CN106632338B - A kind of 9- substitution-N-（2- chlorobenzyl）Purine -6- amine derivant and its preparation method and application - Google Patents
A kind of 9- substitution-N-（2- chlorobenzyl）Purine -6- amine derivant and its preparation method and application Download PDFInfo
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- CN106632338B CN106632338B CN201611168399.XA CN201611168399A CN106632338B CN 106632338 B CN106632338 B CN 106632338B CN 201611168399 A CN201611168399 A CN 201611168399A CN 106632338 B CN106632338 B CN 106632338B
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- chlorobenzyl
- purine
- purin
- amine
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- 238000002360 preparation method Methods 0.000 title claims description 10
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 title abstract description 5
- 241001597008 Nomeidae Species 0.000 title abstract 2
- -1 methoxyl group Chemical group 0.000 claims abstract description 35
- 239000002260 anti-inflammatory agent Substances 0.000 claims abstract description 8
- 238000006467 substitution reaction Methods 0.000 claims abstract description 5
- 150000001875 compounds Chemical class 0.000 claims description 78
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 21
- 238000006243 chemical reaction Methods 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 14
- ZKBQDFAWXLTYKS-UHFFFAOYSA-N 6-Chloro-1H-purine Chemical compound ClC1=NC=NC2=C1NC=N2 ZKBQDFAWXLTYKS-UHFFFAOYSA-N 0.000 claims description 11
- 239000000203 mixture Substances 0.000 claims description 10
- JOKWOTYVMPTGJL-UHFFFAOYSA-N n-[(2-chlorophenyl)methyl]-7h-purin-6-amine Chemical compound ClC1=CC=CC=C1CNC1=NC=NC2=C1NC=N2 JOKWOTYVMPTGJL-UHFFFAOYSA-N 0.000 claims description 10
- KDDNKZCVYQDGKE-UHFFFAOYSA-N (2-chlorophenyl)methanamine Chemical compound NCC1=CC=CC=C1Cl KDDNKZCVYQDGKE-UHFFFAOYSA-N 0.000 claims description 8
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- PVRBGBGMDLPYKG-UHFFFAOYSA-N 6-benzyl-7h-purine Chemical class N=1C=NC=2N=CNC=2C=1CC1=CC=CC=C1 PVRBGBGMDLPYKG-UHFFFAOYSA-N 0.000 description 1
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
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- COGLJXZAQUPABI-UHFFFAOYSA-N n-(3-chlorophenyl)-7h-purin-6-amine Chemical compound ClC1=CC=CC(NC=2C=3NC=NC=3N=CN=2)=C1 COGLJXZAQUPABI-UHFFFAOYSA-N 0.000 description 1
- AXPCHVXFDHPTBW-UHFFFAOYSA-N n-(3-methoxyphenyl)-7h-purin-6-amine Chemical compound COC1=CC=CC(NC=2C=3NC=NC=3N=CN=2)=C1 AXPCHVXFDHPTBW-UHFFFAOYSA-N 0.000 description 1
- URMMHFZNJDOMHC-UHFFFAOYSA-N n-(4-methoxyphenyl)-7h-purin-6-amine Chemical compound C1=CC(OC)=CC=C1NC1=NC=NC2=C1NC=N2 URMMHFZNJDOMHC-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D473/00—Heterocyclic compounds containing purine ring systems
- C07D473/26—Heterocyclic compounds containing purine ring systems with an oxygen, sulphur, or nitrogen atom directly attached in position 2 or 6, but not in both
- C07D473/32—Nitrogen atom
- C07D473/34—Nitrogen atom attached in position 6, e.g. adenine
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a kind of 9- substitution-N-（2- chlorobenzyl）Purine -6- amine derivant, shown in structure such as formula (I), R is selected from hydrogen atom, C1~C4One of alkyl, substitution or unsubstituted benzyl, wherein the substituent group on benzyl is independently selected from halogen ,-CF3、C1~C4Alkoxy, C1~C4One or more in alkyl and nitro；R4To replace or unsubstituted phenyl or benzyl, wherein the substituent group on phenyl or benzyl is one or more in halogen or methoxyl group.Test result shows, compared with gold ion (inhibitor 3) is complexed in 6- benzyl purine compound, 9- substitution-N- (2- chlorobenzyl) purine -6- amine derivative of the invention is higher to the inhibiting rate of inflammatory factor, with better anti-inflammatory activity, there are the potentiality as anti-inflammatory drug.
Description
Technical Field
The invention belongs to the field of medicinal chemistry, and particularly relates to an anti-inflammatory 9-substituted-N- (2-chlorobenzyl) purine-6-amine derivative, and a preparation method and application thereof.
Background
Inflammation (inflammation) is a defense response of the body to pathogenic stimuli, centered on the vascular response, with regeneration of parenchymal and stromal cells to repair damaged tissues. Generally, inflammation is a self-limiting process, and as inflammatory factors disappear, proinflammatory mediators and anti-inflammatory mediators reach equilibrium and inflammation subsides, but in some special cases, the inflammatory factors are continuously stimulated at low intensity to make the inflammatory response continuously progress and turn into chronic inflammation (chronic inflammation), and the continuously existing and unrelenting inflammation is also called non-controllable inflammation (non-refractory). Moderate inflammatory reaction plays a role in protecting the body, and chronic non-controllable inflammation causes much more damage to the body than inflammation caused by inflammatory factors, and can cause substance metabolism disorder, cell degeneration and necrosis. In recent years, it has been found that under the influence of some factors, body inflammation can also induce cardiovascular diseases and cancer, which are serious health risks.
the 6-benzyl purine compound complex alloy ion (inhibitor 3) has strong anti-inflammatory activity, can obviously reduce the expression quantity of a plurality of inflammatory factors generated by macrophages induced by LPS, including TNF- α, IL-1 β and the like, and the generated anti-inflammatory effect can reach the indomethacin level used clinically at present, but after the compound inhibitor3 is subjected to structural analysis, the inhibitor3 has some defects, specifically, the inhibitor3 contains gold ions, so that the production cost of the medicine is increased, the treatment burden of a patient is increased, the accumulation of gold elements in a body can be caused if the compound inhibitor is taken for a long time, the side effect of the medicine is induced, the treatment of chronic inflammation is limited, the compound contains heavy metal elements, the polarity of the purine compound is changed, the solubility and the bioavailability of the compound in the body are not high, and based on the analysis, the inhibitor3 can be subjected to targeted design and structural optimization (see figure 1), so that a compound with high efficiency and good anti-inflammatory effect is obtained.
Disclosure of Invention
The invention provides a 9-substituted-N- (2-chlorobenzyl) purine-6-amine derivative, a preparation method and application thereof, wherein the 9-substituted-N- (2-chlorobenzyl) purine-6-amine derivative has good anti-inflammatory activity and has potential as an anti-inflammatory drug.
A9-substituted-N- (2-chlorobenzyl) purine-6-amine derivative has a structure shown in formula (I):
in the formula (I), R is selected from hydrogen atom and C1～C4One of alkyl and substituted or unsubstituted benzyl, wherein the substituents on the benzyl are independently selected from halogen, -CF3、C1～C4Alkoxy radical, C1～C4One or more of alkyl and nitro;
R4is substituted or unsubstituted phenyl or benzyl, wherein the substituent on the phenyl or benzyl is one or more of halogen or methoxy.
Test results show that compared with 6-benzyl purine compound complex alloy ions (inhibitor 3), the 9-substituted-N- (2-chlorobenzyl) purine-6-amine derivative has higher inhibition rate on inflammatory factors, better anti-inflammatory activity and potential as an anti-inflammatory drug.
Preferably, R is4Is composed ofWhereinIndicating the connection location.
Namely, the structure of the 9-substituted-N- (2-chlorobenzyl) purine-6-amine derivative is as follows:
preferably, the 9-substituted-N- (2-chlorobenzyl) purine-6-amine derivative has a structure shown in one of formulas (II) to (IV):
in the formula (II), R1Independently selected from H, -CH3；
In the formula (III), R2Independently selected from F, Cl, -C (CH)3)3Isobutyl, -CF3、-OCH3and-NO2One or more of the above.
Preferably, the compound represented by the formula (II) is L1～L2In (1)A compound of the formula (III) is L3～L10The kind and position of the substituent are shown in table 1 in the detailed description.
As a further preference, the 9-substituted-N- (2-chlorobenzyl) purine-6-amine derivative is the following specific compound
One of (1):
n- (2-chlorobenzyl) -9-methyl-9H-purin-6-amine, having the following chemical structure:
n- (2-chlorobenzyl) -9- (4-chlorobenzyl) -9H-purin-6-amine having the following chemical structure:
n- (2-chlorobenzyl) -9- (3, 5-dimethoxybenzyl) -9H-purin-6-amine, having the following chemical structure:
the invention also provides a preparation method of the 9-substituted-N- (2-chlorobenzyl) purine-6-amine derivative, which comprises the following steps
Step 1: 6-chloropurine is taken as a raw material to be substituted and reacted with o-chlorobenzylamine to prepare N- (2-chlorobenzyl) -9H-purine-6-amine.
Step 2: and (2) introducing a 9-bit substituent by using the N- (2-chlorobenzyl) -9H-purine-6-amine prepared in the step (1) as an intermediate through a substitution reaction.
Further, the method specifically comprises the following steps:
step 1: 80mg of 6-chloropurine is weighed into a 25mL round-bottom flask, 5mL of n-butanol is added, ultrasonic dissolution is carried out, 147mg of 2-chlorobenzylamine is added, the molar ratio of the 2-chlorobenzylamine to the 6-chloropurine is 2:1, and 50 mu L of triethylamine is added as a catalyst. Heating and refluxing in 110 deg.C oil bath for 5 hr, collecting reaction solution, performing TLC running, observing complete reaction under ultraviolet fluorescence, cooling, vacuum filtering to obtain solid, and recrystallizing to obtain 6- (2-chlorobenzylamine) purine.
Step 2: 90mg of 6- (2-chlorobenzylamino) purine and L are weighed out2-L9Halogenated hydrocarbons of corresponding different structures are put in a proper amount of round-bottom flask, and the molar ratio of the halogenated hydrocarbon to the 6- (2-chlorobenzylamino) purine is 2:1, measuring a proper amount of solvent acetonitrile, and adding 80mg of potassium carbonate as a catalyst. Heating in oil bath at 80 deg.C, refluxing, stirring for 24 hr, and vacuum drying. Adding appropriate amount of water, vacuum filtering the obtained solid, taking out, extracting the liquid with ethyl acetate, spin-drying to obtain sand, mixing all solids, and purifying by column chromatography. And R is selected from methyl substituted or unsubstituted benzyl.
The structure is shown in formulas (I) to (III) to synthesize 10 compounds, L1-10Anti-inflammatory Activity L1It is preferable that
The invention also provides application of the 9-substituted-N- (2-chlorobenzyl) purine-6-amine derivative in preparing anti-inflammatory drugs.
Preferably, the anti-inflammatory drug comprises a therapeutically effective amount of the 9-substituted-N- (2-chlorobenzyl) purine-6-amine derivative and a pharmaceutical excipient.
The dosage form of the anti-inflammatory drug is injection, tablet, capsule, aerosol, suppository, membrane, dripping pill, ointment, controlled release agent, sustained release agent or nano preparation.
Compared with the prior art, the invention has the beneficial effects that:
the 9-substituted-N- (2-chlorobenzyl) purine-6-amine derivative has certain inhibition effect on cells for tests and shows certain anti-inflammatory activity. Synthesized Compound L1、L4The inhibition rate of the inflammatory factors is generally higher than that of the positive factorsThe contrast agent 6-benzyl purine compound is complexed with alloy ions (inhibitor 3), so that the compound has better anti-inflammatory effect.
Drawings
FIG. 1 is a diagram illustrating a method for optimizing a structure of a 6-benzylpurine complex alloy ion (inhibitor 3) in the prior art;
FIG. 2 is a schematic representation of the toxicity test of all target compounds of the present invention;
FIG. 3 is a graph showing the inhibition rate of all target compounds of the present invention acting on the inhibition of inflammatory factors;
FIG. 4 is a graph showing the inhibition rate of all target compounds of the present invention acting on the inhibition of inflammatory factors;
FIG. 5 is a graph showing the inhibition of NO by all target compounds of the present invention;
FIG. 6 is a schematic diagram showing the effect of all target compounds of the present invention on COX and iNOS inhibition.
Detailed Description
The invention will be further described with reference to the following figures and examples. It should be noted that technical features or combinations of technical features described in the following embodiments should not be considered as being isolated, and they may be combined with each other to achieve better technical effects.
Synthesis of compounds
Examples 1-2L 1-L2 Synthesis
Step 1: 80mg of 6-chloropurine is weighed into a 25mL round-bottom flask, 5mL of n-butanol is added, ultrasonic dissolution is carried out, 147mg of 2-chlorobenzylamine is added, the molar ratio of the 2-chlorobenzylamine to the 6-chloropurine is 2:1, and 50 mu L of triethylamine is added as a catalyst. Heating and refluxing in 110 deg.C oil bath for 5 hr, monitoring by TLC plate running, observing reaction under ultraviolet fluorescence, cooling, vacuum filtering to obtain solid, and recrystallizing to obtain 6- (2-chlorobenzylamine) purine.
Step 2: 90mg of 6- (2-chlorobenzylamino) purine and methyl chloride were weighed into a suitable amount of round bottom flask, the molar ratio of methyl chloride to 6- (2-chlorobenzylamino) purine was 2:1, measuring a proper amount of solvent acetonitrile, and adding 80mg of potassium carbonate as a catalyst. Heating in oil bath at 80 deg.C, refluxing, stirring for 24 hr, and vacuum drying. Adding appropriate amount of water, vacuum filtering the obtained solid, extracting the liquid with ethyl acetate, spin-drying to obtain sand, mixing all solids, and purifying by column chromatography to obtain L1-L2The pure product of the compound. The product structure and molecular weight are shown in table 1.
The reaction formula is as follows:
examples 3 to 10L3-L10Synthesis of (2)
Step 1: 80mg of 6-chloropurine is weighed into a 25mL round-bottom flask, 5mL of n-butanol is added, ultrasonic dissolution is carried out, 147mg of 2-chlorobenzylamine is added, the molar ratio of the 2-chlorobenzylamine to the 6-chloropurine is 2:1, and 50 mu L of triethylamine is added as a catalyst. Heating and refluxing in 110 deg.C oil bath for 5 hr, collecting reaction solution, performing TLC plate running, observing complete reaction under ultraviolet fluorescence, cooling, and vacuum filtering to obtain solid for recrystallization to obtain 6- (2-chlorobenzylamine) purine.
Step 2: 90mg of 6- (2-chlorobenzylamino) purine and L are weighed out3-L10Halogenated hydrocarbons of corresponding different structures are put in a proper amount of round-bottom flask, and the molar ratio of the halogenated hydrocarbon to the 6- (2-chlorobenzylamino) purine is 2:1, measuring a proper amount of solvent acetonitrile, and adding 80mg of potassium carbonate as a catalyst. Heating in oil bath at 80 deg.C, refluxing, stirring for 24 hr, and vacuum drying. Adding a proper amount of water into the mixture,and (3) filtering the obtained solid, taking out the liquid, extracting the liquid by using ethyl acetate, then spin-drying the extracted liquid to prepare sand, combining all the solids, and purifying by using column chromatography. The product structure and molecular weight are shown in table 1.
The reaction formula is as follows:
R＝F、Cl、-C(CH3)3、-CF3、-OCH3or-NO2
Example 11W1Synthesis of (2)
1mmol of 6-chloropurine and 2mmol of p-fluorobenzylamine are weighed into a 25mL round-bottom flask, and the molar ratio of the two raw materials is 1:2, adding 5mL of solvent n-butanol, adding 50 mu L of triethylamine as a catalyst, heating and refluxing for 5h in an oil bath at 110 ℃, taking reaction liquid, performing TLC running, observing complete reaction under ultraviolet fluorescence, cooling, performing suction filtration, and recrystallizing to obtain W1The product structure and molecular weight are shown in Table 2.
The reaction formula is as follows:
examples 12 to 18W2-W8Synthesis of (2)
1mmol of 6-chloropurine and 2mmol of W were weighed2-W8The corresponding aniline compound is put in a 25mL round-bottom flask, and the molar ratio of two raw materials is 1:2, adding 5mL of solvent n-butanol, adding 50 mu L of triethylamine as a catalyst, heating and refluxing for 5h in an oil bath at 110 ℃, taking reaction liquid, performing TLC running, observing complete reaction under ultraviolet fluorescence, cooling, performing suction filtration, and recrystallizing to obtain W2-W8. The product structure and molecular weight are shown in Table 2.
The reaction formula is as follows:
R＝F、Cl、OH、-OCH3(ii) a Wherein,indicates the substitution position
Examples 19 to 20W9-W10Synthesis of (2)
1mmol of 6-chloropurine and 2mmol of W were weighed9-W10The corresponding amine compound was placed in a 25mL round bottom flask with a molar ratio of the two starting materials of 1:2, adding 5mL solvent n-butanol, adding 50 mul triethylamine as catalyst, heating and refluxing in 110 ℃ oil bath for 5h, taking reaction liquid, performing TLC running, observing complete reaction under ultraviolet fluorescence, cooling, performing suction filtration, and recrystallizing to obtain W9-W10。
The reaction formula is as follows:
wherein,indicates the substitution position
TABLE 1 Compound L1-L10Is connected withStructuring physical data
TABLE 2 Compound W1-W10Structure of (1) and physical data
Compound L1-L10、W1-W10The ESI-MS of (A-Si) of (A-Si),1H-NMR and13C-NMR data
N-(2-chlorobenzyl)-9H-purin-6-amine(L1)
White powder,Yield：85.6％,Mp:156.1-158.9℃,ESI-MS[M+H]+:260. 2,1H-NMR(600MHz,DMSO-d6):δ(ppm):12.987(m,1H,9-Purine-H),8.140-8.1 57(m,3H,2,8-Purine-H+NH),7.432-7.439(m,1H,3'-2-Cl-Ph-H),7.252-7.283(m, 3H,4',5',6'-2-Cl-Ph-H),4.760(s,2H,CH2).13C-NMR(600MHz,DMSO-d6):152.34 3,139.107,131.711,129.020,128.226,127.970,127.052.
N-(2-chlorobenzyl)-9-methyl-9H-purin-6-amine(L2)
White powder,Yield:50.5％；Mp:120.1-123.2℃；ESI-MS[M+H]+:274.3；1H-NMR(600MHz,CD3Cl):δ(ppm):8.414(s,1H,2-Purine-H),7.752(s,1H,8-P urine-H),7.473-7.487(m,1H,3-Ph-H),7.370-7.385(m,1H,6-Ph-H),7.201-7.260 (m,2H,4,5-Ph-H),6.474(s,1H,NH),4.975(s,2H,CH2),4.226(s,3H,9-CH3).13C-N MR(600MHz,CD3Cl):δ(ppm):154.828,153.181,139.476,136.192,133.816,1 29.689128.912,127.083,120.112,38.954,15.722.
N-(2-chlorobenzyl)-9-(4-fluorobenzyl)-9H-purin-6-amine(L3)
Light Yellow powder,Yield:45.2％；Mp:150.1-152.9℃；ESI-MS[M+H]+:368.2；1H-NMR(600MHz,CD3Cl):δ(ppm):8.435(s,1H,2-Purine-H),7.678(s,1H, 8-Purine-H),7.493-7.502(m,1H,3'-2-Cl-Ph-H),7.363-7.366(m,1H,6'-2-Cl-Ph- H),7.260-7.286(m,2H,4',5'-2-Cl-Ph-H),7.193-7.213(m,2H,2',6'-4-F-Ph-H),7.01 3-7.041(m,2H,3',5'-4-F-Ph-H),6.459(s,1H,NH),5.319(s,2H,9-CH2),4.926(s,2H, NHCH2).13C-NMR(600MHz,CD3Cl):163.612,161.971,154.872,139.684,136.0 95,133.853,131.658,129.956,129.756,128.974,127.100,116.235,46.635,32.075, 29.507.
9-(4-(tert-butyl)benzyl)-N-(2-chlorobenzyl)-9H-purin-6-amine(L4)
Yellow powder,Yield:45.2％；Mp:140.1-142.8℃；ESI-MS[M+H]+:406.2；1H-NMR(600MHz,CD3Cl):δ(ppm):8.440(s,1H,2-purine-H),7.673(s,1H,8-pur ine-H),7.192-7.484(m,8H,Ph-H),6.465(s,1H,NH),5.434(s,2H,9'-CH2),4.966(s,2 H,N-CH2),1.257(s,9H,CH3).13C-NMR(600MHz,CD3Cl):154.916,153.709,139. 803,139.671,133.894,130.029,130.723,129.747,129.035,128.039,127.126,126. 196,46.752.
N-(2-chlorobenzyl)-9-(4-chlorobenzyl)-9H-purin-6-amine(L5)
Green powder,Yield:56.2％；Mp:149.2-152.8℃；ESI-MS[M+H]+:384.6；1H-NMR(600MHz,CD3Cl):δ(ppm):8.412(s,1H,2-Purine-H),7.672(s,1H,8-Pur ine-H),7.474-7.484(m,1H,3'-2-Cl-Ph-H),7.335-7.365(m,1H,6'-2-Cl-Ph-H),7.30 2-7.326(m,2H,4',5'-2-Cl-Ph-H),7.183-7.236(m,4H,2',3',5',6'-4-Cl-Ph-H),6.456 (s,1H,NH),5.316(s,2H,9-CH2),4.964(s,2H,N-CH2).13C-NMR(600MHz,CD3C l):154.891,153.652,145.231,139.710,136.027,133.176,130.419,129.3 94,129.099,128.721,128.929,127.142,125.915,46.651,29.835.
N-(2-chlorobenzyl)-9-(3-nitrobenzyl)-9H-purin-6-amine(L6)
Yellow powder,Yield：46.9％；Mp:160.8-162.7℃；ESI-MS[M+H]+:395. 1；1H-NMR(600MHz,CD3Cl):δ(ppm):8.425(s,1H,2-Purine-H),8.177(s,1H,2' -3-NO2Ph-H),8.163(m,1H,4'-3-NO2Ph-H),7.744(s,1H,8-Purine-H),7.599-7.612 (m,1H,2'-2-Cl-Ph-H),7.481-7.536(m,2H,5',6'-3-NO2Ph-H),7.360-7.375(m,1H,6 '-2-Cl-Ph-H),7.199-7.215(m,2H,4',5'-2-Cl-Ph-H),6.456(s,1H,NH),5.316(s,2H,9 -CH2),4.964(s,2H,N-CH2).13C-NMR(600MHz,CD3Cl):154.921,153.728,148.73 2,139.484,137.947,135.928,130.332,130.032,129.724,129.014,123.527,122.73 0,46.501.
N-(2-chlorobenzyl)-9-(4-methoxybenzyl)-9H-purin-6-amine(L7)
Light Yellow powder，Yield:47.3％；Mp:164.3-166.9℃；ESI-MS[M+H]+:380.7；1H-NMR(600MHz,CD3Cl):δ(ppm):8.445(s,1H,2-Purine-H),7.6 75(s,1H,8-Purine-H),7.498-7.513(m,1H,3'-2-Cl-Ph-H),7.362-7.373(m,1H,6'-2- Cl-Ph-H),7.210-7.289(m,2H,4',5'-2-Cl-Ph-H),7.191-7.248(m,2H,2,6'-4-OCH3- Ph-H),6.860-6.886(m,2H,3',5'-4-OCH3-Ph-H),6.350(s,1H,NH),5.277(s,2H,9-C H2),4.615(s,2H,NHCH2),3.801(s,3H,OCH3).13C-NMR(600MHz,CD3Cl):159.8 31,154.802,154.402,139.833,133.864,129.974,129.721,137.102,114.547,55.42 6,46.922.
N-(2-chlorobenzyl)-9-(3,5-dimethoxybenzyl)-9H-purin-6-amine(L8)
White powder,Yield:45.6％；Mp:175.2-178.1℃；ESI-MS[M+H]+:410.1；1H -NMR(600MHz,CD3Cl):δ(ppm):8.432(s,1H,2-Purine-H),7.665(s,1H,8-Pu rine-H),7.464-7.578(m,1H,3'-2-Cl-Ph-H),7.352-7.365(m,1H,6'-2-Cl-Ph-H),7.1 74-7.210(m,2H,4',5'-2-Cl-Ph-H),6.515-6.525(m,1H,4'-3,5-OCH3-Ph-H),6.404 (s,2H,2',6'-3,5-OCH3-Ph-H),6.388(s,1H,NH),5.268(s,2H,9-CH2),4.905(s,2H,N- CH2),3.737(s,6H,OCH3).13C-NMR(600MHz,CD3Cl):161.441,154.862,153.439, 139.934,137.936,136.123,133.822,129.914,129.694,128.943,127.033,106.022, 100.132,55.512,47.313.
N-(2-chlorobenzyl)-9-(4-(trifluoromethyl)benzyl)-9H-purin-6-amine(L9)
White powder,Yield:81.3％；Mp:170.7-172.5℃；ESI-MS[M+H]+:418.3；1H -NMR(600MHz,CD3Cl)：δ(ppm)：8.343(s,1H,2-Purine-H),7.874(s,1H,8-Pur ine-H),7.593-7.606(d,2H,3',5'-4-CF3-Ph-H,J＝7.8Hz),7.480-7.495(m,1H,3'-2-Cl -Ph-H),7.369-7.383(m,3H,4',5',6'-2-Cl-Ph-H),7.191-7.229(m,2H,2',6'-4-CF3-Ph -H),6.251(s,1H,NH),5.464(s,2H,9-CH2),4.872(s,2H,N-CH2).13C-NMR(600MH z,CD3Cl):154.922,153.714,139.805,139.676,133.892,130.203,129.735,129.034, 128.034,127.143,126.204,46.745.
N-(2-chlorobenzyl)-9-(3-methoxybenzyl)-9H-purin-6-amine(L10)
White powder,Yield：47.3％；Mp:145.2-147.1℃；ESI-MS[M+H]+:380.4；1H-NMR(600MHz,CD3Cl):δ(ppm):8.423(s,1H,2-Purine-H),7.654(s,1H,8- Purine-H),7.489-7.532(m,1H,3'-2-Cl-Ph-H),7.384-7.396(m,1H,6'-2-Cl-Ph-H),7. 195-7.213(m,2H,4',5'-2-Cl-Ph-H),6.819-6.855(m,4H,2',4',5',6'-3-OCH3-Ph-H), 6.591(s,1H,NH),5.834(s,2H,9-CH2),4.956(s,2H,N-CH2),3.873(s,3H,-OCH3).13C-NMR(600MHz,CD3Cl):160.223,154.821,153.452,139.953,137.251,136.121, 133.833,130.237,129.492,129.751,128.954,127.106,120.130,113.823,55.243, 47.244,42.446.
N-(4-fluorobenzyl)-9H-purin-6-amine(W1)
Yellow powder,Yield：80.1％；Mp:155.2-157.1℃；ESI-MS[M+H]+:244.2；1H-NMR(600MHz,DMSO-d6):δ(ppm):12.961(s,1H,9-Purine-H),8.653(s,1 H,2-Purine-H),8.093(s,1H,8-Purine-H),7.285-7.337(d,2H,2,6-Ph-H,J＝8.0Hz),7. 124-7.243(d,2H,3,5-Ph-H,J＝8.0Hz),6.721(s,1H,NH),5.901(s,2H,N-CH2).13C-N MR(600MHz,DMSO-d6):154.787,152.314,151.236,139.154,128.546,126.784, 119.414,43.024.
N-(3-methoxyphenyl)-9H-purin-6-amine(W2)
Yellow powder,Yield/％:76.5；Mp:146.2-149.1℃；ESI-MS[M+H]+:242.3；1H-NMR(600MHz,DMSO-d6):δ(ppm):10.891(s,1H,9-Purine-H),8.621(s,1H, 2-Purine-H),8.232(s,1H,8-Purine-H),7.209-7.232(m,1H,6-Ph-H),7.120-7.134 (m,2H,4,5-Ph-H),6.825-6.825(m,1H,2-Ph-H),6.571(s,1H,NH),3.762(s,3H,CH3).13C-NMR(600MHz,DMSO-d6):162.121,152.914，150.346,143.734,141.236, 129.456,119.224,109.523,108.455,100.235,56.348.
N-(3-chlorophenyl)-9H-purin-6-amine(W3)
Green powder,Yield：86.2％；Mp:135.1-137.9℃；ESI-MS[M+H]+:246.7；1H-NMR(600MHz,DMSO-d6):δ(ppm):10.842(s,1H,9-Purine-H),8.622(s,1H, 2-Purine-H),7.802(s,1H,8-Purine-H),7.509-7.472(m,1H,6-Ph-H),7.220-7.324(d, 2H,4,5-Ph-H),6.927-7.125(d,1H,2-Ph-H),6.651(s,1H,NH).13C-NMR(600MHz, DMSO-d6):153.965,152.673,144.984,139.761,136.128,131.245,121.786,118.7 45,117.563.
N-(4-chloro-3-fluorophenyl)-9H-purin-6-amine(W4)
White powder,Yield:73.9％；Mp:135.1-138.2℃；ESI-MS[M+H]+:264.6；1H -NMR(600MHz,DMSO-d6):δ(ppm):10.636(s,1H,9-Purine-H),8.552(s,1H,2- Purine-H),7.792(s,1H,8-Purine-H),7.420-7.424(m,1H,6-Ph-H),7.217-7.255(m, 1H,5-Ph-H),7.127-7.225(m,1H,2-Ph-H),6.672(s,1H,NH).13C-NMR(600MHz,D MSO-d6):164.39,151.93,150.73,144.96,139.83,131.49,118.18,109.84,107.39. N-(4-chlorophenyl)-9H-purin-6-amine(W5)
White powder,Yield：73.9％；Mp:156.7-158.2℃；ESI-MS[M+H]+:246. 2；1H-NMR(600 MHz,DMSO-d6):δ(ppm)：12.531(s,1H,9-Purine-H),8.141(s, 1H,2-Purine-H),7.562(s,1H,8-Purine-H),7.485-7.524(d,2H,2,6-Ph-H,J＝7.6Hz), 6.934-7.143(d,2H,3,5-Ph-H,J＝8.0Hz),6.456(s,1H,NH).13C-NMR(600MHz,DM SO-d6):153.965,152.674,144.984,139.763,136.124,131,245,121.784,118.74 1,117.568.
N-(furan-2-ylmethyl)-9H-purin-6-amine(W6)
White powder,Yield:76.4％；Mp:162.2-164.8℃；ESI-MS[M+H]+:216.2；1H-NMR(600 MHz,DMSO-d6):δ(ppm)：12.942(s,1H,9-Purine-H),8.113-8.20 2(s,3H,2,8-Purine-H+NH),7.538(s,1H,4'-Furan-H),6.358(s,1H,3'-Furan-H),6.2 30(s,1H,2'-Furan-H),4.694(s,2H,N-CH2).13C-NMR(600MHz,DMSO-d6):154.8 46,152.795,151.592,146.713,143.575,137.414,119.723,113.454,107.232,39.73 4.
N-(4-methoxyphenyl)-9H-purin-6-amine(W7)
Grey powder,Yield:74.8％；Mp:161.9-164.2℃；ESI-MS[M+H]+:242.2；1H-NMR(600 MHz,DMSO-d6):δ(ppm):11.291(s,1H,9-Purine-H),8.742(s, 1H,2-Purine-H),8.033(s,1H,8-Purine-H),7.585-7.624(d,2H,2,6-Ph-H,J＝8.0Hz), 7.034-7.123(d,2H,3,5-Ph-H,J＝8.0Hz),6.744(s,1H,NH),3.963(s,3H,-CH3).13C-N MR(600MHz,DMSO-d6):153.756,153.746,149.243,136.794,133.722,129.563, 122.135,119.233,114.483,60.235.
N-((4-((9H-purin-6-yl)amino)phenyl)sulfonyl)acetamide(W8)
Grey powder,Yield：74.3％；Mp:200.1-202.7℃；ESI-MS[M+H]+:333. 4；1H-NMR(600MHz,DMSO-d6):δ(ppm):12.052(s,1H,9-Purine-H),11.461(s, 1H,NH-C＝O),8.843(s,1H,2-Purine-H),8.731(s,1H,8-Purine-H),7.475-7.524(d,2 H,3,5-Ph-H,J＝8.0Hz),7.204-7.243(d,2H,2,6-Ph-H,J＝7.8Hz),6.632(s,1H,NH),1. 923(s,3H,-CH3).13C-NMR(600MHz,DMSO-d6):172.095,154.283,152.863,1 44.264,143.791,139.855,129.986,119.778,111.793,28.372.
4-((9H-purin-6-yl)amino)phenol(W9)
White powder，Yield：77.3％；Mp:156.1-157.3℃；ESI-MS[M+H]+:228. 1；1H-NMR(600MHz,DMSO-d6):δ(ppm):12.091(s,1H,9-Purine-H),8.78 2(s,1H,2-Purine-H),7.892(s,1H,8-Purine-H),7.285-7.324(d,2H,2,6-Ph-H,J＝7.6 Hz),6.934-7.143(d,2H,3,5-Ph-H,J＝8.0Hz),6.561(s,1H,NH),5.534(s,1H,OH).13C -NMR(600MHz,DMSO-d6):153.236,151.837,143.924,140.713,138.242,131.2 32,129.652,123.522,119.235.
6-(pyrrolidin-1-yl)-9H-purine(W10)
White powder,Yield：85.2％；Mp:162.1-164.2℃；ESI-MS[M+H]+:19 0.2；1H-NMR(600MHz,DMSO-d6):δ(ppm):13.241(s,1H,9-Purine-H),8.742 (s,1H,2-Purine-H),8.121(s,1H,8-Purine-H),3.786(m,4H,N-CH2+N-CH2),1.879 (m,4H,CH2CH2).13C-NMR(600MHz,DMSO-d6):159.122,155.124,153.527,149. 238,138.955,127.944,125.734,117.684,60.235,42.824.
The properties and solubility of the target compound synthesized by the present invention are as follows:
compound L1-2，L8-10，W4-6，W9-10As a white solid. L is3，L7Is light yellow, W1-2， L4，L6Is yellow, L5，W3Is green, W7-8In grey. Is easily dissolved in DMSO and DMF.
The compounds synthesized by the invention are tested by mass spectrometry, and the positive electrodes all show [ M +1 ]]+And the signal is stronger, the compound synthesized by the invention is carried out1H-NMR and13C-NMR measurement of1H-NMR and13the hydrogen signal of the compound and the chemical shift thereof can be clearly seen on the C-NMR spectrum.
II, anti-inflammatory activity data of the compound
1. Cytotoxicity assays for synthetic Compounds
(1) Cell plating: 1) calculating the density of cells required by an experiment, carefully sucking the culture solution in a culture bottle, adding a certain amount of PBS (phosphate buffer solution) to wash the culture solution clean, dissolving the cells growing adherently for 3min by using a pancreatin solution (0.25%), adding 10% serum into the culture medium to stop the pancreatin from continuously digesting the cells, blowing the bottle wall to ensure that the cells growing adherently break away from the bottle wall and uniformly disperse in the culture solution, adding 1640 culture solution into the culture bottle, and diluting the cells to the required density of 44000 cells/mL; 2) uniformly adding the prepared cell solution into a 96-hole enzyme-labeled hole according to the standard that the concentration of each hole is 90 mul/well, and filling a marginal hole with sterile PBS; 3) and taking out the 96-hole enzyme-labeled culture plate, and reasonably setting the experimental group and the control group according to the requirements. The inhibitor3 is used as a positive control drug, one compound is used in each group of the experimental group, each compound is respectively set into 9 identical concentration gradients, each gradient concentration is provided with three multiple holes for repeated experiments, the accuracy and reliability of the experiments are ensured, each hole in the control group is directly inoculated with 100 mu l of cell suspension, no substance is added, and whether the experiments are polluted or not can be detected; 4) finally, the cells are cultured under suitable conditions, particularly with 5% CO2And incubating for 24h at 37 ℃.
(2) Test compound addition: 1) both the synthesized test compound and the positive control were dissolved in 100% DMSO to prepare a solution with a concentration of 200 ×; 2) accurately pipetting 7 μ l of the prepared solution into 133 μ l of complete medium by using a pipette (i.e., diluting 20 times to obtain 10 Xsolution); 3) sucking 10. mu.L of the diluted solution, uniformly adding the solution into a 96-well enzyme label plate (finally obtaining a 1x solution), and 4) finally culturing the cells under proper conditions, wherein the specific requirement is that 5% CO is adopted2And incubating for 24h at 37 ℃.
(3) And measuring results: 1) taking out the 96-well enzyme label plate incubated for 24 hours in the step (2), and placing the plate at room temperature for 30min for later use according to requirements; 2) according to the previously calculated amount, 30 μ l of Cell Titer-Glo reagent was added to each well and shaken for 10min to induce cells and induce cytolysis; 3) standing at room temperature for 2min to stabilize the fluorescence signal; 4) the absorbance of each well at 570nm was measured by ELISA and the results were recorded accurately. And finally, calculating the inhibition rate of the compound according to a given formula. The specific formula is as follows: inhibition (%) - (control well OD value-experimental well OD value)/control well OD value X100%.
The experimental results are as follows: FIG. 2 is a schematic diagram of the toxicity test of all target compounds of the present invention. As can be seen from fig. 2: the toxicity of the synthesized compound is generally lower than that of a positive control medicament inhibitor 3.
2. ELISA method for detecting TNF- α and IL-1 β content
The operation method comprises the following steps:
1) preparation of broth for stimulating mouse to produce large amount of macrophage
Weighing 0.15g of beef extract, 0.5g of peptone, 0.25g of NaCl and 3g of soluble starch, boiling the broth until the broth is transparent, and cooling to room temperature for intraperitoneal injection.
2) Mice were injected with 2mL of broth and three days later the cells were extracted (during which time the compounds could be formulated and dissolved in DMSO).
3) Cell lifting and plating: the mouse is subjected to eye picking, bloodletting to reduce erythrocytes in abdominal cavity, and then broken marrow is killed, the body is sterilized by 75% alcohol, and the mouse is positioned on foam in an upward mode, and the head, the four limbs and the tail are fixed. At the middle position of the median line of the thorax, a diaphragm is clamped by a pair of forceps, 3-4 ml of RPMI-1640 culture medium (not containing FBS) is injected into the abdominal cavity by a syringe, and the abdomen is gently kneaded to uniformly mix the macrophages and the culture medium. The middle diaphragm is clamped by a pair of forceps, surgical scissors (taken in the vertical direction) are used for cutting the diaphragm, the liquid in the abdominal cavity is sucked up by a liquid transfer gun and is transferred into a 15ml centrifugal tube, the centrifugation is carried out for 1100r for 5min, and the supernatant is discarded. About 2ml of a FBS-containing medium was added to the centrifuged cells, and the cells were repeatedly gently blown into a cell suspension.The cells were counted (20. mu.L/well) and cultured in six well plates (1 ml; 5X 10)5Per well) at 37 ℃ 5% CO2And (5) culturing. And after 4-6 h, taking out, washing with PBS, and replacing the solution.
4) Mu.l of the synthetic compound was added at a concentration of 10. mu. mol, and after 30min 1. mu.l of LPS (0.5. mu.g/ul) was added for stimulation, and the mixture was divided into three groups: isotypes + LPS, LPS (stimulated group), con (control group, cells only with DMSO).
5) And after the treatment is finished, putting the mixture into an incubator for incubation for more than 24 hours.
The detection method comprises the following steps:
(1) coating: the incubated medium was removed, 40. mu.l of primary antibody (capure antibody) and 1mL of coating buffer (10X) were added, the mixture was blown and mixed well with a pipette, the mixture was added to an ELISA plate, 100. mu.l of each well was added, and the ELISA plate was embedded with a preservative film overnight at 4 ℃. During this time, the PBST is configured: 500. mu.l of Tween 20 were dissolved in 1000mL of PBS solution.
(2) AD was added for blocking: pouring out the culture medium after overnight culture, washing with PBST for three times, adding 250 mu l of the culture medium into each hole, blowing and beating for 1-2 min with a pipette gun each time, pouring out the culture medium for the last time, slightly beating the culture medium on paper, adding an AD solution into each hole according to 200 mu l, wrapping the culture medium with a preservative film after adding the solution, and sealing the culture medium on a shaking table for 1 h. After the culture medium is sealed, the PBST solution is continuously washed for three times, 250 mu l of PBST solution is still added each time, and the PBST solution is lightly blown for 1-2 min by a pipette. During the above-mentioned sealing process, the supernatant may be removed and thawed.
(3) Sample adding: a certain amount of mixed solution was added in an amount of 100. mu.l per well, wherein the sample accounted for 5%, AD accounted for 95%, wrapped with a preservative film and incubated on a shaker for 2 h.
(4) Adding a secondary antibody: and (3) taking out 40 mu l of secondary antibody and AD100mL, fully and uniformly mixing, adding 100 mu l of secondary antibody into each hole, continuously wrapping by using a preservative film, and continuously incubating for 1h on a shaking table.
(5) Adding an enzyme: and (3) taking out the ELISA plate, washing the plate with PBST for three times, adding 250 mu l of the enzyme into each hole, blowing and beating the plate for 1-2 min with a pipette gun each time, adding HRP enzyme, continuously wrapping the plate with a preservative film, and then putting the plate on a shaking table for continuous incubation for 1h, wherein the volume of each hole is 100 mu l.
(6) Adding a color developing agent: washing with PBST for three times, adding 250 mul into each hole, blowing and beating for 1-2 min with a pipette each time, adding a color developing agent TMP according to 100 mul into each hole under the condition of keeping out of the sun, and developing to blue.
(7) And (3) terminating the reaction: after the ELISA plate was removed, 50. mu.l of H was added to each well at a concentration of 2mol/L2SO4。
(8) Measuring an OD value: the absorbance of the solution at a wavelength of 450nm was measured and calculated according to the formula.
The experimental results are as follows: FIG. 3 is a graph showing the inhibition rate of all target compounds of the present invention acting on the inhibition of inflammatory factors. As can be seen from fig. 3: synthesized Compound L1，L4，W2，W4The inhibition rate of the inflammatory factors is higher than that of a positive control medicament inhibitor3, so the compounds have better anti-inflammatory effect.
By comparing the target compound with the positive control drug, it can be found that: compound L1，L4，W2， W4The inhibition rate of inflammatory factors TNF-a and IL-6 is over 50 percent, and the maximum inhibition rate is about 80 percent, and the 2, 9-disubstituted purine derivatives have certain potential in the research and development of anti-inflammatory drugs. Further study of the anti-inflammatory Activity of 4 preferred Compounds
the compound L is obtained through the preliminary screening result of the inhibition effect of the first part of the compound on the content of TNF- α and IL-1 β induced by LPS under the concentration of 10 mu M1，L4，W2And W4therefore, 4 compounds are selected in the section to further study the anti-inflammatory activity.
3. ELISA method for detecting contents of TNF- α, IL-1 β, PGE2 and IL-6
The specific experimental operating steps are as follows:
(1) coating: the incubated medium was removed, 40. mu.l of primary antibody (capure antibody) and 1mL of coating buffer (10X) were added, the mixture was blown and mixed well with a pipette, the mixture was added to an ELISA plate, 100. mu.l of each well was added, and the ELISA plate was embedded with a preservative film overnight at 4 ℃. During this time, the PBST is configured: 500. mu.l of Tween 20 were dissolved in 1000mL of PBS solution.
(2) Adding AD for sealing: pouring out the culture medium after overnight culture, washing with PBST for three times, adding 250 mu l of the culture medium into each hole, blowing and beating for 1-2 min with a pipette gun each time, pouring out the culture medium for the last time, slightly beating the culture medium on paper, adding an AD solution into each hole according to 200 mu l, wrapping the culture medium with a preservative film after adding the solution, and sealing the culture medium on a shaking table for 1 h. After the culture medium is sealed, the PBST solution is continuously washed for three times, 250 mu l of PBST solution is still added each time, and the PBST solution is lightly blown for 1-2 min by a pipette. During the above-mentioned sealing process, the supernatant may be removed and thawed.
(3) And sample adding: a certain amount of mixed solution was added in an amount of 100. mu.l per well, wherein the sample accounted for 5%, AD accounted for 95%, wrapped with a preservative film and incubated on a shaker for 2 h.
(4) And adding a secondary antibody: and (3) taking out 40 mu l of secondary antibody and AD100mL, fully and uniformly mixing, adding 100 mu l of secondary antibody into each hole, continuously wrapping by using a preservative film, and continuously incubating for 1h on a shaking table.
(5) And adding an enzyme: and (3) taking out the ELISA plate, washing the plate with PBST for three times, adding 250 mu l of the enzyme into each hole, blowing and beating the plate for 1-2 min with a pipette gun each time, adding HRP enzyme, continuously wrapping the plate with a preservative film, and then putting the plate on a shaking table for continuous incubation for 1h, wherein the volume of each hole is 100 mu l.
(6) Adding a color developing agent: washing with PBST for three times, adding 250 mul into each hole, blowing and beating for 1-2 min with a pipette each time, adding a color developing agent TMP according to 100 mul into each hole under the condition of keeping out of the sun, and developing to blue.
(7) And terminating the reaction: after the ELISA plate was removed, 50. mu.l of H was added to each well at a concentration of 2mol/L2SO4。
(8) And measuring an OD value: the absorbance of the solution at a wavelength of 450nm was measured and calculated according to the formula.
The experimental results are as follows: FIG. 4 is a graph showing the inhibition rate of all target compounds of the present invention acting on the inhibition of inflammatory factors. As can be seen from fig. 4: synthesized Compound L1，L4，W2And W4The inhibition rate of the compounds on several inflammatory factors is generally close to or higher than that of a positive control medicament inhibitor3, and the compounds have certain dose dependence, so the compounds have better anti-inflammatory effect.
By comparing the target compound with the positive control drug, it can be found that: compound L1，L4，W2And W4The inhibition rate of a plurality of inflammatory factors is generally close to or higher than that of a positive control medicament inhibitor3, and the 2, 9-disubstituted purine derivative has certain potential in research and development of anti-inflammatory medicaments by preliminary inference.
4. Determination of the NO content
The specific experimental operating steps are as follows:
(1) RAW264.7 cell culture: the cell strain was purchased from a cell bank of Shanghai academy of sciences of China, and the culture conditions were as follows: double antibiotics (streptomycin and penicillin) and 10% fetal bovine serum in DMEM complete medium at 37 deg.C and 5% CO2Culturing in a culture environment.
(2) Detecting the content of NO by a Griess method: taking RAW264.7 cells in log phase for experimental operation, adding LPS, culturing for 30min, adding 2.5,5,10,20 μmol of medicine, and continuing culturing overnight. Collecting cell culture solution, centrifuging (6000rpm,5min), collecting 10 μ l cell supernatant, adding Griess reagent (2% sulfanilamide)With 5% H3PO4Prepared, 0.2 percent of naphthylethylenediamine) is mixed with 100 mul of naphthylethylenediamine), kept stand for 10min in a dark place at room temperature, the absorbance value is measured at the wavelength of 540nm by a microplate reader, and the NO content in the sample is calculated.
Results of inhibition of NO content: FIG. 5 is a graph showing the inhibition rate of all target compounds of the present invention against NO. As can be seen from fig. 5: synthetic Compounds synthetic Compound L1，L4，W2And W4The inhibition rate of the compounds on several inflammatory factors is generally close to or higher than that of a positive control medicament inhibitor3, and the compounds have certain dose dependence, so the compounds have better anti-inflammatory effect.
By comparing the target compound with the positive control drug, it can be found that: compound L1，L4，W2And W4The inhibition rate of the derivatives on several inflammatory factors is generally close to or higher than that of a positive control medicament inhibitor3, and the derivatives have certain dose dependence, so that the 2, 9-disubstituted purine derivatives have certain potential in research and development of anti-inflammatory medicaments.
5. Determination of RT-PCR
The specific experimental operating steps are as follows:
(1) and determination of RNA content: extracting RNA by TRIzol method, adding appropriate amount of TRIzol reagent, mixing, standing for 5min, adding chloroform 5:1, standing for 3min, centrifuging at 12000rpm and 4 deg.C for 15min, and sucking RNA-containing water layer into new centrifuge tube; adding isopropanol according to the amount of 1:2 of TRIzol, standing for 10min, then centrifuging at 12000rpm at 4 ℃ for 10min, leaving precipitate, and removing supernatant; trizol: 75% ethanol (DEPC configuration), centrifuged and the supernatant carefully discarded.
(2) Determination of RNA concentration and purity: the integrity and concentration of the extracted RNA was checked by agarose gel electrophoresis and spectrophotometer, respectively.
(3)、RT-PCR:
Reaction system:
the primer sequence is as follows: qmOX 2(74 bp):
Forward Primer5'TGAGCAACTATTCCAAACCAGC3'
Reverse Primer5'GCACGTAGTCTTCGATCACTATC3'
qmGAPDH(149bp)：
Forward Primer5'TATGTCGTGGAGTCTACTGGT 3'
Reverse Primer 5'GAGTTGTCATATTTCTCGTGG 3'
qmiNOS(127bp)：
Forward Primer5'GTTCTCAGCCCAACAATACAAGA3'
Reverse Primer 5'GTGGACGGGTCGATGTCAC 3'
the experimental results are as follows: FIG. 6 is a schematic diagram showing the effect of all target compounds of the present invention on COX and iNOS inhibition. As can be seen from fig. 6: synthesized Compound L1，L4，W2And W4The inhibition rate of COX and iNOS is generally close to or higher than that of a positive control medicament inhibitor3, and the inhibition rate of the COX and iNOS has certain dose dependence, so that the compounds have good anti-inflammatory effect.
By comparing the target compound with the positive control drug, it can be found that: compound L1，L4，W2And W4The inhibition rate of the derivatives on several inflammatory factors is generally close to or higher than that of a positive control medicament inhibitor3, and the derivatives have certain dose dependence, so that the 2, 9-disubstituted purine derivatives have certain potential in research and development of anti-inflammatory medicaments.
Summary of anti-inflammatory Activity of Compounds
the method comprises the steps of firstly testing the inhibition effect of the synthesized 20 6, 9-disubstituted purine derivatives on TNF- α and IL-1 β by an ELISA method under the condition of 10 mu M concentration, and primarily screening out compounds with good anti-inflammatory activity, then finding that compounds L1, L4, W2 and W4 show better inhibition effect on inflammatory factors, and then starting to carry out intensive anti-inflammatory activity research on the 4 compounds, wherein the intensive research on the anti-inflammatory activity is mainly characterized in that the concentrations of the compounds are increased firstly, the range of the tested concentrations are from 10 mu M to 5,2.5 mu M, and the influence of the compound concentrations on the anti-inflammatory activity is discussed, the concentrations of the compounds L1, L4, W2 and W4 are in a linear relation with the inhibition effect, namely, the anti-inflammatory capability of the compounds is enhanced with the increase of the compound concentrations, the types of the inflammatory factors are increased secondly, the levels of the two inflammatory factors (TNF- α and IL-1 β) which are primarily screened are increased to five inflammatory factors including IL-6, PGE2 and NO, the anti-inflammatory factors L1, the compounds are tested in a simple test on mRNA, and mRNA levels of the inhibition effect of the compounds are considered to be increased.
Claims (6)
1. A9-substituted-N- (2-chlorobenzyl) purine-6-amine derivative is characterized by being one of the following specific compounds:
n- (2-chlorobenzyl) -9-methyl-9H-purin-6-amine, having the formula:
n- (2-chlorobenzyl) -9- (4-chlorobenzyl) -9H-purin-6-amine having the formula:
n- (2-chlorobenzyl) -9- (3, 5-dimethoxybenzyl) -9H-purin-6-amine, having the following structural formula:
2. a process for the preparation of a 9-substituted-N- (2-chlorobenzyl) purin-6-amine derivative according to claim 1, comprising the steps of:
(1) under the action of alkali, 6-chloropurine and 2-chlorobenzylamine have substitution reaction to obtain 6- (2-chlorobenzylamino) purine;
(2) under the action of alkali, 6- (2-chlorobenzyl) purine obtained in the step (1) reacts with halogenated hydrocarbon, and after the reaction is finished, the 9-substituted-N- (2-chlorobenzyl) purine-6-amine derivative is obtained through post treatment.
3. The process for producing a 9-substituted-N- (2-chlorobenzyl) purin-6-amine derivative according to claim 2, wherein the base in step (1) is triethylamine, and the base in step (2) is potassium carbonate.
4. Use of the 9-substituted-N- (2-chlorobenzyl) purin-6-amine derivative of claim 1 in the preparation of anti-inflammatory agents.
5. The use of a 9-substituted-N- (2-chlorobenzyl) purin-6-amine derivative according to claim 4, wherein said anti-inflammatory agent comprises a therapeutically effective amount of a 9-substituted-N- (2-chlorobenzyl) purin-6-amine derivative and a pharmaceutically acceptable excipient.
6. The use of the 9-substituted-N- (2-chlorobenzyl) purin-6-amine derivative of claim 5, wherein the anti-inflammatory agent is in the form of injection, tablet, capsule, aerosol, suppository, membrane, drop pill, ointment, controlled release formulation, sustained release formulation, or nanometer preparation.
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