroxide 0.02mol, back flow reaction 12～20h, methyl alcohol underpressure distillation is filtered, and residue is dissolved in water and with ethyl acetate washing, water layer with hcl acidifying to separating out nitrobenzyl pyrazoles-4-formic acid.
Above-mentioned 8-pyrazoles substituted xanthine class A
2Badenosine receptor antagonists is applied in the medicine for the treatment of asthma.
Beneficial effect of the present invention is:
1, synthesized first 8-(nitrobenzyl-1
h-pyrazoles)-1,3-dimethyl xanthine, reactions steps is relatively less, is easy to suitability for industrialized production, cost, environmental friendliness, total recovery is more than 89.3%.
2, nitrobenzyl pyrazoles-4-formic acid is prepared voluntarily, and has prepared rear direct input 8-(nitrobenzyl-1
h-pyrazoles)-1, in 3-dimethyl xanthine synthetic, needn't store again, yield is improved, cost.
3, in oxyethyl group methylene radical ethyl cyanoacetate synthetic, in the molar ratio United States Patent (USP) 3515715 of triethyl orthoformate and ethyl cyanoacetate, be reported as 1:3, yield is 93～94%, but has solvent recuperation, the loaded down with trivial details problem of aftertreatment.It is 1～1:1.5 that the present invention adopts the molar ratio of triethyl orthoformate and ethyl cyanoacetate, and adopts minute two sections of heat temperature raisings, in reaction process, in time lower boiling resultant is steamed.Because start excess Temperature, cause triethyl orthoformate to decompose, material reduces, and temperature-gradient method can make the ethoxymethyl ethyl cyanoacetate dealcoholysis generating generate cyanoacetic acid, and fractionation plant has been guaranteed not losing of material; And temperature can not, higher than 140 ℃, if steam higher than 140 ℃ of ethyl cyanoacetates, reduce yield on the contrary.After completion of the reaction, by placing 12 hours, the prolongation reaction times can make yield be significantly improved, otherwise understand some oxyethyl group methylene radical ethyl cyanoacetate, is dissolved in solution.In 3-amino-pyrazol-4-ethyl formate synthetic, document Ackermann Otto and Bretzinger. 2'-Phenylhydrazino-2-cyanoacrylic acid esters and herbicides containing these compounds:DE, 2635841[P], 1978. middle report temperature of reaction is reflux temperature, although the reaction times has shortened, but because hydrazine is unstable, yield is not high, this experiment has adopted the method at room temperature stirring, avoided the generation of side reaction, 3-amino-pyrazol-4-ethyl formate direct filtration can obtain a part, decompression filtrate recycling ethanol has obtained again a part, make like this yield bring up to more than 92%.In pyrazoles-4-ethyl formate synthetic, to stir fully to guarantee that pyrazoles-4-ethyl formate can be dissolved in acetic acid completely, and then with nitrite generation addition reaction, to avoid hydrochloric acid directly to react with Sodium Nitrite, and control temperature of reaction at-5～0 ℃, avoid reaction violent, generate a large amount of nitric oxide gas, both contaminate environment, damaged operator's health again.
4, in 8-(nitrobenzyl-1
h-pyrazoles)-1, in 3-dimethyl xanthine synthetic, adopted in microwave and has reacted, and reaction fast, has been saved 8h left and right than the Traube reaction times.
5,8-of the present invention (nitrobenzyl-1
h-pyrazoles)-1,3-dimethyl xanthine, selects pyrazole group to replace in 8-position, because 8-pyrazoles replaces, has stronger A
2Breceptor affinity and selectivity.And the methyl of 1,3-position has good hydrophobic interaction, the 1-position of pyrazoles with between nitrobenzyl increased hydrogen bond action while replacing, a position replaces and makes it have fat-soluble structure with nitro.
6, in the present invention, synthetic compound is the medicinal application of up-to-date treatment asthma aspect, is domestic first with A
2Badenosine receptor antagonists treatment asthma is research direction, has synthesized 8-(nitrobenzyl-1 under the guidance of combinatorial chemistry and bioisosterism
h-pyrazoles)-1,3-dimethyl xanthine.
7, the present invention synthesizes 8-(nitrobenzyl-1 with basic chemical feedstocks by series of chemical
h-pyrazoles)-1,3-dimethyl xanthine, proves that by rat pharmacological evaluation this medicine has anti-asthma effect, is applicable to treat bronchial asthma.
Accompanying drawing explanation
Fig. 1 is 8-(adjacent nitrobenzyl-1H-pyrazoles)-xanthic
1h NMR collection of illustrative plates;
Fig. 2 is 8-(adjacent nitrobenzyl-1H-pyrazoles)-xanthic LC-MS collection of illustrative plates;
Fig. 3 is 8-(base benzyl-1H-pyrazoles between neighbour)-xanthic
1h NMR collection of illustrative plates;
Fig. 4 is 8-(nitrobenzyl-1H-pyrazoles)-xanthic LC-MS collection of illustrative plates;
Fig. 5 is 8-(to nitrobenzyl-1H-pyrazoles)-xanthic
1h NMR collection of illustrative plates;
Fig. 6 is 8-(to nitrobenzyl-1H-pyrazoles)-xanthic LC-MS collection of illustrative plates;
Fig. 7 is 1-methyl-8-(adjacent nitrobenzyl-1H-pyrazoles)-xanthic
1h NMR collection of illustrative plates;
Fig. 8 is 1-methyl-8-(adjacent nitrobenzyl-1H-pyrazoles)-xanthic LC-MS collection of illustrative plates;
Fig. 9 is 1-methyl-8-(nitrobenzyl-1H-pyrazoles)-xanthic
1h NMR collection of illustrative plates;
Figure 10 is 1-methyl-8-(nitrobenzyl-1H-pyrazoles)-xanthic LC-MS collection of illustrative plates;
Figure 11 is 1-methyl-8-(to nitrobenzyl-1H-pyrazoles)-xanthic
1h NMR collection of illustrative plates;
Figure 12 is 1-methyl-8-(to nitrobenzyl-1H-pyrazoles)-xanthic LC-MS collection of illustrative plates;
Figure 13 is 1,3-dimethyl-8-(adjacent nitrobenzyl-1H-pyrazoles)-xanthic
1h NMR collection of illustrative plates;
Figure 14 is 1,3-dimethyl-8-(adjacent nitrobenzyl-1H-pyrazoles)-xanthic LC-MS collection of illustrative plates;
Figure 15 is 1,3-dimethyl-8-(nitrobenzyl-1H-pyrazoles)-xanthic
1h NMR collection of illustrative plates;
Figure 16 is 1,3-dimethyl-8-(nitrobenzyl-1H-pyrazoles)-xanthic LC-MS collection of illustrative plates;
Figure 17 is 1,3-dimethyl-8-(to nitrobenzyl-1H-pyrazoles)-xanthic
1h NMR collection of illustrative plates;
Figure 18 is 1,3-dimethyl-8-(to nitrobenzyl-1H-pyrazoles)-xanthic LC-MS collection of illustrative plates;
Figure 19 is normal group segmental bronchus and alveolar structure figure;
Figure 20 is model group segmental bronchus and alveolar structure figure;
Figure 21 is positive controls segmental bronchus and alveolar structure figure;
Figure 22 is medication therapy groups 1 segmental bronchus and alveolar structure figure;
Figure 23 is medication therapy groups 2 segmental bronchuses and alveolar structure figure;
Figure 24 is medication therapy groups 3 segmental bronchuses and alveolar structure figure;
Figure 24 is medication therapy groups 4 segmental bronchuses and alveolar structure figure;
Figure 26 is medication therapy groups 5 segmental bronchuses and alveolar structure figure;
Figure 27 is medication therapy groups 6 segmental bronchuses and alveolar structure figure;
Figure 28 is medication therapy groups 7 segmental bronchuses and alveolar structure figure;
Figure 29 is medication therapy groups 8 segmental bronchuses and alveolar structure figure;
Figure 30 is medication therapy groups 9 segmental bronchuses and alveolar structure figure;
Figure 31 is the column diagram of the level of the IL-8 in serum.
Embodiment
Embodiment 1
R
1, R
2for hydrogen, R
3for adjacent nitrobenzyl pyrazoles-synthetic 8-(adjacent nitrobenzyl-1H-pyrazoles)-xanthine
(1) the adjacent nitrobenzyl-1H-pyrazoles-4-of synthesis material formic acid
1. synthesizing pyrazole-4-ethyl formate
In the 250mL reaction flask of agitator, thermometer, dropping funnel and fractional column is housed, add triethyl orthoformate 0.12mol, cyanogen ethyl ester ethyl ester 0.10mol, diacetyl oxide 0.4g and zinc chloride catalyst 0.1g, stir, 110 ℃ of backflow 1h, continue to be heated to 132 ℃, and steam from fractional column is suitable for reading the low boilers that when reaction generates; Reaction solution, through cooling crystallization, filters and uses washing with alcohol, obtains faint yellow crystallization oxyethyl group methylene radical ethyl cyanoacetate, Mp:50～51 ℃; 0.1mol oxyethyl group methylene radical ethyl cyanoacetate and 0.10mol hydrazine hydrate stir 12h in 400mL ethanol, and vacuum rotary steam is removed ethanol, obtain faint yellow solid 3-amino-pyrazol-4-ethyl formate, Mp:102 ℃; 3-amino-pyrazol-4-ethyl formate is dissolved in 100mL glacial acetic acid, successively adds rare HCl solution and the 0.2mol sodium nitrite solution of 0.15mol under stirring, reaction mixture stirs 15min at 0 ℃, add 200mL ethanol, backflow 1h, vacuum rotary steam is removed ethanol, residue washes with water, filter, use the dichloromethane extraction aqueous solution, use dried over mgso organic phase, filter, concentrating under reduced pressure obtains beige solid pyrazoles-4-ethyl formate, yield: 99.5%, Mp:69 ℃;
2. synthesize adjacent nitrobenzyl-1H-pyrazoles-4-formic acid
Pyrazoles-4-ethyl formate is dissolved in acetone soln, adds 0.05mol salt of wormwood and 0.12mol o-nitrobenzyl bromide, 50 ℃ of heating 16h, remove by filter salt of wormwood, add solid potassium hydroxide 0.02mol, reflux 18h, methyl alcohol underpressure distillation is filtered, residue is dissolved in water and washs by ethyl acetate, water layer hcl acidifying, separates out the adjacent nitrobenzyl-1H-pyrazoles-4-of light yellow solid formic acid, quality: 20.8g, yield: 85%, Mp:175～177 ℃;
(2) synthetic 8-(adjacent nitrobenzyl-1H-pyrazoles)-xanthine
Adjacent nitrobenzyl-4-the pyrazole carboxylic acid of 1.0mmol and 1.0mmol carbodiimide hydrochloride are dissolved in 22mL methyl alcohol, stir and add 5 of 1.0mmol lower minute 5 times, 6-diaminourea pyrimidine, stir 8 hours, filter, except desolventizing, washing, be dissolved in the sodium hydroxide solution of 15mL methyl alcohol and 15mL, in 300 W microwaves, react 50 min, be cooled to room temperature, hydrochloric acid with 6mol/L is acidified to pH=5 in ice bath, separate out yellow solid 8-(adjacent nitrobenzyl-1H-pyrazoles)-xanthine, quality: 30.1g, yield: 83.1%, Mp:189～192 ℃, purity: > 95%.
1h NMR (DMSO-d6):
1h NMR (DMSO-d6): 13.35 (s, 1H), 10.09 (s, 1H), 8.07 (m; 1H), 7.84 (s, 1H), 7.68 (s, 1H) 7.28-7.43 (m; 3H), 6.12 (s, 1H), 5.09 (s, 2H) (as Fig. 1); LC-MS (m/z): 354 [M+] (as Fig. 2).
Embodiment 2
R
1, R
2for hydrogen, R
3for a nitrobenzyl pyrazoles-synthetic 8-(nitrobenzyl-1H-pyrazoles)-xanthine
Between 1.0mmol, nitrobenzyl-4-pyrazole carboxylic acid and 1.0mmol carbodiimide hydrochloride are dissolved in 22mL methyl alcohol, stir and add 5 of 1.0mmol lower minute 5 times, 6-diaminourea pyrimidine, stir 12 hours, filter, except desolventizing, washing, be dissolved in the sodium hydroxide solution of 15mL methyl alcohol and 15mL, reaction 30 min in microwave (360 W), be cooled to room temperature, hydrochloric acid with 6mol/L is acidified to pH=4 in ice bath, separate out yellow solid 8-(nitrobenzyl-1H-pyrazoles)-xanthine, quality: 31.5g, yield 89.3%., Mp:196～198 ℃, purity: > 95%.
1h NMR (DMSO-d6): 13.39 (s, 1H), 10.01 (s, 1H), 7.99-8.03 (m, 2H), 7.81 (s, 1H), 7.62 (s, 1H) 7.45-7.40 (m, 2H), 6.08 (s, 1H), 5.01 (s, 2H) (as Fig. 3); LC-MS (m/z): 354 [M+] (as Fig. 4).
Embodiment 3
R
1, R
2for hydrogen, R
3for to nitrobenzyl pyrazoles-synthetic 8-(to nitrobenzyl-1H-pyrazoles)-xanthine
1.0mmol is dissolved in 22mL methyl alcohol nitrobenzyl-4-pyrazole carboxylic acid and 1.0mmol carbodiimide hydrochloride, stir and add 5 of 1.0mmol lower minute 5 times, 6-diaminourea pyrimidine, stir 10 hours, filter, except desolventizing, washing, be dissolved in the sodium hydroxide solution of 15mL methyl alcohol and 15mL, reaction 20 min in microwave (350 W), be cooled to room temperature, hydrochloric acid with 6mol/L is acidified to pH=2 in ice bath, separate out faint yellow solid 8-(to nitrobenzyl-1H-pyrazoles)-xanthine, quality: 28.4g, yield 82.8%, Mp:208～210 ℃, purity: 97.4%.
1h NMR (DMSO-d6): 13.39 (s, 1H), 10.02 (s, 1H), 8.07 (m, 2H); 7.61 (s, 1H), 7.32 (m, 2H), 7.43 (s; 1H), 6.08 (s, 1H), 5.01 (s, 2H) (as Fig. 5); LC-MS (m/z): 354 [M+] (as Fig. 6).
Embodiment 4
R
1for hydrogen, R
2for methyl, R
3for adjacent nitrobenzyl pyrazoles-synthetic 1-methyl-8-(adjacent nitrobenzyl-1H-pyrazoles)-xanthine
(1) the adjacent nitrobenzyl-1H-pyrazoles-4-of synthesis material formic acid
1. synthesizing pyrazole-4-ethyl formate
In the 250mL reaction flask of agitator, thermometer, dropping funnel and fractional column is housed, add triethyl orthoformate 0.10mol, cyanogen ethyl ester ethyl ester 0.10mol, diacetyl oxide 1g and zinc chloride catalyst 0.15g, stir, 120 ℃ of backflow 1.5h, continue to be heated to 130 ℃, and steam from fractional column is suitable for reading the low boilers that when reaction generates; Reaction solution, through cooling crystallization, filters and uses washing with alcohol, obtains faint yellow crystallization oxyethyl group methylene radical ethyl cyanoacetate, Mp:50～51 ℃; 0.1mol oxyethyl group methylene radical ethyl cyanoacetate and 0.10mol hydrazine hydrate stir 10h in 400mL ethanol, and vacuum rotary steam is removed ethanol, obtain faint yellow solid 3-amino-pyrazol-4-ethyl formate, Mp:102 ℃; 3-amino-pyrazol-4-ethyl formate is dissolved in 100mL glacial acetic acid, successively adds rare HCl solution and the 0.15mol sodium nitrite solution of 0.2mol under stirring, reaction mixture stirs 30min at-2 ℃, add 200mL ethanol, backflow 0.5h, vacuum rotary steam is removed ethanol, residue washes with water, filter, use the dichloromethane extraction aqueous solution, use dried over mgso organic phase, filter, concentrating under reduced pressure obtains beige solid pyrazoles-4-ethyl formate, yield: 89.1%, Mp:69 ℃;
2. synthesize adjacent nitrobenzyl-1H-pyrazoles-4-formic acid
Pyrazoles-4-ethyl formate is dissolved in acetone soln, adds 0.05mol salt of wormwood and 0.10mol o-nitrobenzyl bromide, 40 ℃ of heating 20h, remove by filter salt of wormwood, add solid potassium hydroxide 0.02mol, reflux 12h, methyl alcohol underpressure distillation is filtered, residue is dissolved in water and washs by ethyl acetate, water layer hcl acidifying, separates out the adjacent nitrobenzyl-1H-pyrazoles-4-of light yellow solid formic acid, quality: 19.1g, yield: 78%, Mp:175～177 ℃;
(2) synthetic 1-methyl-8-(adjacent nitrobenzyl-1H-pyrazoles)-xanthine
In 250mL reaction flask, by the adjacent nitrobenzyl-1H-pyrazoles-4-of 0.1mol formic acid, 0.1mol carbodiimide hydrochloride is dissolved in 150mL methyl alcohol, divides and adds 0.1mol 1-methyl-5 for 5 times, and 6 diaminourea pyrimidines, stir 9 hours, removal of solvent under reduced pressure, washing; Put in 250mL reaction flask, the sodium hydroxide solution that adds 75mL methyl alcohol and 75mL, reaction 10 min in the rearmounted microwave of stirring and dissolving (300 W), cooling, with the hydrochloric acid of 6mol/L, in ice bath, be acidified to pH=2, separate out faint yellow solid 1-methyl-8-(adjacent nitrobenzyl-1H-pyrazoles)-xanthine, quality: 30.4g, yield 81.9%, Mp:219～221 ℃, purity: 97.5%.
1h NMR (DMSO-d6): 13.37 (s, 1H), 8.09 (m, H), 7.81 (s; 1H), 7.64 (s, 1H) 7.52-7.33 (m, 3H), 6.08 (s; 1H), 5.01 (s, 2H), 3.18 (s, 3H) (as Fig. 7); LC-MS (m/z): 368 [M+] (as Fig. 8).
Embodiment 5
R
1for hydrogen, R
2for methyl, R
3for a nitrobenzyl pyrazoles-synthetic 1-methyl-8-(nitrobenzyl-1H-pyrazoles)-xanthine
In 250mL reaction flask by 0.1mol between nitrobenzyl-1H-pyrazoles-4-formic acid, 0.1mol carbodiimide hydrochloride solution, in 150mL methyl alcohol, minute adds 0.1mol 1-methyl-5 for 5 times, 6 diaminourea pyrimidines, stir 13 hours, removal of solvent under reduced pressure is washed; Put in 250mL reaction flask, the sodium hydroxide solution that adds 75mL methyl alcohol and 75mL, reaction 50 min in the rearmounted microwave of stirring and dissolving (400 W), cooling, hydrochloric acid with 6mol/L is acidified to pH=3 in ice bath, separates out faint yellow solid 1-methyl-8-(nitrobenzyl-1H-pyrazoles)-xanthine, quality: 32.4g yield 87.9%, Mp:235～237 ℃, purity: 98.6%.
1h NMR (DMSO-d6): 13.32 (s, 1H), 8.03-7.99 (m, 2H), 7.81 (s, 1H), 7.64 (s, 1H) 7.50-7.43 (m, 2H), 6.02 (s, 1H), 5.03 (s, 2H), 3.14 (s, 3H) (as Fig. 9); LC-MS (m/z): 368 [M+] (as Figure 10).
Embodiment 6
R
1for hydrogen, R
2for methyl, R
3for to nitrobenzyl pyrazoles-synthetic 1-methyl-8-(to nitrobenzyl-1H-pyrazoles)-xanthine
In 250mL reaction flask by 0.1mol to nitrobenzyl-1H-pyrazoles-4-formic acid, 0.1mol carbodiimide hydrochloride is dissolved in 150mL methyl alcohol, minute adds 0.1mol 1-methyl-5 for 5 times, 6 diaminourea pyrimidines, stir 8 hours, removal of solvent under reduced pressure is washed; Put in 250mL reaction flask, the sodium hydroxide solution that adds 75mL methyl alcohol and 75mL2mol/L, reaction 40 min in the rearmounted microwave of stirring and dissolving (300 W), cooling, with the hydrochloric acid of 6mol/L, in ice bath, be acidified to pH=2, separate out faint yellow solid 1-methyl-8-(to nitrobenzyl-1H-pyrazoles)-xanthine, quality: 30. 5g, yield 82.9%, Mp:214～216 ℃, purity: 98.3%.
1h NMR (DMSO-d6): 13.31 (s, 1H), 8.07 (m, 2H), 7.75 (s; 1H), 7.48 (s, 1H) 7.32 (m, 2H), 6.02 (s; 1H), 5.00 (s, 2H), 3.17 (s, 3H) (as Figure 11); LC-MS:(m/z): 368 [M+] (as Figure 12).
Embodiment 7
R
1, R
2for methyl, R
3for adjacent nitrobenzyl pyrazoles-synthetic 1,3-dimethyl-8-(adjacent nitrobenzyl-1H-pyrazoles)-xanthine
(1) the adjacent nitrobenzyl-1H-pyrazoles-4-of synthesis material formic acid
1. synthesizing pyrazole-4-ethyl formate
In the 250mL reaction flask of agitator, thermometer, dropping funnel and fractional column is housed, add triethyl orthoformate 0.15mol, cyanogen ethyl ester ethyl ester 0.10mol, diacetyl oxide 0.1g and zinc chloride catalyst 0.2g, stir, 100 ℃ of backflow 2h, continue to be heated to 140 ℃, and steam from fractional column is suitable for reading the low boilers that when reaction generates; Reaction solution, through cooling crystallization, filters and uses washing with alcohol, obtains faint yellow crystallization oxyethyl group methylene radical ethyl cyanoacetate, Mp:50～51 ℃; 0.1mol oxyethyl group methylene radical ethyl cyanoacetate and 0.10mol hydrazine hydrate stir 14h in 400mL ethanol, and vacuum rotary steam is removed ethanol, obtain faint yellow solid 3-amino-pyrazol-4-ethyl formate, Mp:102 ℃; 3-amino-pyrazol-4-ethyl formate is dissolved in 120mL glacial acetic acid, successively adds rare HCl solution and the 0.1mol sodium nitrite solution of 0.1mol under stirring, reaction mixture stirs 10min at-5 ℃, add 200mL ethanol, backflow 1.5h, vacuum rotary steam is removed ethanol, residue washes with water, filter, use the dichloromethane extraction aqueous solution, use dried over mgso organic phase, filter, concentrating under reduced pressure obtains beige solid pyrazoles-4-ethyl formate, yield: 88.6%, Mp:69 ℃;
2. synthesize adjacent nitrobenzyl-1H-pyrazoles-4-formic acid
Pyrazoles-4-ethyl formate is dissolved in acetone soln, adds 0.05mol salt of wormwood and 0.15mol o-nitrobenzyl bromide, 70 ℃ of heating 12h, remove by filter salt of wormwood, add solid potassium hydroxide 0.02mol, reflux 20h, methyl alcohol underpressure distillation is filtered, residue is dissolved in water and washs by ethyl acetate, water layer hcl acidifying, separates out the adjacent nitrobenzyl-1H-pyrazoles-4-of light yellow solid formic acid, quality: 19.4g, yield: 80%, Mp:175～177 ℃;
(2) synthetic 1,3-dimethyl-5,6-diaminourea pyrimidine
By dimethyl urea 0.18 mol, cyanoacetic acid 0.16mol, at 95 ℃, in 25ml diacetyl oxide, stir 2h, remove acetic acid, diacetyl oxide under reduced pressure, residuum adds water, add activated carbon decolorizing, filter, cooling, with NaOH, adjust pH=10, suction filtration, obtains light yellow solid 1,3 – dimethyl-6-amino-uracil; 1,3 – dimethyl-6-amino-uracil is dissolved in water and is stirred, and 5 Sodium Nitrite 0.16mol of average mark, are cooled to room temperature after adding, continue to stir 1h, filter, collecting precipitation, washing, dry, obtain purple solid 1,3-dimethyl-5-nitroso-group-6-Urea,amino-pyrimidine; 1,3-dimethyl-5-nitroso-group-6-Urea,amino-pyrimidine is dissolved in ammoniacal liquor, and average mark adds 0.18mol V-Brite B 5 times, the solution look that first reddens then becomes green and finally becomes light yellowly, is down to room temperature and continue stirs 10 minutes, and reaction mixture volume reduces half, cooling in ice bath, filter collecting precipitation, wash to obtain faint yellow solid 1,3-dimethyl-5,6-diaminourea pyrimidine, quality: 25.9g, yield: 95%, Mp:210～213 ° C;
(3) synthetic 1,3-dimethyl-8-(adjacent nitrobenzyl-1H-pyrazoles)-xanthine
In 250mL reaction flask, by the adjacent nitrobenzyl-1H-pyrazoles-4-of 0.1mol formic acid, 0.1mol carbodiimide hydrochloride is dissolved in 150mL methyl alcohol, divides and adds 0.1mol 15 times, 3-dimethyl-5,6 diaminourea pyrimidines, stir 10 hours, removal of solvent under reduced pressure, washing; Put in 250mL reaction flask, the sodium hydroxide solution that adds 75mL methyl alcohol and 75mL, reaction 30 min in the rearmounted microwave of stirring and dissolving (350 W), cooling, hydrochloric acid with 6mol/L is acidified to pH=5 in ice bath, separates out faint yellow solid 1,3-dimethyl-8-(adjacent nitrobenzyl-1H-pyrazoles)-xanthine, quality: 31.6g, yield 83.1%.Mp:264～266 ℃, purity: 98.5%.
1h NMR (DMSO-d6): 13.37 (s, 1H), 8.07 (m, H), 7.73 (s; 1H), 7.41 (s, 1H) 7.45-7.32 (m, 3H), 5.04 (s; 2H), 3.11 (s, 3H), 3.07 (s, 3H) (as Figure 13); LC-MS (m/z): 382 [M+] (as Figure 14).
Embodiment 8
R
1, R
2for methyl, R
3for a nitrobenzyl pyrazoles-synthetic 1,3-dimethyl-8-(nitrobenzyl-1H-pyrazoles)-xanthine
(1) synthetic 1,3-dimethyl-5,6-diaminourea pyrimidine
By dimethyl urea 0.18 mol, cyanoacetic acid 0.15mol, at 90 ℃, in 25ml diacetyl oxide, stir 1h, remove acetic acid, diacetyl oxide under reduced pressure, residuum adds water, add activated carbon decolorizing, filter, cooling, with NaOH, adjust pH=9, suction filtration, obtains light yellow solid 1,3 – dimethyl-6-amino-uracil; 1,3 – dimethyl-6-amino-uracil is dissolved in water and is stirred, and 5 Sodium Nitrite 0.15mol of average mark, are cooled to room temperature after adding, continue to stir 2h, filter, collecting precipitation, washing, dry, obtain purple solid 1,3-dimethyl-5-nitroso-group-6-Urea,amino-pyrimidine; 1,3-dimethyl-5-nitroso-group-6-Urea,amino-pyrimidine is dissolved in ammoniacal liquor, and average mark adds 0.18mol V-Brite B 5 times, the solution look that first reddens then becomes green and finally becomes light yellowly, is down to room temperature and continue stirs 20 minutes, and reaction mixture volume reduces half, cooling in ice bath, filter collecting precipitation, wash to obtain faint yellow solid 1,3-dimethyl-5,6-diaminourea pyrimidine, quality: 24.6g, yield: 88%, Mp:210～213 ° C;
(2) synthetic 1,3-dimethyl-8-(nitrobenzyl-1H-pyrazoles)-xanthine
In 250mL reaction flask by 0.1mol between nitrobenzyl-1H-pyrazoles-4-formic acid, 0.1mol carbodiimide hydrochloride is dissolved in 150mL methyl alcohol, minute adds 0.1mol 15 times, 3-dimethyl-5,6 diaminourea pyrimidines, stir 16 hours, removal of solvent under reduced pressure, washing; Put in 250mL reaction flask, the sodium hydroxide solution that adds 75mL methyl alcohol and 75mL, reaction 50 min in the rearmounted microwave of stirring and dissolving (360 W), cooling, hydrochloric acid with 6mol/L is acidified to pH=3.6 in ice bath, separates out yellow solid 1,3-dimethyl-8-(nitrobenzyl-1H-pyrazoles)-xanthine, quality: 33. 6g, yield: 89.3%.Mp:284～287 ℃, purity: 99.1%.
1hNMR (DMSO-d6): 13.33 (s, 1H), 8.01-7.98 (m, 2H), 7.69 (s; 1H), 7.57 (s, 1H) 7.45-7.39 (m, 2H), 5.05 (s; 2H), 3.02 (s, 3H), 3.03 (s, 3H) (as Figure 15); LC-MS (m/z): 382 [M+] (as Figure 16).
Embodiment 9
R
1, R
2for methyl, R
3for to nitrobenzyl pyrazoles-1,3-dimethyl-8-(to nitrobenzyl-1H-pyrazoles)-xanthic synthetic
(1) synthetic 1,3-dimethyl-5,6-diaminourea pyrimidine
By dimethyl urea 0.18 mol, cyanoacetic acid 0.20mol, at 110 ℃, in 25ml diacetyl oxide, stir 1.5h, remove acetic acid, diacetyl oxide under reduced pressure, residuum adds water, adds activated carbon decolorizing, filter, cooling, with NaOH, adjust pH=8, suction filtration, obtain light yellow solid 1,3 – dimethyl-6-amino-uracil; 1,3 – dimethyl-6-amino-uracil is dissolved in water and is stirred, and 5 Sodium Nitrite 0.18mol of average mark, are cooled to room temperature after adding, continue to stir 3h, filter, collecting precipitation, washing, dry, obtain purple solid 1,3-dimethyl-5-nitroso-group-6-Urea,amino-pyrimidine; 1,3-dimethyl-5-nitroso-group-6-Urea,amino-pyrimidine is dissolved in ammoniacal liquor, and average mark adds 0.3mol V-Brite B 5 times, the solution look that first reddens then becomes green and finally becomes light yellowly, is down to room temperature and continue stirs 30 minutes, and reaction mixture volume reduces half, cooling in ice bath, filter collecting precipitation, wash to obtain faint yellow solid 1,3-dimethyl-5,6-diaminourea pyrimidine, quality: 26.2g, yield: 92%, Mp:210～213 ° C;
(2) synthetic 1,3-dimethyl-8-(to nitrobenzyl-1H-pyrazoles)-xanthine
In 250mL reaction flask by 0.1mol to nitrobenzyl-1H-pyrazoles-4-formic acid, 0.1mol carbodiimide hydrochloride is dissolved in 150mL methyl alcohol, minute adds 0.1mol 15 times, 3-dimethyl-5,6 diaminourea pyrimidines, stir 15 hours, removal of solvent under reduced pressure, washing; Put in 250mL reaction flask, the sodium hydroxide solution that adds 75mL methyl alcohol and 75mL, reaction 20 min in the rearmounted microwave of stirring and dissolving (400 W), cooling, hydrochloric acid with 6mol/L is acidified to pH=2 in ice bath, separates out yellow solid 1,3-dimethyl-8-(to nitrobenzyl-1H-pyrazoles)-xanthine, quality 31.6g, yield 82.8%.Mp:254～256 ℃, purity: 99.0%.
1hNMR (DMSO-d6): 13.33 (s, 1H), 8.07 (m, 2H), 7.65 (s, 1H), 7.54 (s, 1H) 7.32 (m, 2H), 5.03 (s, 2H), 3.08 (s, 3H), 3.06 (s, 3H) (as Figure 17); LC-MS (m/z): 382 [M+] (as Figure 18).
Clinical experiment report-anti-asthma activity experiment
The compound 8-synthetic to embodiment 1～embodiment 9 (adjacent nitrobenzyl-1H-pyrazoles)-xanthine (compound 1), 8-(nitrobenzyl-1H-pyrazoles)-xanthine (compound 2), 8-(to nitrobenzyl-1H-pyrazoles)-xanthine (compound 3), 1-methyl-8-(adjacent nitrobenzyl-1H-pyrazoles)-xanthine (compound 4), 1-methyl-8-(nitrobenzyl-1H-pyrazoles)-xanthine (compound 5), 1-methyl-8-(to nitrobenzyl-1H-pyrazoles)-xanthine (compound 6), 1, 3-dimethyl-8-(adjacent nitrobenzyl-1H-pyrazoles)-xanthine (compound 7), 1, 3-dimethyl-8-(nitrobenzyl-1H-pyrazoles)-xanthine (compound 8), 1, pharmacological evaluation is carried out in 3-dimethyl-8-(to nitrobenzyl-1H-pyrazoles)-xanthic synthetic (compound 9) grouping, and pass through the bronchial wall of experimental rat model of asthma and airway smooth muscle thickness, and the level of the IL-8 in serum is analyzed, be intended to observe A
2BaR antagonist 8-(nitrobenzyl-1
hthe intervention effect of the Rat Asthma Model that-pyrazoles) xanthine excites Protalbinic acid (Ovalbumin), inquires into 8-(nitrobenzyl-1
h-pyrazoles)-xanthic pharmacological action is clinical application A
2BaR antagonist 8-(nitrobenzyl-1
h-pyrazoles)-xanthine treatment asthma provides foundation.
Specific experiment step is as follows:
1, build experimental model
Random choose age-sex is identical, 120 of the rats of body weight 150～200g.Be divided at random 12 groups: Normal group (10), model group (10), positive (8-(m-trifluoromethyl benzyl-1H-pyrazoles)-1,3-dimethyl xanthine) control group (10), medication therapy groups (every group 10 of medication therapy groups 1～medication therapy groups 9, totally 90), normal group is not done any processing.Model group: reference literature [1, Kips JC, Brussekke GJ, Joos GF, et al.Interleukin-12 inhibits antigen-induced airway hyperresponsiveness in mice Am J Respir Crit Care Med, 1996,153 (2): 535-539; 2, Liu Yuhui, Zhang Shaodan, Cheng Yanrui etc., Adenosine Receptors and Treating Bronchial Asthma New research progress, international magazine, 2006,2 (1): 64-65 of breathing; 3, thank to scorching Son, Tong Wancheng, atomization sucks the therapeutic action of triptolide to Experimental Asthma In Guinea-pigs airway reactivity, international medical and health Leader, 2008,14 (17): 20-22.] slightly change, with ovum protein suspension (0.1mg/ml) lml intraperitoneal injection sensitization.After 15 days, with 10% ovum protein atomization, suck to lure and breathe heavily, allow animal suck voluntarily, bring out air flue spasm, occur that respiratory rate deepens, accelerates and the motion of nodding, have difficulty in breathing, cough, fall until asthma attack, 1 time on the 1st, lasting 7 days.Medication therapy groups: process animal by the method identical with model group.After 15 days before 10% ovum protein brings out 30 minutes, with synthetic compound 1～compound 9, get 1ml suspension (8ug/ml) intraperitoneal injection respectively, 1 time on the 1st, continue 7 days.Positive controls: process animal by the method identical with model group.Within after 15 days 30 minutes before 10% ovum protein brings out, with 8-(m-trifluoromethyl benzyl-1H-pyrazoles)-1,3-dimethyl xanthine 1ml suspension (8ug/ml) intraperitoneal injection, 1 time on the 1st, continues 7 days.
2, measurement index
(1) preparation of light microscopy specimen
1. draw materials and fixing fixedly rat, exposed chest, takes out the four groups of bilateral lobe of the lung and main bronchuss that rat is complete, with 10% formalin solution, fixes;
2. embedding and section preparation input 0.01molL dewater
-1in PBS phosphate buffered saline buffer (PH=7.4), soak 12 hours, liquid is changed twice in centre; Drop into afterwards 70% alcohol, 80% alcohol, 90% alcohol, 100% alcohol I, 100% alcohol II, gradient alcohol dehydration; Dimethylbenzene is transparent; Routine paraffin wax embedding; With paraffin slicing machine, carry out continuous paraffin section, thick 5 microns;
3. HE staining procedure section routine dewaxes to dimethylbenzene I, dimethylbenzene II, 100% alcohol I, 100% alcohol II, each 10 minutes, 95% alcohol, 90% alcohol, 80% alcohol, 70% alcohol each 10 minutes, distilled water flushing; Haematoxylin dyeing 1 minute, tap water rinses; 1% hcl acidifying, tap water rinses; Yihong dyeing 2 minutes; Conventional gradient alcohol dehydration 70% alcohol, 80% alcohol, 90% alcohol, 95% alcohol, each 2 minutes, 100% alcohol I, 100% alcohol II, each 10 minutes; Dimethylbenzene is transparent; Neutral gum mounting;
4. observe lung tissue after HE dyeing, section is placed under the microscope that connects computer (amplifying 400 times), find complete lung entobronchus transverse section, measure lung entobronchus wall thickness and airway smooth muscle thickness.
(2) mensuration of IL-8
Every group get 2 after last atomization 24h extract right eye ball and get blood 3mL, with 4000 revs/min centrifugal, 5 minutes, leave and take supernatant liquor, be kept in the refrigerator of-20 ℃ to be measured.Get and respectively organize Mouse Blood supernatant liquor 100 μ l and standard substance 100 μ l; From balance, to the sealing bag of room temperature, take out lath; Except blank well, by sample and standard substance (100 μ l/ hole), add in respective aperture respectively; 37 ℃ of incubators are hatched 90 minutes; Wash plate 4 times; Except blank well, add biotinylated antibody (100 μ l/ hole), with shrouding gummed paper, seal reacting hole, 37 ℃ of incubators are hatched 60 minutes; Wash plate 4 times; Except blank well, add enzyme conjugates (10 μ l/ hole), seal plate hole; 37 ℃ of incubators are hatched 30 minutes; Wash plate 4 times; Add developer 100 μ l/ holes, 37 ℃, lucifuge colour developing 15 minutes; Add stop buffer 100 μ l/ holes, after mixing, at once measure OD450 value.
3, the pharmacological results
(1) normal group of observing under microscope, model group, positive controls and medication therapy groups segmental bronchus and alveolar full figure (Figure 19～Figure 30) and the lung entobronchus thickness recording by inverted phase contrast microscope and photographic system and airway smooth muscle thickness are as table 1:
Table 1
| Grouping | Lung entobronchus wall thickness (μ m) | Airway smooth muscle thickness (μ m) |
| Normal group | 38.71±18.02 ☆☆◇◇ | 13.70±6.67 ☆☆◇◇ |
| Model group | 93.52±22.99 △△◇◇ | 19.18±4.34 △△◇◇ |
| Positive controls | 68.78±18.68 △☆ | 16.45±2.98 △☆ |
| Compound 1 treatment group | 81.04±15.68 △☆ | 18.91±2.12 △☆ |
| Compound 2 treatment groups | 68.03±16.01 △☆ | 16.91±3.32 △☆ |
| Compound 3 treatment groups | 80.05±19.76 △☆ | 17.45±2.34 △☆ |
| Compound 4 treatment groups | 45.89±12.16 △☆ | 14.89±3.61 △☆ |
| Compound 5 treatment groups | 42.67±11.89 ☆ | 14.32±4.07 ☆ |
| Compound 6 treatment groups | 47.62±12.87 △☆ | 14.67±4.54 △☆ |
| Compound 7 treatment groups | 51.16±13.30 △☆ | 15.22±2.57 △☆ |
| Compound 8 treatment groups | 48.16±14.15 △☆ | 14.53±2.85 △☆ |
| Compound 9 treatment groups | 53.88±12.39 △☆ | 14.58±3.18 △☆ |
Note: 1, with normal group comparison:
△ p<0.05,
△ △ p<0.01
2, with model group comparison:
☆ p<0.05,
☆ ☆ p<0.01
3, with positive controls comparison:
◇ p<0.05,
◇ ◇ p<0.01
Application SPSS13.0 software carries out data processing, numerical variable result
± s represents, relatively adopts the random packet t method of inspection between two groups, relatively adopts variance analysis between many groups, with
p<0.01 or
p<0.05 is criterion.Experiment shows, experimental rat model of asthma compared with normal induced lung entobronchus wall thickness and airway smooth muscle thickness obviously increase, with normal group relatively there were significant differences (
p<0.01).Compound 1 treatment group～compound 9 treatment groups, its tracheal wall thickness and unstriated muscle thickness obviously reduce, with model group have significance (
p<0.05).
(2) level of the IL-8 in serum is as table 2:
Table 2
| Grouping | Investigation of plasma interleukin-8 levels (ng/L) |
| Normal group | 8.23±0.72 ☆☆◇◇ |
| Model group | 50.32±0.83 △△◇◇ |
| Positive controls | 17.65±0.55 △☆☆ |
| Compound 1 treatment group | 14.34±0.68 △☆☆ |
| Compound 2 treatment groups | 12.45±0.83 △☆☆ |
| Compound 3 treatment groups | 14.12±0.58 △☆☆ |
| Compound 4 treatment groups | 14.12±0.70 △☆☆ |
| Compound 5 treatment groups | 10.03±0.81 ☆☆ |
| Compound 6 treatment groups | 14.28±0.57 △☆☆ |
| Compound 7 treatment groups | 17.31±0.71 △☆ |
| Compound 8 treatment groups | 18.82±0.39 △☆ |
| Compound 9 treatment groups | 19.98±0.64 △☆ |
Note: 1, with normal group comparison:
△ p<0.05,
△ △ p<0.01
2, with model group comparison:
☆ p<0.05,
☆ ☆ p<0.01
3, with positive controls comparison:
◇ p<0.05,
◇ ◇ p<0.01
Application SPSS13.0 software carries out data processing, numerical variable result
± s represents, relatively adopts the random packet t method of inspection between two groups, relatively adopts variance analysis between many groups, with
p<0.01 or
p<0.05 is criterion.Experiment shows, experimental rat model of asthma compared with normal rat blood serum IL-8 level obviously increases, by statistics Epidemiological Analysis and normal group there were significant differences (
p<0.01).Compound 1 treatment group～compound 9 treatment groups, its Investigation of plasma interleukin-8 levels obviously reduces, with model group have significance (
p<0.05).
By rat pharmacological evaluation, the anti-asthma activity of synthetic compound is verified, but as can be seen from Figure 31 the structure of compound is different, and its antasthmatic degree is not identical yet.Depend primarily on charge distribution and the lipotropy of synthetic compound, after 1-methyl substituted, the lipotropy of compound strengthens, and 1, although it is stronger that 3-dimethyl replaces lipotropy, but charge distribution is different from 1-methyl substituted, experimental results show that the anti-asthma activity of position nitro substitution compound between 1-methyl substituted is the strongest.
Claims (3)
1. a 8-pyrazoles substituted xanthine class A
2Badenosine receptor antagonists, is characterized in that, structural formula is as follows:
R wherein
1, R
2for hydrogen or methyl, and R
1, R
2different; R
3for nitrobenzyl.
2. a kind of 8-pyrazoles substituted xanthine class A as claimed in claim 1
2Bthe synthetic method of adenosine receptor antagonists, is characterized in that,
The reaction formula of preparing nitrobenzyl pyrazoles-4-formic acid is:
Add 0.1mol ethyl cyanoacetate, 0.1～0.15mol triethyl orthoformate, diacetyl oxide 0.1～1g and zinc chloride catalyst 0.1～0.2g, stir, 100～120 ℃ of 1～2h that reflux, by Claisen ester condensation, continue to be heated to 130～140 ℃ and make it to react completely, and steam from fractional column is suitable for reading the low boilers that when reaction generates; Reaction solution, through cooling crystallization, filters and uses washing with alcohol, obtains oxyethyl group methylene radical ethyl cyanoacetate, Mp:50～51 ℃; 0.1mol oxyethyl group methylene radical ethyl cyanoacetate and 0.1mol hydrazine hydrate stir 10～14h in being dissolved in ethanol, and vacuum rotary steam is removed ethanol, obtain 3-amino-pyrazol-4-ethyl formate, Mp:102 ℃; 3-amino-pyrazol-4-ethyl formate is dissolved in glacial acetic acid, adds rare HCl solution and the 0.1～0.3mol sodium nitrite solution of 0.1～0.2mol under stirring, reaction mixture stirs 10～30min at-5～0 ℃, add ethanol, backflow 0.5～1.5h, vacuum rotary steam is removed ethanol, and residue washes with water, filter, use the dichloromethane extraction aqueous solution, use dried over mgso organic phase, filter, concentrating under reduced pressure obtains pyrazoles-4-ethyl formate, Mp:69 ℃; Pyrazoles-4-ethyl formate is dissolved in acetone soln, add salt of wormwood and 0.1～0.15mol nitrobenzyl bromine, at 40～70 ℃, reaction 12～20h, removes by filter salt of wormwood, adds solid potassium hydroxide 0.02mol, back flow reaction 12～20h, methyl alcohol underpressure distillation is filtered, and residue is dissolved in water and with ethyl acetate washing, water layer with hcl acidifying to separating out nitrobenzyl pyrazoles-4-formic acid;
Preparation 8-pyrazoles substituted xanthine class A
2Badenosine receptor antagonists reaction formula is:
Nitrobenzyl pyrazoles-4-formic acid and the carbodiimide hydrochloride of equimolar amount are dissolved in methyl alcohol, divide the formula 2 adding for 5 times with nitrobenzyl pyrazoles-4-formic acid equimolar amount, stir 8～16h, filter, except desolventizing, washing, is dissolved in methyl alcohol and sodium hydroxide solution, makes mixing solutions PH=8～12, in stirring and dissolving postposition 300～400W microwave, react 10～50min, be cooled to room temperature, with hydrochloric acid, in ice bath, be acidified to pH=2～5, separate out 8-pyrazoles substituted xanthine class A
2Badenosine receptor antagonists.
3. 8-pyrazoles substituted xanthine class A as claimed in claim 1
2Badenosine receptor antagonists is applied in the medicine of preparation treatment asthma.
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