CN114605334A - 2-aminopyrimidine compound, preparation method, application and biomembrane inhibitor - Google Patents

Abstract

The invention relates to the technical field of biology, and particularly relates to a 2-aminopyrimidine compound, a preparation method, application and a biofilm inhibitor. The compound has good anti-biofilm activity on acinetobacter baumannii, and also has good anti-biofilm activity on methicillin-resistant staphylococcus, staphylococcus epidermidis, escherichia coli and the like. The compound is based on natural marine alkaloids with anti-biofilm activity, and 2-aminoimidazole is mostly used as a parent nucleus, so that the problem that AHL analogues are easily hydrolyzed and inactivated is solved. Meanwhile, the compound provides more chemical entities for a novel treatment means of the anti-infective drug, and the compound has the advantages of easily obtained raw materials, simple steps, low reaction cost and high efficiency, and can be prepared and produced in large quantities. The compound is used as a biological membrane inhibitor, has stronger activity and has good biological membrane inhibition effect. The compound can also be used for preparing antibacterial drugs or biological materials.

Description

A kind of 2-aminopyrimidine compound, preparation method, application and biofilm inhibitor

technical field

The invention relates to the field of biotechnology, in particular to a 2-aminopyrimidine compound, a preparation method, an application and a biofilm inhibitor.

Background technique

With the continuous increase of bacterial resistance, especially the emergence of multidrug-resistant superbugs, it has brought huge potential dangers to the health of human society. A common defense mechanism observed in multidrug-resistant and other bacteria is the formation of biofilms. In recent years, clinical practice and research results at home and abroad have proved that bacterial drug resistance, chronic infectious diseases are difficult to cure and repeated attacks are closely related to bacterial biofilms. Due to the barrier function of the biofilm and the low metabolism of bacteria in the biofilm, once the bacterial biofilm is formed, its drug resistance can be increased by hundreds to thousands of times, and the bacteria can evade the immune system attack, making the infection chronic and difficult to control. . Bacterial biofilm infection is an important reason for the persistence of clinical infection and the difficulty in removing pathogenic bacteria completely.

Acinetobacter baumannii (A. baumannii, Ab) is a non-fermenting Gram-negative bacterium, its significant multidrug resistance and high incidence make it one of the most troublesome nosocomial pathogens. The World Health Organization has also listed Acinetobacter baumannii as one of the most resistant bacteria, posing a particularly great threat to human health.

For the biofilm formation of Acinetobacter baumannii, studies have shown that quorum sensing (QS) system can be used to interfere. This is because, genes involved in biofilm formation are also regulated by the QS system. The study found that interfering with the QS system of Ab will not affect its individual growth, but it can block the group communication between Abs, making it unable to regulate the formation of biofilms as a group, thereby avoiding the shielding of biofilms against antibiotic production. Action Because this process does not kill bacteria directly, it reduces the possibility of bacteria developing resistance to it.

The QS system of Acinetobacter baumannii includes self-induced synthase (abaI), acylhomoserine lactone (AHL) signaling molecule and abaR receptor, which belongs to the typical LasR-LasI population behavior control system. Bacteria synthesize one or more autoinducers (AI) through abaI synthase and release them into the external environment. The abaR receptor senses the concentration of AI to determine the bacterial population density and changes in the surrounding environment. When the autoinducer concentration reaches a certain level After the threshold of , the flora initiates a series of corresponding gene expression to regulate the formation of biofilm.

QS inhibitors in the prior art include natural or synthetic ones, and most of these inhibitors are AHL analogs, receptor inhibitors or synthase inhibitors. However, these inhibitors often suffer from a number of problems that limit their usefulness in practical applications.

For example, the research group of Philip N. Rathe found that the autoinducer AHL and its derivatives of the Ab M2 strain had a certain inhibitory effect on the biofilm of Ab, but the inactivation was caused by the easy hydrolysis of the lactone ring of AHL and its derivatives.

N-pyrrole substituted p-orotidine derivatives discovered by Christian Melander's group, natural marine alkaloids and their derivatives discovered by HelenE.Blacwell's group, and polysubstituted 2-aminoimidazoles discovered by Rupinder K.Gill's group The marine alkaloids of the backbone have all reported biofilm inhibitory activity against Acinetobacter baumannii, and even the anti-biofilm activity IC ₅₀ is as low as 26.8 μM, but the extraction and synthesis of natural macromolecules are difficult.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a 2-aminopyrimidine compound, a preparation method, an application and a biofilm inhibitor.

The technical scheme that the present invention solves the above-mentioned technical problems is as follows:

The present invention provides a 2-aminopyrimidine compound. The general structural formula of the compound is shown in general formula (1):

Wherein, R ₁ represents substituted or unsubstituted C ₂ -C ₁₄ alkyl, C ₂ -C ₁₄ cycloalkyl, C ₂ -C ₁₄ carboxyl, and substituted or unsubstituted phenyl, naphthyl, penta One of the membered heterocyclic aryl groups;

R ₂ represents one of substituted or unsubstituted C ₂ -C ₁₄ alkyl, alkoxy, X, and R ₃ ; X is a halogen atom; R ₃ is an N-containing group;

The value of n is 0, 1, 2 or 3.

Further, the value of n is 1, 2 or 3.

Further, R ₁ represents unsubstituted C ₂ -C ₁₄ alkyl, F-substituted alkyl, cyclobutanyl, pentylcarboxy, substituted or unsubstituted phenyl, alkyl-substituted phenyl, alkoxy One of substituted phenyl, alkenyl substituted phenyl, nitro substituted phenyl, naphthyl, thienyl; R ₂ represents methyl, methoxy, Cl, Br, F, amino, methylamino , one of dimethylamino, piperazinyl and morpholino.

Further, the specific structural formula of the compound is:

中的一种。

one of the.

The present invention provides a preparation method of the above-mentioned 2-aminopyrimidine compounds. When the value of n is 2, and one R ₂ is represented as X, and the other R ₂ is represented as R ₃ , the reaction equation that occurs in the preparation process is:

The specific reaction process is:

Under nitrogen protection at room temperature, the acid chloride substrate was added to a dry toluene solution containing 2-aminopyrimidine raw material and N,N-diisopropylethylamine, and the reaction was carried out at 25 °C or 110 °C; wait for the 2-aminopyrimidine raw material to react completely Then, spin dry the solvent, extract with ethyl acetate and hydrochloric acid solution, extract the organic layer with saturated sodium bicarbonate solution, pass through column chromatography with anhydrous sodium sulfate, and obtain compound 1;

Wherein, the molar concentration ratio of acid chloride substrate, 2-aminopyrimidine raw material and N,N-diisopropylethylamine is 1.5:1:2;

Under nitrogen protection, compound 1, amine raw material, and triethylamine were sequentially added to methanol, and reacted at 25 °C for 1-2 h; after complete reaction, the solvent was spin-dried, extracted with ethyl acetate and hydrochloric acid solution, and then saturated with carbonic acid. After the organic layer is extracted with sodium hydrogen solution, the target compound is obtained by column chromatography through anhydrous sodium sulfate;

Wherein, the molar concentration ratio of compound 1, amine raw material and triethylamine is 1:1.1:1.5;

The structure of the amine starting material is _R3 -H.

The present invention provides the use of the above-mentioned 2-aminopyrimidine compounds as biofilm inhibitors.

The present invention provides a biofilm inhibitor, the active ingredient of which is the above-mentioned 2-aminopyrimidine compound.

Further, the biofilm inhibitor can inhibit the formation of biofilm, and the biofilm is formed by one of Acinetobacter baumannii, methicillin-resistant Staphylococcus, Staphylococcus epidermidis, and Escherichia coli.

The present invention provides an anti-infective drug, which is characterized by comprising the above-mentioned 2-aminopyrimidine compound and auxiliary materials.

The present invention provides a biological material whose surface is coated with the above-mentioned biofilm inhibitor.

The beneficial effects of the present invention are:

1) The 2-aminopyrimidine compounds of the present invention, based on the natural marine alkaloids with anti-biofilm activity, mostly use 2-aminoimidazole as the parent nucleus, which avoids the problem that AHL analogs are easily hydrolyzed and inactivated.

2) The 2-aminopyrimidine compounds of the present invention have good anti-biofilm activity against Acinetobacter baumannii, and also have good anti-biofilm activity against methicillin-resistant Staphylococcus, Staphylococcus epidermidis, Escherichia coli, etc. Biofilm activity.

3) The 2-aminopyrimidine compounds of the present invention provide more chemical entities for novel therapeutic methods of anti-infective drugs.

3) The preparation method of the 2-aminopyrimidine compounds of the present invention has the advantages of easy availability of raw materials, simple steps, low reaction cost and high efficiency, so that the 2-aminopyrimidine compounds can be prepared and produced in large quantities.

4) The biofilm inhibitor of the present invention adopts the 2-aminopyrimidine compound of the present invention as an active ingredient, which has stronger activity and can effectively inhibit the formation of Acinetobacter baumannii biofilm, so that it has a good biofilm inhibitory effect .

5) The antibacterial drug of the present invention contains the 2-aminopyrimidine compound of the present invention, which can effectively inhibit bacterial infection.

6) The surface of the biological material of the present invention is coated with the biofilm inhibitor of the present invention, and the biological material can be used to make medical instruments, so that the medical instruments have good antibacterial properties.

Description of drawings

Fig. 1 is the 2-aminopyrimidine compounds of the present invention, in Example 2, the results of biofilm inhibitory activity of compounds 3a-8;

Figure 2 is the 2-aminopyrimidine compound of the present invention, in Example 3, the SEM result of compounding on the silica gel sheet, wherein, Figure a is the positive control, b is the compound 3ab, c is the compound 3ac, d is the compound 3ad, e is compound 3x;

Fig. 3 is the 2-aminopyrimidine compound of the present invention, in Example 4, the bar chart of the measurement result of exopolysaccharide content;

Figure 4 is a 2-aminopyrimidine compound of the present invention, in Example 7, a cross-sectional view of the biofilm of the compound under a laser confocal microscope, wherein A is the solvent control of DMSO; B is the ACBA.S1 under the action of compound 3ac Biofilm; C is the biofilm of ACBA.S1 under the action of compound 8d;

Figure 5 is the 2-aminopyrimidine compounds of the present invention, in Example 8, the results of the compound Swaring exercise; wherein, A is the DMSO positive control; B is the effect of the compound 3ac; C is the effect of the compound 3aj; D is the blank without DMSO Control; E is the effect of compound 8d; F is the effect of compound 8e;

6 is the 2-aminopyrimidine compound of the present invention, in Example 9, the biofilm SEM change diagram of sustained release 1d, wherein a is PLG-DMSO control, b is PLG-3ac, c is PLG-8d, d is PLGA-DMSO control, e is PLGA-3ac, f is PLGA-8d;

Figure 7 2-aminopyrimidine compounds of the present invention, in Example 9, the biofilm SEM change diagram of sustained release 7d, wherein a is PLG-DMSO control, b is PLG-3ac, c is PLG-8d, and d is PLGA-DMSO control, e is PLGA-3ac, f is PLGA-8d;

Figure 8 2-aminopyrimidine compounds of the present invention, in Example 9, the biofilm SEM change diagram of sustained release 14d, wherein a is PLG-DMSO control, b is PLG-3ac, c is PLG-8d, and d is PLGA-DMSO control, e is PLGA-3ac, f is PLGA-8d.

Detailed ways

The principles and features of the present invention will be described below with reference to the accompanying drawings. The examples are only used to explain the present invention, but not to limit the scope of the present invention.

Pyrimidine structure is also an important part of many endogenous substances, which is the advantage of pyrimidine derivatives interacting with biological macromolecules such as genetic material and enzymes in cells. Therefore, the present invention uses 2-aminopyrimidine as the core to design and synthesize a series of 2-aminopyrimidine derivatives, and obtain a biofilm inhibitor with stronger activity, which provides more chemical entities for new treatment methods of anti-infective drugs.

The general structural formula of the 2-aminopyrimidine compounds of the present invention is shown in the general formula (1):

Wherein, R ₁ represents a substituted or unsubstituted C ₂ -C ₁₄ alkyl group, a C ₂ -C ₁₄ cycloalkyl group, a C ₂ -C ₁₄ carboxyl group, a substituted or unsubstituted phenyl group, a naphthyl group, a penta One of the membered heterocyclic aryl groups; R ₂ represents one of alkyl, alkoxy, X, and R ₃ ; X is a halogen atom; R ₃ is a N-containing group; the value of n is 0, 1, 2 or 3.

Preferably, the value of n is 1, 2 or 3.

R ₁ represents unsubstituted C ₂ -C ₁₄ alkyl, F substituted alkyl, cyclobutanyl, pentylcarboxy, substituted or unsubstituted phenyl, alkyl substituted phenyl, alkoxy substituted One of phenyl, alkenyl-substituted phenyl, nitro-substituted phenyl, naphthyl, and thienyl.

R ₂ represents one of methyl, methoxy, Cl, Br, F, amino, methylamino, dimethylamino, piperazinyl, and morpholino.

The above-mentioned 2-aminopyrimidine compound structure of the present invention is obtained by modifying the structure of the autoinducer OH-dDHL of Acinetobacter baumannii, using 2-aminopyrimidine, different substituted acyl chlorides, etc. as raw materials, to acyl homoserine It is designed by replacing the lactone ring, or changing the length of the acyl carbon chain, changing the carbon chain structure, or changing the side chain substituent.

The present invention provides 41 kinds of specific compound structures, the specific structures are as follows: the specific structural formula of the compound is:

The preparation methods of compounds 3a～3u are existing, and the specific reaction process is:

Under nitrogen protection at room temperature, the 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). The reaction was carried out at room temperature (25°C) or 110°C, and the progress of the reaction was monitored by TLC. After the 2-aminopyrimidine was completely reacted, the solvent was spin-dried by a rotary evaporator, extracted with ethyl acetate and 1M hydrochloric acid solution, and then the organic layer was extracted with saturated sodium bicarbonate solution, passed through anhydrous sodium sulfate, and passed through the column layer. The corresponding target compounds 3a-3u were obtained by analysis.

For compounds 3v to 3ah, the reaction conditions can be adjusted on the basis of the above-mentioned preparation methods of compounds 3a to 3u, and the target compounds 3v to 3ah can be obtained by reacting at 1110°C.

The preparation methods of compounds 8a-8e are as follows: on the basis of the above-mentioned preparation methods of compounds 3a-3u, further react with amine raw materials, and the specific reaction process is as follows:

Under nitrogen protection, compound 3af (1 mmol), amine starting material (1.1 mmol), and triethylamine (1.5 mmol) were sequentially added to methanol (2 mL), and the reaction was carried out at room temperature (25° C.) for 1-2 h. The reaction progress was monitored by TLC. After the complete reaction, the solvent was dried by rotary evaporator, extracted with ethyl acetate and 1M hydrochloric acid solution, and then the organic layer was extracted with saturated sodium bicarbonate solution, and then passed through anhydrous sodium sulfate and column chromatography to obtain the corresponding compound. Target compounds 8a-8e.

The structure of the amine starting material is _R3 -H.

It should be noted that, in the structures of compound 3ac and compound 3aj, the pyrimidine ring has amino substitution and halogen atom substitution. These two compounds can be directly synthesized by existing methods or obtained by the preparation methods of compounds 8a to 8e. . However, considering the cost saving and preparation efficiency, these two compounds were obtained by the preparation method of compounds 3a-3u and adjusting the reaction conditions.

The following are the structural characterizations of compounds 3v~3af, 8a~8e:

Compound 3v, white solid, yield 97%, mp: 68～71°C. ¹ H NMR (600 MHz, CDCl ₃ ) δ 8.45 (d, J=5.1 Hz, 1H), 6.85 (d, J=5.1 Hz, 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). ¹³ C NMR (150MHz, CDCl ₃ )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 C ₁₉ H ₃₃ N3O[ _M +H] ⁺ : 320.2702, found: 320.2703.

Compound 3w, white solid, yield 47%, mp: 141～147℃. ¹ H NMR (600MHz, DMSO) δ 9.75(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). ¹³ C NMR (150MHz, CDCl ₃ ) δ 164.0, 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 C ₁₈ H ₃₂ N ₄ O[M+H] ⁺ :321.2654,found:321.2646.

Compound 3x, white solid, 16% yield, mp: 93-97°C. ¹ H NMR (600 MHz, CDCl ₃ ) δ 8.50 (d, J=5.2 Hz, 1H), 7.03 (d, J=5.3 Hz, 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). ¹³ C NMR (150MHz, CDCl ₃ )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 C ₁₈ H ₃₀ ClN ₃ O[M+H] ⁺ : 340.2156, found: 340.2163.

Compound 3y, white solid, 8% yield, mp: 96～101°C. ¹ H NMR (600 MHz, CDCl ₃ ) δ 8.64 (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). ¹³ C NMR(150MHz, CDCl ₃ )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 C ₁₈ H ₃₀ BrN ₃ O[M+H] ⁺ :384.1651 found:384.1651.

Compound 3z, white solid, yield 31%, mp: 89～93°C. ¹ H NMR (600 MHz, CDCl ₃ ) δ 8.55 (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). ¹³ C NMR (150MHz, CDCl ₃ )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 forC ₁₈ H ₃₀ ClN ₃ O[M+H] ⁺ :340.2156,found :340.2155.

Compound 3aa, white solid, yield 49%, mp: 162～168℃. ¹ H NMR (600 MHz, CDCl ₃ ) δ 6.02(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). ¹³ C NMR (150MHz, CDCl ₃ )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 C _{19 H33N3O2} [ _{M +} H] ⁺ : 336.2651 found: 336.2657.

Compound 3ab, white solid, yield 16%, mp: 53～55℃. ¹ H NMR (600 MHz, CDCl ₃ ) δ 5.74 (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 ). ¹³ C 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 C _{20 H35N3O3 [ M} +H] ⁺ : 366.27571 found: 366.2759.

Compound 3ac, white solid, yield 10%, mp: 94～98℃. ¹ H NMR (600MHz, DMSO) δ 10.08(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). ¹³ C NMR (150 MHz, DMSO) δ 172.1, 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 forC ₁₈ H ₃₁ ClN ₄ O[M+H] ⁺ :355.2265, found: 355.2277.

Compound 3ad, white solid, yield 33%, mp: 88～94℃. ¹ H NMR (600 MHz, CDCl ₃ ) δ 7.87(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=7.0 Hz, 3H). ¹³ C NMR (150 MHz, CDCl ₃ ) δ 173.4, 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 C ₁₉ H ₃₂ ClN ₃ O ₂ [M+H] ⁺ : 370.2261 found: 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 C ₁₉ H ₃₂ ClN ₃ O[M+H] ⁺ : 354.2312, found: 354.2313.

Compound 3af, white solid, yield 75%, mp: 67～70°C. ¹ H NMR (600MHz, CDCl ₃ )δ7.05(s, 1H), 2.75(t, J=7.5Hz, 2H), 2.35( ¹³ C NMR (150MHz, CDCl ₃ )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 C ₁₈ H _{29 Cl2N3O} [ _M +H] ⁺ : 374.1766, found: 374.1766.

Compound 3ag, white solid, yield 84%, mp: 129～131°C. ¹ H NMR (600 MHz, CDCl ₃ ) δ 8.01 (s, 1H), 2.72 (t, J=7.5 Hz, 2H), 1.72– 1.68(m, 2H), 1.41-1.36(m, 2H), 1.27(m, 18H), 0.88(t, J=7.0Hz, 3H). ¹³ C NMR (150MHz, CDCl ₃ )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 forC ₁₈ H ₂₉ Cl ₂ N ₃ O[M+H] ⁺ :374.1766 , found: 374.1765.

Compound 3ah, white solid, yield 76%, mp: 118～120°C. ¹ H NMR (600 MHz, CDCl ₃ ) δ 2.49-2.43 (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). ¹³ C NMR (150MHz, CDCl ₃ , DMSO) δ 181.6, 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 C ₁₈ H ₂₈ Cl ₃ N ₃ O[M+H] ⁺ :408.1376,found:408.1373.

Compound 3ai, white solid, yield 79%, mp: 75～76℃. ¹ H NMR (600MHz, DMSO) δ 11.06(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 C ₁₈ H ₂₉ F ₂ N ₃ O[M+H] ⁺ : 342.2357, found: 342.2357.

Compound 3aj, white solid, yield 60%, mp: 119～121℃. ¹ H NMR (600 MHz, DMSO) δ 10.06(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). ¹³ C 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°C. ¹ H NMR (600 MHz, CDCl ₃ ) δ 6.05(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). ¹³ C 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 C ₁₉ H ₃₃ ClN ₄ O[M+H ] ⁺ :369.2421,found:369.2425.

Compound 8b, white solid, yield 39%, mp: 56～60℃. ¹ H NMR (600MHz, MeOD) δ 6.37(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). ¹³ C 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 forC ₂₀ H ₃₅ ClN ₄ O[M+H]+:383.2578,found :383.2587.

Compound 8c, white solid, 16% yield, mp: 94～96°C. ¹ H NMR (600 MHz, CDCl ₃ ) δ 7.71 (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). ¹³ C NMR( For _{C 22H37ClN4O2} [M ₊ H] ₊ : _425.2683 , found: ^425.2690 .

Compound 8d, pale yellow solid, yield 32%, mp: 73～76℃. ¹ H NMR (600MHz, CDCl ₃ )δ 6.18(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). ¹³ C NMR (150MHz, CDCl ₃ )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 C _{22 H38ClN5O} [M+H] ⁺ : _424.2843 , found: 424.2841.

Compound 8e, white solid, yield 56%, mp: 98～100℃ ¹ H NMR (600MHz, DMSO/CDCl ₃ )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). ¹³ C NMR (150MHz, DMSO) δ172.4, 163.1, 159.5, 156.9 (d, J=11.2 Hz), 96.7 (d, J=114.7 Hz), 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 C ₂₂ H ₃₈ FN ₅ O[M+H] ⁺ :408.3139,found:408.3145.

The sources of experimental reagents used in the preparation of the above-mentioned specific compounds in the present invention are:

2-Aminopyrimidine substrates, acyl chloride substrates and other chemical reagents were purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd.; LB medium, MHB medium, LB agar medium, 96-well plate, 24-well plate, 6-well plate, etc. All were purchased from Guiyang Chaoyuanzhicheng Biotechnology Co., Ltd.; the silica gel used in the experimental column chromatography was 200-300 mesh, and the GF254 used for the preparation of thin layers were all products produced by Shandong Qingdao Ocean Chemical Factory; the chemical reaction reagents and chromatography solvents used in the experiment All are analytically pure.

The effects of some specific compounds are verified by the following examples:

The sources of strains used in the examples of the present invention are as follows: A.baumannii S1 strains are all provided by the National University of Singapore; Ps.aeruginosa ATCCC 9027; E.coli Dh5a; S.aureus ATCC 433000; S.epidermidis 102555 were purchased from Beijing North Nanotron Biotechnology Research Institute; AB ATCC19606; AB 1778027; AB1780608; AB 1913061; AB 1925052;

Example 1 Determination of Minimum Inhibitory Concentration (MIC)

According to the American Clinical and Laboratory Standards Institute (CLSI), a loop of bacterial solution was taken from a -80°C refrigerator in a sterile centrifuge tube containing 5mL MHB medium, and incubated in a constant temperature shaking incubator at 37°C for 18-24 hours; Take 200 μL of the activated bacterial liquid into a sterile centrifuge tube containing 5 mL of MHB medium, and incubate it at a constant temperature of 37°C for 3 to 4 hours, then adjust the concentration of the bacterial liquid to 1×10 ⁶ CFU/mL (OD ₆₀₀ =0.1). The concentration was 1×10 ⁸ CFU/mL). The compound to be tested was prepared at 30.72 mg/mL for later use. Label and number the 96-well polystyrene plate, add 10 μL of the above-prepared compound to each well from the third column; add 150 μL MHB to each well; add 150 μL and 140 μL of MHB to the first and third columns, respectively; After mixing the 3rd column, pipette 150μL of the mixture into the 4th column, dilute it to the 12th column in equal times, and then discard 150μL of the mixture in the 12th column; add 150μL of the above prepared mixture to the 2nd to 12th columns respectively. Bacterial fluid. The first column is the blank control group, and the second column is the negative control group; the final concentration of the drug is 1024, 512, 256, 128, 64, 32, 16, 8, 4, 2 (μg/mL), and the final concentration of the bacterial solution was 1×10 ⁶ CFU/mL, and the total volume was 300 μL. After culturing the 96-well plate in a 37°C constant temperature incubator for 18-24 hours, compare the turbidity of the medium in the observation wells, and it can be observed that the clear concentration in one well is the lowest inhibitor concentration against the bacteria.

After testing, the MICs of compounds 3a-3ah and 8a-8e against Acinetobacter baumannii were all greater than 256 μg/mL, indicating that the compounds had no inhibitory effect on bacterial growth, and could avoid bacterial stress and produce drug resistance.

Example 2 Anti-biofilm activity assay

According to CLSI, take a loop of bacterial solution from the -80 °C refrigerator in a sterile centrifuge tube containing 5 mL of LB medium, and incubate it in a constant temperature shaking incubator at 37 °C for 18-24 hours; then take 200 μL of the activated bacterial solution in a In a sterile centrifuge tube of 5mL LB medium, after incubating at 37°C for 3-4h, the concentration of the bacterial solution was adjusted to 1×10 ⁶ CFU/mL (when OD ₆₀₀ = 0.1, the bacterial concentration was 1×10 ⁸ CFU/mL ); the compound to be tested was prepared at 0.03 mmol/mL and then diluted 15 times to 2 μmol/mL before use. The 96-well plate was labeled and numbered, the first column was used as a solvent control, and 190 μL of LB broth and 10 μL of DMSO solution were added; the second column was used as a negative control, and 190 μL of bacterial solution without inhibitor and 10 μL of DMSO solution were added; Add 10 μL of the above-prepared compound to each of the 12 columns, and then add 190 μL of bacterial solution to the 3rd to 12th wells respectively, and the compound concentration reaches 100 μM, which is the target concentration. Each compound was tested in 8 groups in parallel; the 96-well plate was placed in an electrothermal constant temperature incubator at 37°C for static culture for 18-24 h, and the cultured biofilm was quantitatively determined by crystal violet staining. Aspirate the bacterial solution in the 96-well plate and carefully rinse twice with double-distilled water to remove planktonic bacteria; add 95% ethanol solution, fix the biofilm for 30 min, and after natural air-drying, add 200 μL of 0.1% crystal violet staining solution to each well. After staining for 10 minutes, rinse off the excess dye solution; dry at room temperature or in a 37°C oven until completely dry, add 200 μL of 33% acetic acid solution to each well; after the stained biofilm is fully dissolved, use ELX800 type The OD value in the well was measured by an enzyme-linked immunosorbent assay at a wavelength of 590 nm; the biofilm inhibition rate of the compound was calculated.

After testing, at a concentration of 100 μM, using compound A developed by Blackwell’s research group as a positive control, a series of screenings were carried out for the anti-Acinetobacter baumannii biofilm activity of compounds 3a to 8e. The results are shown in Figure 2 and Table 1. Show.

Table 1 Results of biofilm inhibitory activity of compounds 3a-8

The experimental results showed that the compound 3u containing tridecyl group had the best activity; therefore, on the basis of compound 3u, further structure-activity relationship studies were carried out on the substituents on the aromatic ring of 2-aminopyrimidine. Compounds 3v-3aj were synthesized and tested for their preliminary anti-biofilm activity. The experimental results show that the compounds 3ac and 3aj have obvious anti-biofilm activities when the 4-position of the pyrimidine ring is substituted with an amino group and the 6-position of the pyrimidine ring is substituted with a halogen. Therefore, the amino groups of the above two compounds were further modified to synthesize compounds 8a-8e. The results of activity experiments showed that when the 6-position of the pyrimidine ring was introduced into the piperazine ring, the anti-biofilm activities of compounds 8d and 8e were better than that of compound 3ac. At the concentration of 100 μM, the anti-biofilm activity of compound 8d against A. baumanniiS1 was as high as 70.17%.

Example 3 Scanning electron microscope observation of Acinetobacter baumannii biofilm

The modified plate culture method was used to test whether Acinetobacter baumannii could form BF on the silica membrane. Cut the silicone membrane into small squares of (1.0×1.0) cm ² . According to CLSI, the LB broth was prepared to a concentration of 1.0×10 ⁶ CFU/mL bacterial suspension, and the compounds with better activity screened out in the above activity test were prepared to 2 mmol/L. Put the silica gel sterilized with alcohol into the 24-well plate, add 50 μL of the prepared compound, and then add 950 μL of the diluted bacterial solution of 1×10 ⁶ CFU/mL, and leave the bacterial solution without the compound culture as the control group , after culturing in a 37°C biochemical incubator for 24h, rinse with flowing saline to remove planktonic bacteria. After fixing the silica gel sheet with 2.5% glutaraldehyde at low temperature for 12 hours, eluting with 30%, 50%, 70%, 90% and 100% ethanol gradients for 15 minutes respectively, drying at critical point, spraying gold, loading samples, scanning and observing, Send scanning electron microscope observation, photography.

During the activity screening process of each compound, the relatively good compounds 3ab, 3ac, 3ad, and 3x were cultured for 18-24 hours at a concentration of 100 μM and a bacterial concentration of 1×10 ⁶ CFU/mL. The bacterial solution was sucked off, dried, frozen, and plated with gold for preliminary observation by SEM.

The results are shown in Fig. 2. It can be visually observed in the visual field that the bacteria in the visual field containing the compound are significantly reduced than those in the visual field without the compound. Thus, it is further demonstrated that compounds 3ab, 3ac, 3ad, and 3x have certain anti-biofilm activity against A. baumannii S1, in order to reduce the drug resistance of Acinetobacter baumannii in combination with antibiotics.

Example 4 Determination of bacterial exopolysaccharide

Since exopolysaccharide is the main component of biofilm, the inhibition of exopolysaccharide by compound can further detect the inhibitory effect of compound on biofilm. The extracellular polysaccharide content of compounds 3ac, 3aj, 8d and 8e was determined by the phenol-sulfuric acid method at a concentration of 100 μM.

According to CLSI, the LB broth was prepared to a concentration of 1.0×10 ⁶ CFU/mL bacterial suspension, and the compounds with better activity screened out in the above activity test were prepared to 2 mmol/L. Add 50 μL of the prepared compound to the 24-well plate, and then add 950 μL of the diluted bacterial solution of 1×10 ⁶ CFU/mL, and leave the bacterial solution without the compound culture as a control group, and cultivate in a 37°C biochemical incubator. After 24h, the upper bacterial solution was discarded by suction. Rinse the 24-well plate with 0.9% NaCl, add 0.25 mL of 5% phenol and 1.25 mL of concentrated sulfuric acid to the 24-well plate, and incubate it in the dark at 30°C for 1 h; use an ELX 800 enzyme-linked immunosorbent assay at a wavelength of 490 nm. OD value; calculated the effect of compounds on the extracellular polysaccharide content of biofilms.

The results are shown in Figure 3. At the concentration of 100 μM, compounds 3ac, 3aj, 8d and 8e have obvious inhibitory effects on the extracellular polysaccharides in A. baumannii S1 biofilm, and the inhibitory activities are 49.67%, 59.40%, 73.00% and 73.00%, respectively. 60.25%, which has a similar trend with the results of biofilm inhibitory activity; it is further proved that compounds 3ac, 3aj, 8d and 8e have stronger inhibitory effects on A. baumannii.S1 biofilm.

Embodiment 5 chessboard method combined drug test

According to CLSI, the MHB broth was prepared into a bacterial suspension with a concentration of 1.0×10 ⁶ CFU/mL, and the compounds 3ac, 3aj, 8d and 8e with better activity screened out in the above activity tests were prepared at 30.72 mg/mL, respectively. Combined with various antibiotics, their inhibitory effect on A. baumannii S1 was observed.

The specific test method is to label and number the 96-well plate, add twice the MIC concentration of antibiotics to the horizontal wells, and then dilute them by two times in turn, add the prepared compound at 30.72 mg/mL to the vertical wells, and sequentially Double-diluted back to establish a checkerboard; the first well was used as a blank control, and 200 μL of MHB broth was added; the second well was used as a solvent control, and 190 μL of bacterial solution and 10 μL of DMSO solution were added; 18-24h; observe the turbidity in the comparative wells, obtain the MIC of the compound, and calculate the fractional inhibitory concentration index (FICI).

Synergy of compounds with various antibiotics was assessed by a checkerboard titration assay on microplates as recommended by the 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), that is, the MIC when the A drug is used in combination/the MIC when the A drug is used alone + the MIC/B drug when the B drug is used in combination MIC for single use. When FICI≤0.5, A and B have synergistic effect; when 1≥FICI>0.5, A and B have additive effect; when 2≥FICI>1, there is no interaction between A and B; when FICI>2 A and B have antagonistic effects.

The test results are shown in Table 2:

Table 2. Study on the inhibition of Acinetobacter baumannii by combination drug

The FICI values of compounds 3ac, 3aj, 8d and 8e in combination with gentamicin sulfate, oxacillin, chloramphenicol, erythromycin and meropenem, respectively, were all less than or equal to 0.5, indicating that compounds 3ac, 3aj, 8d and 8e has a certain synergistic effect with aminoglycosides, β-lactams, amido alcohols and macrolide antibiotics. Among them, when used in combination with gentamicin sulfate in aminoglycosides, the MIC decreased by 2 to 8 times; when used in combination with oxacillin in penicillins and chloramphenicol in amide alcohols, the MIC also decreased by 2 ～4 times; when combined with erythromycin and carbapenems in macrolides, the MIC decreased by 2 times.

Example 6 IC ₅₀ test

The anti-biofilm effects of compounds 3ac, 3aj, 8d and 8e on various Acinetobacter baumannii were determined by crystal violet staining. In addition, the anti-biofilm activities of methicillin-resistant Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis were simply screened.

The specific test process is as follows: according to CLSI, the LB broth is configured into a bacterial suspension with a concentration of 1.0×10 ⁶ CFU/mL, and the compounds with better activity screened out in the above activity test are configured to be 2 mmol/L, 0.4 mmol/L and 0.4 mmol/L in turn. L, 0.08mmol/L, 0.016mmol/L, and 0.0032mmol/L for later use. Label the 96-well plate and place a circle around it with LB medium to prevent volatilization. In the second column, add LB medium as a blank control group, and in the third column, add 10 μL DMSO and 190 μL LB bacteria-containing medium as a solvent control group; in the fourth to eighth columns, add 10 μL of the above-prepared compounds according to the concentration gradient; Add 190 μL LB bacteria-containing medium; add LB medium to the remaining wells. The final concentration of the drug was 100, 20, 4, 0.8, 0.16 μM, the final concentration of the bacterial solution was 1×10 ⁶ CFU/mL, and the total volume was 200 μL. The 96-well plate was placed in a 37°C constant temperature incubator for 18-24 hours, and the biofilm was stained by crystal violet staining, and the OD value of the solution in the culture well was measured with an ELX800 enzyme-linked immunosorbent assay at 590 nm. ; Calculate the biofilm inhibition rate of compounds at different concentrations. The _IC50 of the compound was calculated using GraphPad Prism 5 software.

The test results are shown in Table 3:

Table 3 _IC50 results of compounds 3ac, 3aj, 8d and 8e on various bacterial biofilms

According to Table 3, compounds 3ac, 3aj, 8d and 8e also have obvious inhibitory effects on the biofilms of five species of Acinetobacter baumannii except ACBA.S1. In particular, compound 3aj had a strong anti-biofilm effect against the standard strain AB ATCC19606 with an _IC50 as low as 3.82 μM. Excitingly, compounds 8d and 8e also showed good anti-biofilm activity against some Gram-negative and Gram-positive bacteria. Among them, the IC 50 of compound 8d for MRSA20151023070 anti-biofilm activity was as low as 17.04 μM; the IC ₅₀ of compound 8e for E. coli Dh5a anti-biofilm activity was as low as 1.95 μM; the IC ₅₀ of Pa _ATCC9027 was as low as 1.28 μM; _IC50 for SA ATCC 433000 anti-biofilm activity as low as 4.33 μM; IC ₅₀ for Se ATCC 102555 anti-biofilm activity as low as 5.82 μM. Therefore, the strong anti-biofilm activities of compounds 8d and 8e endow the structure with good antibacterial potential.

Example 7 Observation of Acinetobacter baumannii biofilm by laser confocal microscope (LSCM)

According to CLSI, the LB broth was prepared into a bacterial suspension with a concentration of 1.0×10 ⁶ CFU/mL, and the compounds 3ac, 3aj, 8d and 8e were prepared into 2 mmol/L solutions respectively; 100 μL of the prepared compounds were put into the confocal laser In the small dish, add 1900 μL of LB bacteria-containing medium, and the final concentration of the drug is 100 μM; take 100 μL of DMSO solution and put it into the laser confocal small dish, and add 1900 μL of LB bacteria-containing medium as a control group. After culturing at 37°C for 18-24 hours, the culture medium was removed, washed twice with a PBS solution with a pH of 7.4, and the floating bacteria were removed by suction; fixed with 4% formaldehyde in PBS for 20 min; washed twice with PBS, each time 10min, after washing off the fixative, add the prepared Alexa Fluor 488-labeled phalloidin solution, and stain in the dark for 90min; wash with PBS solution twice, 5min each time; after drying, observe under a laser confocal microscope, take pictures and record Biofilm thickness.

With the maximum emission wavelength of 517 nm and the maximum excitation wavelength of 493 nm, the results of laser confocal microscope biofilm thickness are shown in Figure 4: A is only DMSO as a solvent control, and the thickness of ACBA.S1 biofilm under CLSM is 25.91 μm; B is the biofilm of ACBA.S1 under the action of compound 3ac, with a thickness of 16.04 μm; C is the biofilm of ACBA.S1 under the action of compound 8d, with a thickness of 14.68 μm. Using CLSM technology, the change of biofilm thickness was observed more intuitively, which further demonstrated the inhibitory activity of the compound on its biofilm.

Example 8 Swarming Movement Impact Check

In this example, the formation of biofilm is inhibited by inhibiting quorum sensing (QS). However, QS has a certain motility. Therefore, whether the compound has inhibitory effect on bacterial QS was further verified by the motility of bacterial population.

According to CLSI, take a loop of bacterial solution from the -80°C refrigerator in a sterile centrifuge tube containing 5mL MHB medium, and incubate it in a constant temperature shaking incubator at 37°C for 18-24h; then take 200μL of the activated bacterial solution in a In a sterile centrifuge tube of 5mL MHB medium, incubate at 37°C for 3-4 hours before use; the compounds with better activity screened in the above activity test were sequentially configured to 2mmol/L before use; for 6-well plates Mark number, the first well A is used as a solvent control, after adding 100 μL of DMSO, quickly add 1900 μL of semi-solid medium (0.8% NB broth, 0.5% D-glucose and 0.3% agar) and shake well; in the fourth well D, add half 2000μL of solid medium was used as blank control; Compounds 3ac, 3aj, 8d and 8e were added to the 2nd, 3rd, 5th and 6th wells B, C, E and F, respectively, and each compound was added to 100μL of semi-solid at a concentration of 2mmol/L. After adding 1900 μL of medium, shake well to make the final concentration of each well compound reach 100 μM. After the medium was solidified, 5 μL of the above activated bacterial solution was added to each well, and after culturing at 30° C. for 24 h, photographed and recorded.

In the 6-well plate, the results are shown in Figure 5. First, the diameter of the bacterial colony movement in D is larger than that of A, indicating that the solvent DMSO has an inhibitory effect on bacterial QS. Therefore, ensure that the DMSO is below 5% during each experiment. Secondly, it can be observed that the movement diameter of bacterial colonies of B, C, E and F is significantly smaller than that of A, indicating that compounds 3ac, 3aj, 8d and 8e have inhibitory effects on bacterial QS.

Example 9 Drug sustained-release immobilization

Poly(lactic-co-glycolic acid, PLGA) is a biodegradable macromolecule with good biocompatibility, non-toxicity and good film-forming properties. PLG is racemic PLGA, which also has certain film-forming properties. Therefore, in this example, the two compounds were used as loading materials to investigate the inhibitory activity of the compounds on biofilms.

Dissolve 182 mg of PLG in 7.6 mL of dichloromethane for later use; then take 2.5 mL of the above-mentioned spare PLG solution into two sterilized tubes, add compounds 3ac and 8d respectively, and configure them into a 1 mmol/L PLG solution. Cut the biological silica gel sheet into small squares (1.0×1.0) cm ² , put it into a 24-well plate after sterilizing it with alcohol; take 100 μL of the above preparation solution to the washed and sterilized silica gel sheet, and put it in a fume hood to evaporate the solvent. , and then put it in a drying oven under reduced pressure to dry for 12 h, to prepare a silica gel tablet containing 2.4 mg of PLG and 0.1 μmol of compound per well. As described above, silica gel tablets containing 1.2 mg of PLGA and 0.1 μmol of compound per well were prepared. Due to the poor solubility of PLGA, the half load was adopted in this example. According to CLSI, the LBS medium (2% LB medium, 1.5% NaCl, 0.3% glycerol and 50 mM Tris-HCl) was prepared into a bacterial suspension with a concentration of 1.0×10 ⁶ CFU/mL, and 1 mL was added to the prepared 24 wells above. The plates were incubated at a constant temperature of 37°C for 1 d, 7 d and 14 d respectively; the LBS medium was replaced every 24 h; after the incubation, the cells were washed twice with PBS to remove the planktonic bacteria. After fixing the silica gel sheet with 2.5% glutaraldehyde at low temperature for 12 hours, 30%, 50%, 70%, 90% and 100% ethanol gradients were used for elution for 15 minutes respectively, and the critical point was dried and then sprayed with gold. The sustained release results were detected by SEM The results of changes in biofilms are shown in Figures 6-8.

The results showed that when compounds 3ac and 8d were released slowly on 1d and 7d, the anti-biofilm activity was more obvious; the anti-biofilm activity of compounds 14d could also be seen, but the results were poorer than those of 7d. The experimental results once again demonstrate the anti-biofilm activity of compounds 3ac and 8d, and can be loaded on PLGA and PLG materials to achieve long-term biofilm inhibition.

Therefore, compounds 3ac and 8d can be loaded on various medical devices to reduce multidrug-resistant infections in clinical medicine.

Example 10MTT test

Normal cells have vigorous metabolism, and the succinate dehydrogenase in the mitochondria can reduce tetrazolium salts (such as MTT) to purple crystalline substances, which are deposited around the cells, and then read the OD value through a microplate reader. Thus, the state of cell proliferation is detected. Cytotoxicity assays are widely used in basic research and drug discovery to screen libraries of toxic compounds. Compounds that produce cytotoxic responses can be eliminated in subsequent screening; or compounds that target rapidly dividing cells can be selected as candidates for cancer therapy. Therefore, in this example, mouse embryonic fibroblasts 3T3-L1 were used to test the cytotoxicity of compounds 3ac and 8d.

The specific experimental process was as follows: mouse embryonic fibroblasts (3T3-L1) were inoculated in 100 μL of medium at 10,000 cells per well. The 96-well plate was then placed in a humidified incubator, 5% carbon dioxide, 95% air, for 24 h. Thereafter, the medium was removed from the 96-well plate, replaced with compound-containing medium, and incubated for 24 h. After incubation, cytotoxicity was measured by MTT assay. 5 mg/mL MTT 20 μL in warm phosphate buffered saline (PBS) was added to each well and incubated at 37° C. in dark air with 5% carbon dioxide and 95% relative humidity for 4 h. After incubation, pipette MTT and add 0.1 mL of 0.04 mol/L hydrochloric acid isopropanol solution. Stir the plate until completely dissolved. Absorbance was read at 570 nm using a Multiskan-EX spectrophotometer. Six replicates of each compound were tested three times. This example calculates the absorbance and its standard deviation for wells containing the same compound.

The test results are shown in Table 4:

Table 4 Results of cytotoxicity of compounds 3ac and 8d on mouse 3T3-L1

Cytotoxicity was graded according to cell viability compared to controls; non-cytotoxic > 90% cell viability, mild cytotoxicity = 60-90% cell viability, moderate cytotoxicity = 30-59% cell viability, severe Cytotoxicity <30% cell viability.

The results showed that compound 3ac was not cytotoxic. While compound 8d had slight cytotoxicity at 50 μM, its IC ₅₀ for cytotoxicity was 73.63 μM, which was much higher than the IC ₅₀ for its anti-biofilm activity. Therefore, the above two compounds can provide an important guarantee for the development and application of new medical devices and biological materials.

Example 11 Stability test in artificial gastric juice

Using 1-(2-pyrimidinyl)piperazine as the internal standard, the internal standard stock solution with a mass concentration of 0.1 mg/mL was prepared and stored at 4°C. Take 16.4 mL of dilute hydrochloric acid, add 800 mL of water, adjust the pH to 1.3 with 0.1 mol/L hydrochloric acid solution, add water to dilute and dilute to 1000 mL to obtain blank artificial gastric juice.

Take 16.4 mL of dilute hydrochloric acid, add 800 mL of water and 10 g of pepsin, shake well to dissolve it, adjust the pH to 1.3 with 0.1 mol/L hydrochloric acid solution, add water to dilute and make up to 1000 mL to obtain artificial gastric juice.

Precisely measure 1 mL of methanol solution of the target compound with a mass concentration of 1 mg/mL in a 10 mL volumetric flask, dilute to the mark with blank artificial gastric juice and artificial gastric juice respectively (both alcohol content is 1%); 600 μL, shake and mix, and incubate in a 37°C water bath for 0, 6, 12, 24, and 36 h. Take 200 μL of the incubated sample, add 2 μL of the internal standard solution with a mass concentration of 100 μg/mL, vortex for 5 min to mix, centrifuge at 20000×g for 10 min at 4°C, and take the supernatant for measurement.

The chromatographic conditions for the determination of the stability of the compounds in artificial gastric juice are: chromatographic column: WondaSilC18 (250mm×4.6mm, 5μm); mobile phase: methanol (B)-0.1% triethylamine aqueous solution (A), isocratic elution ( 0～15min, 40% B); flow rate: 1mL/min; detection wavelength: 254nm; column temperature: 30°C; injection volume: 20μL.

After injection and analysis, the peak area was recorded, and the mass concentration of the drug in each sample was calculated according to the internal standard method. Each sample was measured 3 times in parallel.

The test results are shown in Table 5:

Table 5 Remaining percentage of compound 8d in artificial gastric juice within 0～72h

The residual percentage of compound 8d at each incubation time point was calculated based on the theoretical mass concentration of compound 8d before incubation (100 μg/mL), and the residual percentage of compound 8d incubated in blank artificial gastric juice at different times was in the range of (106.22±6.38)%～(90.63±2.43) )%; the remaining percentage was (87.14±1.12)% when incubated in artificial gastric juice for 36h, and there was no significant change compared with blank artificial gastric juice.

In this example, the stability of compound 8d within 36 hours of incubation in artificial gastric juice was investigated, which provided an important reference for the subsequent structural modification of similar derivatives, formulation development and combined drug regimens of this compound.

The above-described embodiments are not intended to limit the present invention, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included within the protection scope of the present invention.

Claims (10)

1. a 2-aminopyrimidine compound, is characterized in that, the general structural formula of described compound is as shown in general formula (1):

Wherein, R ₁ represents substituted or unsubstituted C ₂ -C ₁₄ alkyl, C ₂ -C ₁₄ cycloalkyl, C ₂ -C ₁₄ carboxyl, and substituted or unsubstituted phenyl, naphthyl, penta One of the membered heterocyclic aryl groups; R ₂ represents one of substituted or unsubstituted C ₂ -C ₁₄ alkyl, alkoxy, X, and R ₃ ; X is a halogen atom; R ₃ is an N-containing group; The value of n is 0, 1, 2 or 3. 2 . The 2-aminopyrimidine compound according to claim 1 , wherein the value of n is 1, 2 or 3. 3 . 3. a kind of 2-aminopyrimidine compound according to claim 1 is characterized in that, R ₁ represents C ₂ -C ₁₄ alkyl, F-substituted alkyl, cyclobutanyl, pentylcarboxy, phenyl, alkyl-substituted phenyl, alkoxy-substituted phenyl, alkenyl-substituted benzene One of phenyl, naphthyl and thienyl substituted by nitro group; R ₂ represents one of methyl, methoxy, Cl, Br, F, amino, methylamino, dimethylamino, piperazinyl, and morpholino. 4. The 2-aminopyrimidine compound according to any one of claims 1 to 3, wherein the specific structural formula of the compound is:

one of the. 5. A method for preparing a 2-aminopyrimidine compound according to any one of claims 1 to 4, characterized in that, When the value of n is ₂ , and one R2 is represented as X and the other R2 is represented as _R3 , the reaction equation that occurs in the preparation process is _:

The specific reaction process is: Under nitrogen protection at room temperature, the acid chloride substrate was added to the dry toluene solution of 2-aminopyrimidine raw material and N,N-diisopropylethylamine, and the reaction was performed at 25°C or 110°C; after the 2-aminopyrimidine raw material was completely reacted , spin dry the solvent, extract with ethyl acetate and hydrochloric acid solution, extract the organic layer with saturated sodium bicarbonate solution, pass through anhydrous sodium sulfate, and pass column chromatography to obtain compound 1; Wherein, the molar concentration ratio of acid chloride substrate, 2-aminopyrimidine raw material and N,N-diisopropylethylamine is 1.5:1:2; Under nitrogen protection, compound 1, amine raw material, and triethylamine were sequentially added to methanol, and reacted at 25 °C for 1-2 h; after complete reaction, the solvent was spin-dried, extracted with ethyl acetate and hydrochloric acid solution, and then saturated with carbonic acid. After the organic layer is extracted with sodium hydrogen solution, the target compound is obtained by column chromatography through anhydrous sodium sulfate; Wherein, the molar concentration ratio of compound 1, amine raw material and triethylamine is 1:1.1:1.5; The structure of the amine starting material is _R3 -H. 6. The use of the 2-aminopyrimidine compound according to any one of claims 1 to 4 as a biofilm inhibitor. 7 . A biofilm inhibitor, wherein the active ingredient of the biofilm inhibitor is the 2-aminopyrimidine compound according to any one of claims 1 to 4 . 8. a kind of biofilm inhibitor according to claim 7 is characterized in that, described biofilm inhibitor can inhibit biofilm formation, and described biofilm is made of Acinetobacter baumannii, methicillin-resistant Staphylococcus, Formed by Staphylococcus epidermidis and Escherichia coli. 9 . An anti-infective drug, characterized in that it comprises the 2-aminopyrimidine compound according to any one of claims 1 to 4 and an adjuvant. 10 . 10 . A biological material, wherein the surface of the biological material is coated with the biofilm inhibitor according to claim 7 or 8 .

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