CN114605334B - 2-Aminopyrimidine compound, preparation method, application and biological membrane inhibitor - Google Patents
2-Aminopyrimidine compound, preparation method, application and biological membrane inhibitor Download PDFInfo
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- CN114605334B CN114605334B CN202210291411.5A CN202210291411A CN114605334B CN 114605334 B CN114605334 B CN 114605334B CN 202210291411 A CN202210291411 A CN 202210291411A CN 114605334 B CN114605334 B CN 114605334B
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- 239000003112 inhibitor Substances 0.000 title claims abstract description 25
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
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- DMJNNHOOLUXYBV-PQTSNVLCSA-N meropenem Chemical compound C=1([C@H](C)[C@@H]2[C@H](C(N2C=1C(O)=O)=O)[C@H](O)C)S[C@@H]1CN[C@H](C(=O)N(C)C)C1 DMJNNHOOLUXYBV-PQTSNVLCSA-N 0.000 description 1
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- 239000012730 sustained-release form Substances 0.000 description 1
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- 150000003952 β-lactams Chemical class 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
- C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
- C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
- C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
- C07D239/32—One oxygen, sulfur or nitrogen atom
- C07D239/42—One nitrogen atom
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P31/04—Antibacterial agents
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- C07D—HETEROCYCLIC COMPOUNDS
- C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
- C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
- C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
- C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
- C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
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- C07D—HETEROCYCLIC COMPOUNDS
- C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
- C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
- C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
- C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
- C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
- C07D239/48—Two nitrogen atoms
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- C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
- C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
- C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
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Abstract
The invention relates to the technical field of biology, in particular to a 2-aminopyrimidine compound, a preparation method, application and a biological membrane inhibitor. The antibacterial agent has good antibacterial activity on Acinetobacter baumannii, and also has good antibacterial activity on methicillin-resistant Lin Putao cocci, staphylococcus epidermidis, escherichia coli and the like. The compound is based on natural marine alkaloids with anti-biofilm activity, and mostly takes 2-aminoimidazole as a mother nucleus, so that the problem that AHL analogues are easy to hydrolyze and inactivate is avoided. Meanwhile, the compound provides more chemical entities for a novel treatment means of anti-infective drugs, and has the advantages of easily available raw materials, simple steps, low reaction cost, high efficiency and capability of being prepared and produced in a large amount. The compound is used as a biological film inhibitor, has stronger activity and good biological film inhibition effect. The compounds can also be used for preparing antibacterial drugs or biological materials.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a 2-aminopyrimidine compound, a preparation method, application and a biological membrane inhibitor.
Background
With the continuous enhancement of bacterial drug resistance, particularly the emergence of multi-drug resistant superbacteria, great potential hazards are brought to the health of human society. One common defense mechanism observed in multi-drug resistant bacteria and other bacteria is biofilm formation. In recent years, clinical practice and research results at home and abroad prove that the drug resistance of bacteria, the difficult cure of chronic infectious diseases and repeated attacks are closely related to the biological film of bacteria. Due to the barrier effect of the biological film and the low metabolism of bacteria in the biological film, once the bacterial biological film is formed, the drug resistance of the bacterial biological film can be improved by hundreds to thousands times, and the bacteria can escape from the attack of an immune system, so that the infection is chronized and difficult to control. Bacterial biofilm infection is an important reason that clinical infection is not prolonged and pathogenic bacteria are difficult to thoroughly clear.
Acinetobacter baumannii (A.baumannii, ab) is a non-fermented gram-negative bacterium, and its remarkable multi-drug resistance and high incidence make it one of the most troublesome accepted nosocomial pathogenic bacteria. The world health organization also lists acinetobacter baumannii as one of the strongest resistant bacteria, which constitutes a particularly serious threat to human health.
For biofilm formation by acinetobacter baumannii, studies have shown that a quorum sensing (quorum sensing, QS) system can be used to interfere. This is because genes involved in biofilm formation are also regulated by the QS system. It has been found that QS systems interfering with abs do not affect their individual growth, but block population communication between abs, making them unable to act as a population to regulate biofilm formation, thereby avoiding the masking effect of biofilm on antibiotics since this process does not kill bacteria directly, and thus reducing the likelihood of bacteria developing resistance to them.
The QS system of Acinetobacter baumannii comprises self-induced synthetase (abaI), acyl homoserine lactone (Acyl homoserine lactone, AHL) signal molecules and abaR receptors, and belongs to a typical LasR-LasI group behavior control system. Bacteria synthesize one or more self-inducers (Autoinducer, AI) by abaI synthetases and release the agents to the external environment, abaR receptors determine the change of the flora density and the surrounding environment by sensing the AI concentration, and when the concentration of the self-inducers reaches a certain threshold value, the flora starts a series of corresponding gene expression to regulate the formation of a biological film.
Prior art QS inhibitors include natural or synthetic inhibitors, which are mostly analogues of AHL, receptor inhibitors or synthetase inhibitors. However, these inhibitors tend to have a number of problems, resulting in limited utility.
For example, philip n.the task group of the rathe found that the autoinducer AHL of Ab M2 strain and its derivatives have some inhibitory effect on Ab biofilm, but are inactivated due to the easy hydrolysis of the lactone ring of AHL and its derivatives.
N-pyrrole substituted orotidine derivatives found in CHRISTIAN MELANDER, natural marine alkaloids and derivatives thereof found in Helen E.Blacwell, and marine alkaloids with polysubstituted 2-aminoimidazole as skeleton found in Rupinder K.Gill all reported inhibitory activity against Acinetobacter baumannii biofilm, even with anti-biofilm activity IC 50 as low as 26.8 μm, but the extraction and synthesis process of natural macromolecules were difficult.
Disclosure of Invention
The invention aims to provide a 2-aminopyrimidine compound, a preparation method, application and a biological membrane inhibitor.
The technical scheme for solving the technical problems is as follows:
The invention provides a 2-aminopyrimidine compound, the structural general formula of which is shown as the general formula (1):
Wherein R 1 is one of substituted or unsubstituted C 2～C14 alkyl, C 2～C14 cycloalkyl, C 2～C14 carboxyl, substituted or unsubstituted phenyl, naphthyl and five-membered heterocyclic aryl;
R 2 represents one of substituted or unsubstituted C 2～C14 alkyl, alkoxy, X, R 3; x is a halogen atom; r 3 is an N-containing group;
n has a value of 0, 1, 2 or 3.
Further, n has a value of 1,2 or 3.
Further, R 1 represents one of unsubstituted C 2～C14 alkyl, F substituted alkyl, cyclobutylalkyl, pentylcarboxyl, substituted or unsubstituted phenyl, alkyl substituted phenyl, alkoxy substituted phenyl, alkenyl substituted phenyl, nitro substituted phenyl, naphthyl, thienyl; r 2 is one of methyl, methoxy, cl, br, F, amino, methylamino, dimethylamino, piperazinyl and morpholinyl.
Further, the specific structural formula of the compound is as follows:
One of them.
The invention provides a preparation method of the 2-aminopyrimidine compound, when the value of n is 2, one R 2 is represented as X, the other R 2 is represented as R 3, the reaction equation generated in the preparation process is as follows:
The specific reaction process is as follows:
Under the protection of nitrogen at room temperature, adding an acyl chloride substrate into a dry toluene solution containing a 2-aminopyrimidine raw material and N, N-diisopropylethylamine, and reacting at 25 ℃ or 110 ℃; after the 2-aminopyrimidine raw material is completely reacted, spin-drying the solvent, extracting by ethyl acetate and hydrochloric acid solution, extracting the organic layer by saturated sodium bicarbonate solution, and passing through column chromatography by anhydrous sodium sulfate to obtain a compound 1;
wherein, the molar concentration ratio of acyl chloride substrate, 2-aminopyrimidine raw material and N, N-diisopropylethylamine is 1.5:1:2;
Under the protection of nitrogen, adding the compound 1, an amine raw material and triethylamine into methanol in sequence, and reacting for 1-2 h at 25 ℃; after complete reaction, spin-drying the solvent, extracting by ethyl acetate and hydrochloric acid solution, extracting the organic layer by saturated sodium bicarbonate solution, and passing through column chromatography by anhydrous sodium sulfate to obtain the target compound;
wherein, the molar concentration ratio of the compound 1 to the amine raw material to the triethylamine is 1:1.1:1.5;
The structure of the amine raw material is R 3 -H.
The invention provides application of the 2-aminopyrimidine compound as a biological membrane inhibitor.
The invention provides a biological film inhibitor, wherein the effective component of the biological film inhibitor is the 2-aminopyrimidine compound.
Further, the biofilm inhibitor can inhibit the formation of a biofilm formed by one of Acinetobacter baumannii, methicillin-resistant Lin Putao cocci, staphylococcus epidermidis and escherichia coli.
The invention provides an anti-infective drug which is characterized by comprising the 2-aminopyrimidine compound and auxiliary materials.
The present invention provides a biomaterial coated on the surface with a biofilm inhibitor as described above.
The invention has the beneficial effects that:
1) The 2-aminopyrimidine compound of the invention is based on natural marine alkaloids with anti-biofilm activity, and mostly takes 2-aminoimidazole as a mother nucleus, thereby avoiding the trouble that AHL analogues are easy to be hydrolyzed and deactivated.
2) The 2-aminopyrimidine compound has good anti-biological film activity on Acinetobacter baumannii, and also has good anti-biological film activity on methicillin-resistant Lin Putao cocci, staphylococcus epidermidis, escherichia coli and the like.
3) The 2-aminopyrimidine compound provides more chemical entities for novel treatment means of anti-infective drugs.
3) The preparation method of the 2-aminopyrimidine compound has the advantages of easily available raw materials, simple steps, low reaction cost and high efficiency, and enables the 2-aminopyrimidine compound to be prepared and produced in a large quantity.
4) The biological film inhibitor adopts the 2-aminopyrimidine compound as an active ingredient, has stronger activity, can effectively inhibit the formation of the Acinetobacter baumannii biological film, and has good biological film inhibition effect.
5) The antibacterial drug contains the 2-aminopyrimidine compound disclosed by the invention, and can effectively inhibit bacterial infection.
6) The biological material of the invention is coated with the biological film inhibitor of the invention on the surface, and the biological material can be used for manufacturing medical devices, so that the medical devices have good antibacterial performance.
Drawings
FIG. 1 is a graph showing the results of the biofilm inhibition activity of 2-aminopyrimidine compounds of the present invention, examples 2, wherein the compounds 3a to 8 are shown in the following formula;
FIG. 2 is a graph showing SEM results of a silica gel sheet of example 3, wherein FIG. a is a positive control, b is a compound 3ab, c is a compound 3ac, d is a compound 3ad, and e is a compound 3x;
FIG. 3 is a bar chart showing the results of measuring the content of extracellular polysaccharide in example 4 of the 2-aminopyrimidine compound of the present invention;
FIG. 4 is a plan view of a 2-aminopyrimidine compound of the present invention, example 7, showing a biofilm in a confocal laser microscope, wherein A is a solvent control of DMSO; b is a biological film of ACBA.S1 under the action of a compound 3 ac; c is a biological film of ACBA.S1 under the action of a compound 8d;
FIG. 5 is a graph showing the results of the movement of 2-aminopyrimidine compounds of the present invention, example 8, compound Swaring; wherein a is DMSO positive control; b is the result of the action of the compound 3 ac; c is the result of the action of compound 3 aj; d is a DMSO-free blank control; e is the result of the action of compound 8 d; f is the result of the action of the compound 8 e;
FIG. 6 is a SEM change pattern of a slow release 1d biofilm of the 2-aminopyrimidine compound of the present invention in example 9, wherein a is PLG-DMSO control, b is PLG-3ac, c is PLG-8d, d is PLGA-DMSO control, e is PLGA-3ac, and f is PLGA-8d;
FIG. 7 shows SEM change pattern of slow release 7d of 2-aminopyrimidine compound of the present invention in example 9, wherein a is PLG-DMSO control, b is PLG-3ac, c is PLG-8d, d is PLGA-DMSO control, e is PLGA-3ac, and f is PLGA-8d;
FIG. 8 is a SEM change map of a biofilm of slow release 14d in example 9, wherein a is PLG-DMSO control, b is PLG-3ac, c is PLG-8d, d is PLGA-DMSO control, e is PLGA-3ac, and f is PLGA-8d.
Detailed Description
The principles and features of the present invention are described below with reference to the drawings, the examples are illustrated for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention.
Pyrimidine structures may be an important component of endogenous substances, which is the advantage of pyrimidine derivatives interacting with intracellular genetic material, enzymes and other biopolymer substances. Therefore, the invention takes 2-aminopyrimidine as a mother nucleus to design and synthesize a series of 2-aminopyrimidine derivatives, obtains a biological membrane inhibitor with stronger activity, and provides more chemical entities for novel treatment means of anti-infective drugs.
The structural general formula of the 2-aminopyrimidine compound is shown as a general formula (1):
Wherein R 1 is one of substituted or unsubstituted C 2～C14 alkyl, C 2～C14 cycloalkyl, C 2～C14 carboxyl, substituted or unsubstituted phenyl, naphthyl and five-membered heterocyclic aryl; r 2 is one of alkyl, alkoxy, X, R 3; x is a halogen atom; r 3 is an N-containing group; n has a value of 0, 1,2 or 3.
Preferably, n has a value of 1,2 or 3.
R 1 represents one of unsubstituted C 2～C14 alkyl, F substituted alkyl, cyclobutyl, pentylcarboxyl, substituted or unsubstituted phenyl, alkyl substituted phenyl, alkoxy substituted phenyl, alkenyl substituted phenyl, nitro substituted phenyl, naphthyl, thienyl.
R 2 is one of methyl, methoxy, cl, br, F, amino, methylamino, dimethylamino, piperazinyl and morpholinyl.
The structure of the 2-aminopyrimidine compound is designed by modifying the structure of an autoinducer OH-dDHL of Acinetobacter baumannii, taking 2-aminopyrimidine, acyl chloride with different substitutions and the like as raw materials, and replacing an acyl homoserine lactone ring, or changing the length of an acyl carbon chain, the carbon chain structure or the side chain substituent.
The invention provides 41 specific compound structures, which are as follows: the specific structural formula of the compound is as follows:
the preparation method of the compounds 3 a-3 u is existing, and the specific reaction process is as follows:
Acid chloride substrate (1.5 mmol) was slowly added dropwise to a solution of 2-aminopyrimidine starting material (1 mmol) and N, N-diisopropylethylamine (2 mmol) in dry toluene (2 mL) under nitrogen at room temperature, the reaction was monitored by TLC at room temperature (25 ℃) or 110 ℃. After the 2-aminopyrimidine completely reacts, the solvent is dried by spin-drying through a rotary evaporator, the solvent is extracted through ethyl acetate and 1M hydrochloric acid solution, the organic layer is extracted through saturated sodium bicarbonate solution, and the corresponding target compounds 3 a-3 u are obtained through column chromatography through anhydrous sodium sulfate.
The target compounds 3v to 3ah can be obtained by adjusting the reaction conditions based on the above preparation methods of the compounds 3a to 3u and reacting at 1110 ℃.
The preparation method of the compounds 8 a-8 e comprises the following steps: on the basis of the preparation method of the compounds 3 a-3 u, the compound is further reacted with an amine raw material, and the specific reaction process is as follows:
under the protection of nitrogen, compound 3af (1 mmol), amine raw material (1.1 mmol) and triethylamine (1.5 mmol) were added in this order to methanol (2 mL), and the reaction was carried out at room temperature (25 ℃) for 1 to 2 hours, followed by monitoring the progress of the reaction by TLC. After complete reaction, the solvent is dried by spin-drying by a rotary evaporator, extracted by ethyl acetate and 1M hydrochloric acid solution, and the organic layer is extracted by saturated sodium bicarbonate solution, and then the corresponding target compounds 8 a-8 e are obtained by column chromatography by anhydrous sodium sulfate.
The amine raw material has a structure of R 3 -H.
In the structures of the compound 3ac and the compound 3aj, the pyrimidine ring has an amino group substitution and a halogen atom substitution, and these two compounds may be directly synthesized by the existing method or may be obtained by the preparation methods of the compounds 8a to 8 e. But in view of cost saving and production efficiency, the two compounds are obtained by adopting the production methods of the compounds 3a to 3u and adjusting the reaction conditions.
The following are structural characterizations of compounds 3 v-3 af, 8 a-8 e:
compound 3v, white solid, yield 97％,mp:68～71℃.1H NMR(600MHz,CDCl3)δ8.45(d,J＝5.1Hz,1H),6.85(d,J＝5.1Hz,1H),2.71(s,2H),1.75-1.68(m,2H),1.42-1.34(m,2H),1.31-1.21(m,18H),0.87(t,J＝7.0Hz,3H).13C NMR(150MHz,CDCl3)δ173.4,168.8,157.8,157.3,115.8,37.6,31.9,29.7,29.5,29.4,29.4,29.3,25.1,24.1,22.7,14.1.HRMS(ESI)calculated for C19H33N3O[M+H]+:320.2702,found:320.2703.
Compound 3w, white solid, yield 47％,mp:141～147℃.1H NMR(600MHz,DMSO)δ9.75(s,1H),7.89(d,J＝5.8Hz,1H),6.77(s,2H),6.10(d,J＝5.8Hz,1H),2.43(t,J＝7.2Hz,2H),1.55-1.43(m,2H),1.26-1.21(m,20H),0.85(t,J＝7.0Hz,3H).13C NMR(150MHz,CDCl3)δ164.0,157.4,156.5,100.6,37.4,31.9,29.7,29.6,29.5,29.5,29.4,29.3,25.1,22.7,14.1.HRMS(ESI)calculated for C18H32N4O[M+H]+:321.2654,found:321.2646.
Compound 3x, white solid, yield 16％,mp:93～97℃.1H NMR(600MHz,CDCl3)δ8.50(d,J＝5.2Hz,1H),7.03(d,J＝5.3Hz,1H),2.73(t,J＝7.3Hz,2H),1.78-1.67(m,2H),1.41-1.37(m,2H),1.31-1.20(m,18H),0.88(t,J＝7.0Hz,3H).13C NMR(150MHz,CDCl3)δ173.1,161.8,159.4,157.4,116.1,37.6,31.9,29.7,29.7,29.6,29.5,29.4,29.4,29.2,24.9,22.7,14.1.HRMS(ESI)calculated for C18H30ClN3O[M+H]+:340.2156,found:340.2163.
Compound 3y, white solid, yield 8％,mp:96～101℃.1H NMR(600MHz,CDCl3)δ8.64(s,2H),2.70(s,2H),1.72(dt,J＝15.1,7.6Hz,2H),1.42-1.36(m,2H),1.33-1.22(m,18H),0.88(t,J＝7.0Hz,3H).13C NMR(150MHz,CDCl3)δ158.8,155.9,113.1,37.6,31.9,29.7,29.7,29.6,29.5,29.4,29.4,29.3,24.9,22.7,14.1.HRMS(ESI)calculated for C18H30BrN3O[M+H]+:384.1651found:384.1651.
Compound 3z, white solid, yield 31％,mp:89～93℃.1H NMR(600MHz,CDCl3)δ8.55(s,2H),2.69(s,2H),1.83-1.68(m,2H),1.38(dd,J＝15.1,7.4Hz,2H),1.32-1.23(m,18H),0.88(t,J＝7.0Hz,3H).13C NMR(150MHz,CDCl3)δ172.9,156.6,155.5,125.2,37.5,31.9,29.7,29.7,29.6,29.5,29.4,29.4,29.2,24.9,22.7,14.1.HRMS(ESI)calculated for C18H30ClN3O[M+H]+:340.2156,found:340.2155.
Compound 3aa, white solid, yield 49％,mp:162～168℃.1H NMR(600MHz,CDCl3)δ6.02(s,1H),2.51(s,2H),2.23(s,3H),1.69(dt,J＝15.0,7.4Hz,2H),1.35(d,J＝7.2Hz,2H),1.31-1.23(m,18H),0.88(t,J＝7.0Hz,3H).13C NMR(150MHz,CDCl3)δ174.5,166.0,162.8,149.5,107.9,37.3,31.9,29.7,29.6,29.6,29.4,29.4,29.3,29.0,24.8,24.1,22.7,14.1.HRMS(ESI)calculated for C19H33N3O2[M+H]+:336.2651found:336.2657.
Compound 3ab, white solid, yield 16％,mp:53～55℃.1H NMR(600MHz,CDCl3)δ5.74(s,1H),3.90(s,6H),2.89(s,2H),2.34(t,J＝7.5Hz,1H),1.83-1.66(m,2H),1.36(d,J＝7.8Hz,2H),1.25(s,17H),0.87(d,J＝7.1Hz,3H).13C NMR(150MHz,CDCl3)δ171.9,156.2,84.6,54.2,37.4,31.9,29.7,29.7,29.6,29.5,29.5,29.4,29.4,24.7,22.7,14.1.HRMS(ESI)calculated for C20H35N3O3[M+H]+:366.27571found:366.2759.
Compound 3ac, white solid, yield 10％,mp:94～98℃.1H NMR(600MHz,DMSO)δ10.08(s,1H),7.13(s,2H),6.14(s,1H),2.41(t,J＝7.2Hz,2H),1.51(s,2H),1.24(s,20H),0.86(t,J＝6.8Hz,3H).13C NMR(150MHz,DMSO)δ172.1,165.8,158.2,157.8,98.2,36.8,31.8,29.5,29.5,29.5,29.4,29.2,29.2,29.0,25.2,22.6,14.4.HRMS(ESI)calculated for C18H31ClN4O[M+H]+:355.2265,found:355.2277.
Compound 3ad, white solid, yield 33％,mp:88～94℃.1H NMR(600MHz,CDCl3)δ7.87(s,1H),6.44(s,1H),3.98(s,3H),2.81(t,J＝7.2Hz,2H),1.71(dt,J＝15.2,7.6Hz,2H),1.38(dt,J＝15.0,6.6Hz,2H),1.30-1.23(m,18H),0.88(t,J＝7.0Hz,3H).13C NMR(150MHz,CDCl3)δ173.4,171.3,160.9,156.5,101.8,54.7,37.5,31.9,29.7,29.7,29.6,29.5,29.4,29.4,29.3,24.7,22.7,14.1.HRMS(ESI)calculated for C19H32ClN3O2[M+H]+:370.2261found:370.2266.
Compound 3ae, white solid, yield White solid,18％,mp:65～68℃.1H NMR(600MHz,CDCl3)δ8.08(s,1H),6.89(s,1H),2.74(t,J＝7.1Hz,2H),2.46(s,3H),1.72-1.68(m,2H),1.42-1.34(m,2H),1.30-1.22(m,18H),0.87(t,J＝7.0Hz,3H).13C NMR(150MHz,CDCl3)δ173.3,170.4,161.4,156.9,115.3,37.5,31.9,29.7,29.7,29.6,29.5,29.4,29.4,29.3,24.9,24.0,22.7,14.1.HRMS(ESI)calculated for C19H32ClN3O[M+H]+:354.2312,found:354.2313.
Compound 3af, white solid, yield 75％,mp:67～70℃.1H NMR(600MHz,CDCl3)δ7.05(s,1H),2.75(t,J＝7.5Hz,2H),2.35(t,J＝7.5Hz,1H),1.73-1.66(m,2H),1.42-1.36(m,2H),1.34-1.21(m,17H),0.88(t,J＝7.0Hz,3H).13C NMR(150MHz,CDCl3)δ162.4,156.8,115.4,37.5,31.9,29.7,29.7,29.6,29.5,29.4,29.3,29.2,29.1,24.7,22.7,14.1.HRMS(ESI)calculated for C18H29Cl2N3O[M+H]+:374.1766,found:374.1766.
Compound 3ag, white solid, yield 84％,mp:129～131℃.1H NMR(600MHz,CDCl3)δ8.01(s,1H),2.72(t,J＝7.5Hz,2H),1.72–1.68(m,2H),1.41–1.36(m,2H),1.27(m,18H),0.88(t,J＝7.0Hz,3H).13C NMR(150MHz,CDCl3)δ175.4,172.7,160.1,153.2,121.6,37.3,31.9,29.6,29.6,29.6,29.4,29.3,29.3,29.1,24.6,22.6,14.1.HRMS(ESI)calculated for C18H29Cl2N3O[M+H]+:374.1766,found:374.1765.
Compound 3ah, white solid, yield 76％,mp:118～120℃.1H NMR(600MHz,CDCl3)δ2.49–2.43(m,2H),2.29(t,J＝7.4Hz,2H),1.50(dd,J＝14.9,7.5Hz,2H),1.21–1.06(m,18H),0.73(t,J＝6.8Hz,3H).13C NMR(150MHz,CDCl3,DMSO)δ181.6,180.2,174.2,155.2,121.1,38.9,36.5,34.3,34.2,34.2,34.1,34.0,33.9,33.8,29.6,27.3,18.9.HRMS(ESI)calculated for C18H28Cl3N3O[M+H]+:408.1376,found:408.1373.
Compound 3ai, white solid, yield 79％,mp:75～76℃.1H NMR(600MHz,DMSO)δ11.06(s,1H),6.90(s,1H),2.43(t,J＝7.4Hz,2H),1.58–1.48(m,2H),1.30–1.18(m,20H),0.84(t,J＝7.0Hz,3H).13C NMR(150MHz,DMSO)δ173.1(d,J＝21.6Hz),171.8,171.4(d,J＝21.6Hz),157.2(d,J＝21.3Hz),87.0(t,J＝37.9Hz),36.9,31.7,29.5,29.4,29.4,29.3,29.1,29.1,28.9,24.8,22.5,14.4.HRMS(ESI)calculated for C18H29F2N3O[M+H]+:342.2357,found:342.2357.
Compound 3aj, white solid, yield 60％,mp:119～121℃.1H NMR(600MHz,DMSO)δ10.06(s,1H),7.12(s,2H),5.67(s,1H),2.41(t,J＝7.2Hz,2H),1.53–1.48(m,2H),1.28–1.21(m,20H),0.85(t,J＝6.9Hz,3H).13C NMR(151MHz,DMSO)δ172.0,170.4(d,J＝236.5Hz),166.9(d,J＝336.7Hz),157.7(d,J＝21.9Hz),81.1(d,J＝34.5Hz),36.8,31.7,29.5,29.4,29.4,29.4,29.3,29.2,29.1,29.0,25.1,22.5,14.4.HRMS(ESI)calculated for C18H31FN4O[M+H]+:339.2560,found:339.2570.
Compound 8a, white solid, yield 20％,mp:112～114℃.1H NMR(600MHz,CDCl3)δ6.05(s,1H),2.90(s,3H),2.79(s,2H),1.70-1.67(m,2H),1.39-1.34(m,2H),1.31-1.24(m,18H),0.87(t,J＝7.0Hz,3H).13C NMR(150MHz,MeOD)δ174.2,163.6,159.2,156.4,96.2,36.7,36.2,33.6,31.7,29.4,29.4,29.3,29.2,29.1,28.9,24.9,22.4,13.1.HRMS(ESI)calculated for C19H33ClN4O[M+H]+:369.2421,found:369.2425.
Compound 8b, white solid, yield 39％,mp:56～60℃.1H NMR(600MHz,MeOD)δ6.37(d,J＝0.7Hz,1H),3.10(s,6H),2.57(s,2H),1.73-1.63(m,2H),1.33-1.27(m,20H),0.90(t,J＝7.0Hz,3H).13C NMR(150MHz,MeOD)δ174.2,163.6,159.2,156.4,96.2,36.7,36.2,33.6,31.7,29.4,29.4,29.3,29.2,29.1,28.9,24.9,22.4,13.1.HRMS(ESI)calculated for C20H35ClN4O[M+H]+:383.2578,found:383.2587.
Compound 8c, white solid, yield 16％,mp:94～96℃.1H NMR(600MHz,CDCl3)δ7.71(s,1H),6.19(s,1H),3.81-3.72(m,4H),3.62(s,4H),2.67(s,2H),1.73-1.64(m,2H),1.36-1.23(m,20H),0.87(t,J＝7.0Hz,3H).13C NMR(150MHz,CDCl3)δ173.0,163.4,160.5,156.4,96.3,66.4,44.6,37.7,32.0,29.8,29.7,29.7,29.6,29.5,29.4,29.3,24.8,22.76,14.2.HRMS(ESI)calculated for C22H37ClN4O2[M+H]+:425.2683,found:425.2690.
Compound 8d, pale yellow solid, yield 32％,mp:73～76℃.1H NMR(600MHz,CDCl3)δ6.18(s,1H),3.62(s,4H),2.96-2.90(m,4H),2.69(s,2H),1.70-1.65(m,2H),1.35(s,2H),1.29-1.21(m,18H),0.87(t,J＝5.3Hz,3H).13C NMR(150MHz,CDCl3)δ163.1,160.2,156.3,96.2,45.7,45.4,37.6,31.9,29.7,29.7,29.6,29.5,29.4,29.4,29.3,24.8,22.7,14.1.HRMS(ESI)calculated for C22H38ClN5O[M+H]+:424.2843,found:424.2841.
Compound 8e, white solid, yield 56％,mp:98～100℃ 1H NMR(600MHz,DMSO/CDCl3)δ10.11(s,1H),6.43(s,1H),3.59–3.49(m,4H),2.75–2.71(m,4H),2.43(t,J＝7.3Hz,2H),1.72(s,1H),1.53–1.50(m,2H),1.28–1.17(m,20H),0.83(s,3H).13C NMR(150MHz,DMSO)δ172.4,163.1,159.5,156.9(d,J＝11.2Hz),96.7(d,J＝114.7Hz),46.8,46.4,45.7,45.5,37.0,31.7,29.5,29.5,29.5,29.4,29.3,29.3,29.2,29.1,25.0,22.5,14.3.HRMS(ESI)calculated for C22H38FN5O[M+H]+:408.3139,found:408.3145.
The experimental reagent sources adopted for preparing the specific compounds are as follows:
Chemical reagents such as 2-aminopyrimidine substrates, acyl chloride substrates and the like are purchased from Shanghai Bi to get medical science and technology Co., ltd; LB medium, MHB medium, LB agar medium, 96-well plate, 24-well plate, 6-well plate, etc. are all purchased from Guiyang ultra-polygala root Chengshengzhi biotechnology Co., ltd; silica gel used for experimental column chromatography is 200-300 meshes, and the GF254 for preparing the thin layer is a product produced by ocean chemical factory of Qingdao in Shandong province; the chemical reaction reagent and the chromatographic solvent used in the experiment are all analytically pure.
The effect of a part of the specific compounds was verified by the following examples:
the sources of the strains used in the examples of the present invention are specifically, the a.baumannii S1 strains, all provided by singapore national university; aerocosa ATCCC 9027,9027; e.coli Dh5a; aureus ATCC 433000; s. epidemic 102555 is purchased from Beijing North Innovative biological technology institute; AB ATCC19606; AB 1778027; AB 1780608; AB 1913061; AB 1925052; MASA20151023070 was isolated from patients in an affiliated hospital at Guizhou university of medical science.
Example 1 determination of Minimum Inhibitory Concentration (MIC)
According to the American Clinical and Laboratory Standards Institute (CLSI), taking a loop bacterial solution from a refrigerator at-80 ℃ into a sterile centrifuge tube containing 5mL of MHB medium, and culturing the loop bacterial solution in a constant-temperature shaking incubator at 37 ℃ for 18-24 hours; and then 200 mu L of the activated bacterial liquid is taken in a sterile centrifuge tube containing 5mL of MHB culture medium, and after the bacterial liquid is cultured for 3 to 4 hours at the constant temperature of 37 ℃, the bacterial liquid concentration is adjusted to be 1 multiplied by 10 6CFU/mL(OD600 =0.1, and the bacterial concentration is 1 multiplied by 10 8 CFU/mL. The compound to be tested is prepared to be 30.72mg/mL for later use. The 96-well polystyrene plate was labeled and 10. Mu.L of the above-prepared compound was added to each well from column 3; 150. Mu.L of MHB was added to each well; 150. Mu.L and 140. Mu.L of MHB were added to columns 1 and 3, respectively; mixing column 3, sucking 150 μl of the mixture, adding column 4, diluting to column 12 with equal magnification, sucking 150 μl of the mixture in column 12, and discarding; 150. Mu.L of the above-prepared bacterial liquid was added to each of columns 2 to 12. Column 1 is blank control group, column 2 is negative control group; the final concentration of the medicines is 1024, 512, 256, 128, 64, 32, 16, 8, 4 and 2 (mug/mL), the final concentration of the bacterial liquid is 1 multiplied by 10 6 CFU/mL, and the total volume is 300 mu L. After the 96-well plate is placed into a constant temperature incubator at 37 ℃ for culturing for 18-24 hours, turbidity of the culture medium in the observation holes is compared, and the clarified one-hole concentration is the lowest inhibitor concentration for the bacteria.
Through detection, the MIC of the compounds 3 a-3 ah and 8 a-8 e on Acinetobacter baumannii is greater than 256 mug/mL, which shows that the compounds have no inhibition effect on bacterial growth, and can avoid bacterial stress and generate drug resistance.
Example 2 determination of anti-biofilm Activity
According to CLSI, taking a loop of bacteria liquid from a refrigerator at the temperature of minus 80 ℃ into a sterile centrifuge tube containing 5mL of LB culture medium, and culturing in a constant-temperature shaking incubator at the temperature of 37 ℃ for 18-24 hours; then 200 mu L of activated bacterial liquid is taken and cultured in a sterile centrifuge tube containing 5mL of LB culture medium at the constant temperature of 37 ℃ for 3-4 hours, and the bacterial concentration is adjusted to be 1 multiplied by 10 6CFU/mL(OD600 =0.1 when the bacterial concentration is 1 multiplied by 10 8 CFU/mL); the compound to be tested is diluted to 15 times to 2 mu mol/mL after being prepared into 0.03mmol/mL for later use. Labeling the 96-well plates, column 1 as solvent control, add 190 μl of LB broth with 10 μl of DMSO solution; column 2 was used as a negative control, 190. Mu.L of the bacterial liquid without inhibitor and 10. Mu.L of the DMSO solution were added; and respectively adding 10 mu L of the prepared compound into the 3 rd to 12 th columns, and then respectively adding 190 mu L of bacterial liquid into the 3 rd to 12 th holes, wherein the concentration of the compound reaches 100 mu M, thus obtaining the target concentration. 8 groups per compound were assayed in parallel; placing the 96-well plate into an electric heating constant temperature incubator for static culture at 37 ℃ for 18-24 hours, and quantitatively measuring the cultured biological film by adopting a crystal violet staining method. Sucking out bacteria liquid in the 96-well plate, and carefully flushing twice with double distilled water to remove planktonic bacteria; adding 95% ethanol solution, fixing the biomembrane for 30min, naturally air-drying, adding 200 mu L of 0.1% crystal violet dye solution into each hole, dyeing for 10min, and washing the redundant dye solution; after air-drying at room temperature or oven-drying at 37deg.C to complete drying, 200 μl of 33% acetic acid solution is added to each well; after the dyed biological film is fully dissolved, an ELX 800 type enzyme-linked immunosorbent assay instrument is used for measuring the OD value in the hole at 590nm wavelength; calculating the inhibition rate of the compound biological film.
A series of screening for anti-Acinetobacter baumannii biofilm activity of compounds 3a to 8e was performed at a concentration of 100. Mu.M using Compound A developed by Blackwell group as a positive control, and the results are shown in FIG. 2 and Table 1.
Table 1 results of biofilm inhibitory Activity of Compounds 3a to 8
Experimental results show that the activity of the tridecyl-containing compound 3u is optimal; therefore, on the basis of the compound 3u, further structure-activity relationship research is carried out on the substituent group on the 2-aminopyrimidine aromatic ring. Compounds 3 v-3 aj were synthesized and subjected to preliminary anti-biofilm activity tests. The experimental results show that the compounds 3ac and 3aj have more obvious anti-biofilm activity when the 4-position of the pyrimidine ring is substituted by amino and the 6-position is substituted by halogen. Therefore, the amino groups of the above two compounds were further structurally modified to synthesize compounds 8a to 8e. The results of the activity experiments show that when the piperazine ring is introduced into the 6-position of the pyrimidine ring, the anti-biofilm activity of the compounds 8d and 8e is better than that of the compound 3 ac. At a concentration of 100 μm, compound 8d has an anti-biofilm activity of up to 70.17% on a.baumannii S1.
Example 3 scanning electron microscope observation of Acinetobacter baumannii biofilm
The modified plate culture method is adopted to test whether the Acinetobacter baumannii can form BF on the silica gel membrane. The silica gel film was cut into small squares of (1.0X1.0) cm 2. LB broth was prepared at a concentration of 1.0X10 6 CFU/mL of bacterial suspension according to CLSI, and the better active compound screened in the activity test above was prepared at 2mmol/L. Adding silica gel sheet sterilized by alcohol into 24-well plate, adding 50 μl of the compound, adding 950 μl of diluted bacterial liquid of 1× 6 CFU/mL, culturing in biochemical incubator at 37deg.C for 24 hr without adding compound, washing with flowing physiological saline, and removing plankton. Fixing silica gel sheet with 2.5% glutaraldehyde at low temperature for 12 hr, eluting with 30%, 50%, 70%, 90% and 100% ethanol gradient for 15min, drying critical point, spraying gold, loading sample, scanning, observing with scanning electron microscope, and photographing.
In the activity screening process of each compound, the compounds 3ab, 3ac, 3ad and 3x with relatively good activity are cultured for 18-24 hours at the concentration of 100 mu M, wherein the concentration of bacterial liquid is 1X 10 6 CFU/mL. The bacterial liquid was aspirated, dried, frozen, and subjected to SEM preliminary observation after gold plating.
As a result, as shown in FIG. 2, it was intuitively observed that the bacteria in the compound-containing field of view were significantly reduced compared to those in the compound-free field of view. Thus, it is further illustrated that compounds 3ab, 3ac, 3ad, 3x have some anti-biofilm activity against a.baumannii S1, in hope of reducing acinetobacter baumannii resistance in combination with antibiotics.
Example 4 bacterial exopolysaccharide assay
Since the extracellular polysaccharide is the main component of the biological film, the inhibition of the compound to the extracellular polysaccharide can further detect the inhibition effect of the compound to the biological film. The extracellular polysaccharide content of compounds 3ac, 3aj, 8d and 8e was determined using the phenol-sulfuric acid method at a concentration of 100. Mu.M.
LB broth was prepared at a concentration of 1.0X10 6 CFU/mL of bacterial suspension according to CLSI, and the better active compound screened in the activity test above was prepared at 2mmol/L. 50. Mu.L of the prepared compound was added to the 24-well plate, 950. Mu.L of diluted 1X 10 6 CFU/mL of the bacterial liquid was added, the bacterial liquid cultured without the compound was left as a control group, and after 24 hours of culture in a biochemical incubator at 37 ℃, the upper bacterial liquid was removed by suction. Rinsing the 24-well plate with 0.9% NaCl, adding 0.25mL of 5% phenol and 1.25mL of concentrated sulfuric acid into the 24-well plate, and culturing in the dark at 30 ℃ for 1h; using an ELX 800 type enzyme-linked immunosorbent assay (ELISA) to determine the OD value in the hole at the wavelength of 490 nm; the effect of the compound on the extracellular polysaccharide content of the biofilm was calculated.
As a result, as shown in FIG. 3, at a concentration of 100. Mu.M, the compounds 3ac, 3aj, 8d and 8e had a remarkable inhibitory effect on extracellular polysaccharide in the A.baumannii S1 biofilm, with inhibitory activities of 49.67%, 59.40%, 73.00% and 60.25%, respectively, and a similar trend to that of the biofilm inhibitory activity results; it was further demonstrated that compounds 3ac, 3aj, 8d and 8e have a stronger inhibitory effect on a.baumannii.s1 biofilm.
Example 5 chessboard method Combined drug administration test
MHB broth was prepared at a concentration of 1.0X10 6 CFU/mL of bacterial suspension according to CLSI, and the better active compounds 3ac, 3aj, 8d and 8e screened in the activity test above were each prepared at 30.72mg/mL and were combined with various antibiotics to observe their inhibition of A.baumannii S1.
The specific test method comprises the steps of marking and numbering a 96-well plate, adding two times of MIC concentration of antibiotics into a horizontal well, sequentially diluting the two times of antibiotics backwards, adding a prepared compound with the concentration of 30.72mg/mL into a vertical well, sequentially diluting the two times backwards, and establishing a checkerboard; well 1 served as a blank and 200 μl of MHB broth was added; well 2 was used as a solvent control, and 190. Mu.L of bacterial solution and 10. Mu.L of DMSO solution were added; after the plates are paved, the plates are put into the culture medium for 18 to 24 hours at the constant temperature of 37 ℃; turbidity in the comparison wells was observed to give MIC of the compound and Fractional Inhibitory Concentration Index (FICI) was calculated.
The synergy of the compounds with various antibiotics was assessed by checkerboard titration assay on microwell plates as suggested by NCCLS and expressed as the sum of the Fractional Inhibitory Concentration (FIC) indices for each drug. FICI = MIC (comb a)/MIC (alone a) +mic (comb B)/MIC (alone B), i.e., MIC at the time of combination a/MIC at the time of combination B. A, B has synergistic effect when FICI is less than or equal to 0.5; a, B has additive effect when 1 is greater than or equal to FICI > 0.5; when the FICI is more than or equal to 2 and is more than 1, A, B have no interaction; a, B has antagonism when FICI > 2.
The test results are shown in table 2:
TABLE 2 study of inhibition of Acinetobacter baumannii with combination drugs
The FICI values of the compounds 3ac, 3aj, 8d and 8e which are respectively combined with gentamicin sulfate, oxacillin, chloramphenicol, erythromycin and meropenem are all smaller than or equal to 0.5, thus showing that the compounds 3ac, 3aj, 8d and 8e have certain synergistic effect with aminoglycosides, beta-lactams, amide alcohols and macrolide antibiotics. Wherein, when being combined with gentamicin sulfate in aminoglycosides, MIC is reduced by 2-8 times; when the penicillin is respectively combined with chloramphenicol in oxacillin and amidoalcohol in penicillin, the MIC is reduced by 2-4 times; when combined with erythromycin and carbapenems in macrolides, respectively, MIC is reduced by 2-fold.
Example 6IC 50 test
The effect of compounds 3ac, 3aj, 8d and 8e on various acinetobacter baumannii anti-biofilm was determined by crystal violet staining. In addition, simple screening of anti-biofilm activity against methicillin-resistant Staphylococcus aureus, escherichia coli, pseudomonas aeruginosa, staphylococcus aureus was performed.
The specific test process is that LB broth is prepared into bacterial suspension with the concentration of 1.0X10 6 CFU/mL according to CLSI, and the compound with better activity screened in the activity test is prepared into 2mmol/L, 0.4mmol/L, 0.08mmol/L, 0.016mmol/L and 0.0032mmol/L in sequence for later use. The 96-well plates were labeled and placed around with LB medium to prevent evaporation. Adding LB culture medium as blank control group in column 2, adding 10 μl DMSO and 190 μl LB bacteria-containing culture medium as solvent control group in column 3; sequentially adding 10 mu L of the prepared compound according to a concentration gradient in a row 4 to a row 8; 190 mu L of LB containing medium is added into each hole; LB medium was added to the remaining wells. The final concentration of the medicine is 100, 20, 4, 0.8 and 0.16 mu M, the final concentration of the bacterial liquid is 1X 10 6 CFU/mL, and the total volume is 200 mu L. Placing the 96-well plate into a constant temperature incubator at 37 ℃ for culturing for 18-24 hours, adopting a crystal violet staining method to stain a biological film, and measuring the OD value of a solution in the culture hole at 590nm by using an ELX800 type enzyme-linked immunosorbent assay instrument; and calculating the inhibition rate of the compound biological film at different concentrations. IC 50 of the compound was calculated using GRAPHPAD PRISM software.
The test results are shown in table 3:
TABLE 3 IC 50 results of Compounds 3ac, 3aj, 8d and 8e on various bacterial biofilms
According to Table 3, compounds 3ac, 3aj, 8d and 8e all had significant inhibitory effects on the biofilm of 5 Acinetobacter baumannii other than ACBA.S1. In particular, compound 3aj has a strong anti-biofilm effect on standard strain AB ATCC19606, with IC 50 as low as 3.82 μm. It is exciting that compounds 8d and 8e also show better anti-biofilm activity against some gram-negative and gram-positive bacteria. Wherein compound 8d has an IC 50 as low as 17.04 μm for MRSA20151023070 anti-biofilm activity; compound 8e has IC 50 as low as 1.95 μm for e.coli Dh5a anti-biofilm activity; IC 50 was as low as 1.28 μm for P.a ATCC 9027 anti-biofilm activity; IC 50 was as low as 4.33 μm for s.a ATCC 433000 anti-biofilm activity; IC 50 for S.e ATCC 102555 anti-biofilm activity was as low as 5.82 μm. Therefore, the compounds 8d and 8e have strong anti-biofilm activity, so that the structure has better antibacterial potential.
EXAMPLE 7 laser confocal microscopy (LSCM) observation of Acinetobacter baumannii biofilm
LB broth was prepared to a concentration of 1.0X10 6 CFU/mL of bacterial suspension according to CLSI, and compounds 3ac, 3aj, 8d and 8e were prepared as 2mmol/L solutions, respectively; 100 mu L of the prepared compound is put into a laser confocal small dish, 1900 mu L of LB bacteria-containing culture medium is added, and the final concentration of the drug is 100 mu M; 100. Mu.L of DMSO solution was placed in a laser confocal dish and 1900. Mu.L of LB medium was added as a control. Culturing at 37 ℃ for 18-24 hours, sucking the culture solution, washing for 2 times by using PBS solution with pH of 7.4, and sucking the floating bacteria; fixing with 4% formaldehyde PBS for 20min; washing with PBS solution for 2 times, each time for 10min, washing off the fixing solution, adding the prepared Alexa Fluor 488-labeled phalloidin solution, and dyeing for 90min in dark; washing with PBS solution for 2 times and 5min each time; after being dried, the biological film is observed by a laser confocal microscope, photographed and recorded.
The results of laser confocal microscopy biomembrane thickness at a maximum emission wavelength of 517nm and a maximum excitation wavelength of 493nm are shown in fig. 4: a is a control with DMSO alone as a solvent, and the thickness of the ACBA.S1 biofilm under CLSM is 25.91 μm; b is a biological film of ACBA.S1 under the action of the compound 3ac, and the thickness is 16.04 mu m; c is the biological film of ACBA.S1 under the action of the compound 8d, and the thickness is 14.68 mu m. By adopting the CLSM technology, the change of the biological film thickness is more intuitively observed, so that the inhibition activity of the compound on the biological film is further demonstrated.
Example 8Swarming motion influencing inspection
This example is to inhibit biofilm formation by inhibiting quorum sensing (quorum sensing, QS). Whereas QS has a certain motility, it is thus further verified by the motility of the bacterial population whether the compound has an inhibitory effect on bacterial QS.
According to CLSI, taking a loop of bacteria liquid from a refrigerator at the temperature of minus 80 ℃ into a sterile centrifuge tube containing 5mL of MHB culture medium, and culturing in a constant-temperature shaking incubator at the temperature of 37 ℃ for 18-24 hours; then 200 mu L of activated bacterial liquid is taken and put into a sterile centrifuge tube containing 5mL of MHB culture medium, and is cultivated for 3-4 hours at the constant temperature of 37 ℃ for standby; the compounds with better activity screened in the activity test are sequentially configured into 2mmol/L for later use; well A, 1 st well, was used as a solvent control for 6 well plate labeling numbering, and after addition of 100. Mu.L DMSO, 1900. Mu.L of semi-solid medium (0.8% NB broth, 0.5% D-glucose and 0.3% agar) was added rapidly and shaken well; wells 4D add 2000 μl of semi-solid medium as a blank; compounds 3ac, 3aj, 8d and 8e were added to wells B, C, E, F, 2, 3, 5 and 6, respectively, and 100. Mu.L of each compound was added to the semi-solid medium at a concentration of 2mmol/L and shaken well to give a final concentration of 100. Mu.M per well. After the culture medium is solidified, 5 mu L of the activated bacterial liquid is added into each hole, and after the culture medium is cultured for 24 hours at 30 ℃, the record is photographed.
In 6-well plates, the results are shown in FIG. 5, where, first, D has a greater flora movement diameter than A, indicating that solvent DMSO inhibits QS of bacteria. Thus, DMSO was ensured to be below 5% during each experiment. Second, it was observed that B, C, E, F had a significantly smaller flora movement diameter than A, indicating that compounds 3ac, 3aj, 8d and 8e had an inhibitory effect on the QS of the bacteria.
EXAMPLE 9 drug sustained Release immobilization
Polylactic acid-glycolic acid copolymer (PLGA) is a degradable biological macromolecule with good biocompatibility, non-toxicity and good film forming performance. PLG is racemic PLGA and also has a certain film-forming property. Therefore, in this example, both were used as a supporting material for a compound to examine the inhibitory activity of the compound on a biofilm.
182Mg of PLG was dissolved in 7.6mL of dichloromethane for use; then, 2.5mL of the above-mentioned stock PLG solution was taken into two sterilization tubes, and the compounds 3ac and 8d were added, respectively, to prepare a PLG solution of 1 mmol/L. Cutting biological silica gel sheet into small blocks with a length of (1.0X1.0) cm 2, sterilizing with alcohol, and placing into 24-well plate; 100. Mu.L of the above preparation solution was taken respectively to a washed and sterilized silica gel sheet, and the silica gel sheet containing 2.4mg PLG and 0.1. Mu. Mol of the compound per well was prepared by placing the silica gel sheet in a fume hood to volatilize the solvent, and then placing the silica gel sheet in a reduced pressure drying oven to dry for 12 hours. As described above, silica gel sheets containing 1.2mgPLGA and 0.1. Mu. Mol of compound per well were produced. Because of the poor solubility of PLGA, this example uses halving load. Preparing LBS culture solution (2% LB culture medium, 1.5% NaCl, 0.3% glycerol and 50mM Tris-HCl) into bacterial suspension with concentration of 1.0X10 6 CFU/mL according to CLSI, adding 1mL into the prepared 24-well plate, and culturing for 1d, 7d and 14d respectively at constant temperature of 37 ℃; changing LBS culture solution every 24 hours; after the incubation, the cells were washed with PBS 2 times to remove plankton. After the silica gel sheet is fixed for 12 hours at a low temperature by using 2.5% glutaraldehyde, 30%, 50%, 70%, 90% and 100% ethanol gradients are respectively adopted for eluting for 15 minutes, the critical point is dried and then the gold spraying treatment is carried out, and the slow release result adopts SEM to detect the change of the biological film, and the results are shown in figures 6-8.
The results show that when the compounds 3ac and 8d are slowly released for 1d and 7d, the anti-biofilm activity is more obvious; 14d can also be seen with anti-biofilm activity, but the results are poor relative to 7 d. The experimental results again demonstrate the anti-biofilm activity of compounds 3ac and 8d, and can be loaded on PLGA and PLG materials to realize long-acting inhibition of the biofilm.
Thus, compounds 3ac and 8d may be loaded on a variety of medical devices to reduce multiple drug resistant infections in clinical medicine.
Example 10MTT assay
Normal cells are metabolized vigorously, succinic dehydrogenase in mitochondria can reduce tetrazolium salt substances (such as MTT) into purple crystalline substances, the purple crystalline substances are deposited around the cells, and then OD values are read through an enzyme-labeling instrument, so that the cell proliferation state is detected. Cytotoxicity assays are widely used in basic research and drug discovery to screen libraries of toxic compounds. Compounds that can produce a cytotoxic response can be eliminated in subsequent screens; or selecting a compound that targets rapidly dividing cells as a candidate compound for cancer treatment. Thus, this example uses mouse embryonic fibroblasts 3T3-L1 for cytotoxicity testing compounds 3ac and 8 d.
In a specific test procedure, mouse embryonic fibroblasts (3T 3-L1) were seeded at 10000 cells per well in 100. Mu.L of medium. The 96-well plates were then placed in a humid incubator, 5% carbon dioxide, 95% air, for 24 hours. Thereafter, the medium was removed from the 96-well plate, replaced with the compound-containing medium, and cultured for 24 hours. After incubation, cytotoxicity was determined using the MTT method. To each well was added 5mg/mL MTT 20. Mu.L of a solution in warm Phosphate Buffered Saline (PBS) and incubated at 37℃for 4h in dark air with 5% carbon dioxide and 95% relative humidity. After incubation, MTT was aspirated and 0.1mL of 0.04mol/L isopropyl alcohol HCl solution was added. Stir the plate until complete dissolution. Absorbance was read at 570nm using a Multiskan-EX spectrophotometer. Three 6 duplicate wells per compound were tested. The absorbance and standard deviation of wells containing the same compound were calculated in this example.
The test results are shown in table 4:
TABLE 4 cytotoxicity results of Compounds 3ac and 8d on mouse 3T3-L1
Cytotoxicity was graded according to cell viability compared to control; non-cytotoxicity >90% cell viability, slight cytotoxicity = 60-90% cell viability, moderate cytotoxicity = 30-59% cell viability, severe cytotoxicity <30% cell viability.
The results indicated that compound 3ac was non-cytotoxic. While compound 8d was slightly cytotoxic at 50 μm, its cytotoxic IC 50 was 73.63 μm, which was far higher than its anti-biofilm activity IC 50. Therefore, the two compounds can provide an important guarantee for the development and application of novel medical instruments and biological materials.
Example 11 stability test in Artificial gastric juice
1- (2-Pyrimidinyl) piperazine is used as an internal standard substance to prepare an internal standard stock solution with the mass concentration of 0.1mg/mL, and the stock solution is stored at the temperature of 4 ℃. Taking 16.4mL of dilute hydrochloric acid, adding 800mL of water, adjusting the pH to 1.3 by using 0.1mol/L hydrochloric acid solution, and then adding water for dilution and constant volume to 1000mL to obtain the blank artificial gastric juice.
Taking 16.4mL of dilute hydrochloric acid, adding 800mL of water and 10g of pepsin, shaking uniformly to dissolve the dilute hydrochloric acid, regulating the pH to 1.3 by using 0.1mol/L hydrochloric acid solution, adding water to dilute the dilute hydrochloric acid, and fixing the volume to 1000mL to obtain the artificial gastric juice.
Precisely measuring 1mL of methanol solution of a target compound with mass concentration of 1mg/mL in a 10mL volumetric flask, and respectively diluting with blank artificial gastric juice and artificial gastric juice to scale (the alcohol content is 1 percent); and sub-packaging 600 mu L by using an EP tube, shaking and mixing uniformly, and incubating for 0, 6, 12, 24 and 36 hours in a water bath at 37 ℃. 200 mu L of the incubated sample is taken, 2 mu L of an internal standard solution with the mass concentration of 100 mu g/mL is added, the mixture is uniformly mixed by vortex for 5min, the mixture is centrifuged for 10min at 20000 Xg at 4 ℃, and the supernatant is taken for measurement.
The chromatographic conditions for measuring the stability content of the compound in artificial gastric juice are as follows: chromatographic column: wondaSilC18 (250 mm. Times.4.6 mm,5 μm); mobile phase: methanol (B) -0.1% triethylamine aqueous solution (A), isocratic elution (0-15 min,40% B); flow rate: 1mL/min; detection wavelength: 254nm; column temperature: 30 ℃; sample injection amount: 20. Mu.L.
After sample injection analysis, the peak area is recorded, and the mass concentration of the traditional Chinese medicine in each sample is calculated according to an internal standard method. Each sample was assayed in 3 replicates.
The test results are shown in table 5:
TABLE 5 residual percentage of Compound 8d in artificial gastric juice over 0-72 h
Calculating the residual percentages of the medicines at each incubation time point according to the theoretical mass concentration (100 mug/mL) of the compound 8d before incubation, wherein the residual percentages of the compound 8d in the blank artificial gastric juice for different incubation time are all in the range of (106.22 +/-6.38)% (90.63+/-2.43)%; the residual percentage is (87.14 +/-1.12)% when the artificial gastric juice is incubated for 36 hours, and the residual percentage is not obviously changed compared with the blank artificial gastric juice.
The stability of the compound 8d in artificial gastric juice incubated for 36h is examined in the embodiment, and important references are provided for structural transformation of the subsequent similar derivatives, formulation development of the compound and combined medication scheme.
The above embodiments are not intended to limit the present invention, and any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (7)
1. The 2-aminopyrimidine compound is characterized in that the specific structural formula of the compound is one of the following structural formulas:
2. a process for the preparation of 2-aminopyrimidine compounds as claimed in claim 1 wherein the reaction equation for the preparation is:
Wherein X, R 1 and R 3 correspond to substituents at corresponding positions in the specific structural formula set forth in claim 1;
The specific reaction process is as follows:
under the protection of nitrogen at room temperature, adding an acyl chloride substrate into a dry toluene solution of a 2-aminopyrimidine raw material and N, N-diisopropylethylamine, and reacting at 25 ℃ or 110 ℃; after the 2-aminopyrimidine raw material is completely reacted, spin-drying the solvent, extracting by ethyl acetate and hydrochloric acid solution, extracting the organic layer by saturated sodium bicarbonate solution, and passing through column chromatography by anhydrous sodium sulfate to obtain a compound 1;
wherein, the molar concentration ratio of acyl chloride substrate, 2-aminopyrimidine raw material and N, N-diisopropylethylamine is 1.5:1:2;
Under the protection of nitrogen, adding the compound 1, an amine raw material and triethylamine into methanol in sequence, and reacting for 1-2 h at 25 ℃; after complete reaction, spin-drying the solvent, extracting by ethyl acetate and hydrochloric acid solution, extracting the organic layer by saturated sodium bicarbonate solution, and passing through column chromatography by anhydrous sodium sulfate to obtain the target compound;
wherein, the molar concentration ratio of the compound 1 to the amine raw material to the triethylamine is 1:1.1:1.5;
The structure of the amine raw material is R 3 -H.
3. The use of a 2-aminopyrimidine compound as claimed in claim 1 for the preparation of a biofilm inhibitor.
4. A biofilm inhibitor, characterized in that the effective component of the biofilm inhibitor is the 2-aminopyrimidine compound of claim 1.
5. The biofilm inhibitor of claim 4, wherein the biofilm inhibitor inhibits biofilm formation, wherein the biofilm is formed by one of acinetobacter baumannii, methicillin-resistant Lin Putao coccus, staphylococcus epidermidis, and escherichia coli.
6. An anti-infective drug, comprising the 2-aminopyrimidine compound of claim 1 and an adjuvant.
7. A biomaterial characterized in that the surface of the biomaterial is coated with the biofilm inhibitor as claimed in claim 4 or 5.
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