5, 6-dihydroxypyrimidine compound as well as preparation method and application thereof

Abstract

The invention relates to a 5, 6-dihydroxypyrimidine compound as shown in a formula I, a preparation method of the 5, 6-dihydroxypyrimidine compound and application of the 5, 6-dihydroxypyrimidine compound in formation of an antibacterial biological membrane. In-vivo and in-vitro pharmacological verification shows that most of the 5, 6-dihydroxypyrimidine compounds have excellent bacterial biofilm and bacterial iron uptake inhibitory activity, can be used for preparing broad-spectrum antibiotic synergists, and can enhance the antibacterial effect of existing antibiotics. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to a class of 5,6-dihydroxypyrimidine compounds, a preparation method thereof, and an application thereof in the preparation of antibacterial drugs. Background Art

[0002] Epidemiological studies have shown that antibiotic resistance is the leading cause of infectious deaths worldwide, with a death toll far exceeding that of HIV / AIDS and malaria. According to statistics, hundreds of thousands of people die each year from antibiotic resistance worldwide, among which children and newborns are particularly vulnerable to resistant bacteria. One in five deaths from antibiotic resistance occur in children under five years old, and up to 3 million newborns suffer from severe infections leading to sepsis each year. The long-term presence of antibiotic resistance can lead to longer hospital stays and a greater economic burden on infected patients. If effective antibiotics are lacking, this will lead to a more serious global public health crisis. Therefore, there is an urgent need to study new strategies to curb the development of antibiotic resistance.

[0003] Currently, the most difficult-to-treat hospital infections are caused by Gram-negative bacteria, whose persistent infections lead to the development of antibiotic resistance. The formation of bacterial biofilms is one of the main causes of persistent infections. Biofilms associated with medical devices and tissues may lead to the failure of antibiotic treatment and may cause many chronic infections. Biofilms play a key role in the survival and persistence of pathogenic microorganisms, enabling them to evade host immune responses and antimicrobial therapy. The resistance of biofilm-associated bacteria to antimicrobial drugs stems from multiple mechanisms, including low metabolic rate, the protective effect of the biofilm matrix, and the development of specific resistance mechanisms within individual cells. Clinical studies have shown that biofilm infections can be treated with a combination of traditional antibiotics and biofilm-disrupting substances, and show excellent therapeutic prospects. However, the current research and development of biofilm inhibitors is only in the clinical research stage, and they cannot be marketed mainly due to reasons such as therapeutic doses and therapeutic instability. Therefore, the study of new anti-biofilm drugs is of great significance to overcome antibiotic resistance. On the other hand, iron is an indispensable trace element for bacteria. In addition to maintaining normal physiological activities, the availability of iron ions has also been shown to be involved in the development of bacterial resistance. Inhibitors of bacterial iron uptake and reducing the availability of iron in bacteria can increase bacterial sensitivity to traditional antibiotics and curb the development of antibiotic resistance. At present, research in this area has made some progress, and the combination of gentamicin and EDTA conducted by the Pasteur Institute has entered clinical research. However, as a strong iron chelator, EDTA has strong toxic side effects, which limits its therapeutic effect.

[0004] Therefore, in the field of anti-biofilm resistance, more effective treatment strategies need to be developed. The present invention combines anti-biofilm and iron uptake strategies to develop a dual-effect inhibitor, which is expected to be used to reduce the occurrence of bacterial antibiotic resistance. Summary of the invention

[0005] In order to overcome the above-mentioned shortcomings and deficiencies of the prior art, the present invention has found a class of 5,6-dihydroxypyrimidine compounds, which have a stable chemical structure and have been verified by activity experiments to have anti-biofilm and anti-iron uptake effects, and have the potential to become a new antibiotic synergist.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned 5,6-dihydroxypyrimidine compounds.

[0007] Another object of the present invention is to provide the use of the above 5,6-dihydroxypyrimidine compounds as anti-biofilm drugs, anti-bacterial iron uptake drugs or antibiotic synergists.

[0008] The purpose of the present invention is achieved through the following solutions:

[0009] In one aspect, the present invention provides a class of 5,6-dihydroxypyrimidine compounds or pharmaceutically acceptable salts, medically acceptable variants, mesomorphs, racemates, stereoisomers, metabolites, metabolic precursors, prodrugs or solvates thereof, characterized by having a chemical structure as shown in Formula I:

[0010]

[0011] Wherein R1 is selected from one of C1-C4 straight chain or branched saturated or unsaturated hydrocarbon groups, aryl groups, benzyl groups, and 5-6 membered heteroaryl groups;

[0012] R2 is selected from C3-C6 straight chain or branched saturated or unsaturated hydrocarbon group, C3-C6 cyclic hydrocarbon group, 3-6 membered heterocyclic group, aryl group, One of arylethyl, arylsulfonyl, 5- to 6-membered heteroaryl and 5- to 6-membered heteroarylmethyl.

[0013] R3 is selected from halogen, hydroxyl, amino, cyano, C1-C3 straight chain or branched saturated or unsaturated hydrocarbon group, C1-C3 straight chain or branched saturated or unsaturated alkoxy group, trifluoromethyl, trifluoromethoxy, aryl, aryloxy group.

[0014] In the present invention, the "straight-chain or branched saturated or unsaturated hydrocarbon group" includes C1-C4 alkyl or C1-C3 alkyl, such as methyl, ethyl, propyl, butyl and the like.

[0015] In the present invention, the aryl group includes phenyl, naphthyl and the like.

[0016] In the present invention, the heteroaryl group includes pyrrolyl, imidazolyl, oxazolyl, thiazolyl, thienyl, furanyl, triazolyl, isoxazolyl, isothiazolyl, pyrazolyl, pyridyl, pyrimidinyl, triazinyl and the like.

[0017] In the present invention, the heterocyclic group includes tetrahydropyrrolyl, tetrahydrofuranyl, tetrahydrothiophenyl, piperidinyl, piperazinyl and the like.

[0018] The 5,6-dihydroxypyrimidine aromatic compound with bacterial biofilm and bacterial iron homeostasis inhibitory activity described in the present invention can inhibit the formation of Pseudomonas aeruginosa biofilm and inhibit its iron uptake pathway to disrupt bacterial iron homeostasis, can be used to prepare bacterial biofilm inhibitors and iron uptake inhibitors, and has a certain effect of enhancing the efficacy of antibiotics.

[0019] Preferably, the 5,6-dihydroxypyrimidine compound or its pharmaceutically acceptable salt, medically acceptable variant isomer, mesomorph, racemate, stereoisomer, metabolite, metabolic precursor, prodrug or solvate is selected from:

[0020]

[0021]

[0022]

[0023]

[0024] Preferably, the pharmaceutically acceptable salt of the 5,6-dihydroxypyrimidine compound is an acid addition salt or a base addition salt of the 5,6-dihydroxypyrimidine compound, including but not limited to: hydrochloride, hydrobromide, hydroiodide, phosphate, sulfate, nitrate, ethanesulfonate, toluenesulfonate, benzenesulfonate, acetate, maleate, tartrate, succinate, citrate, benzoate, ascorbate and salicylate, malonate, adipate, caproate, arginine salt, fumarate, nicotinate, phthalate, oxalate, lithium salt, sodium salt, potassium salt, barium salt, calcium salt, magnesium salt, aluminum salt, iron salt, ferrous salt, copper salt, zinc salt, or any one or a combination of at least two of the salts of the 5,6-dihydroxypyrimidine compound and morpholine, diethylamine, triethylamine, isopropylamine, trimethylamine, lysine or histidine.

[0025] In another aspect, the present invention provides a method for preparing the 5,6-dihydroxypyrimidine compound, characterized by comprising the following steps:

[0026] Step (1.1) dissolving N-hydroxyacetamidine or N-hydroxyisobutylamidine or N-hydroxybenzamidine and dimethyl butynedioate in methanol, reacting at 0°C, then removing methanol and refluxing in xylene for reaction, and purifying the reaction solution obtained after the reaction to obtain compound 1;

[0027] Step (1.2) Compound 1, benzyl bromide and potassium carbonate are placed in an acetonitrile solvent and reacted at 80° C. The obtained reaction solution is purified to obtain Compound 2;

[0028] Step (1.3) Compound 2 and lithium hydroxide aqueous solution are placed in a methanol solvent for reaction at 40° C. The obtained reaction solution is purified to obtain Compound 3;

[0029] Step (1.4) Compound 3, an amine compound, benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (PyBop) and N,N-diisopropylethylamine (DIPEA) are reacted in tetrahydrofuran (THF) solvent at room temperature, and the obtained reaction solution is purified to obtain Compound 4;

[0030] Step (1.5): Compound 4 is dissolved in methanol, a palladium-carbon catalyst is added, hydrogen is introduced and the mixture is reacted at room temperature. The obtained reaction solution is purified to obtain Compound 5.

[0031] The synthetic route is:

[0032]

[0033] Preferably, the reaction conditions are: (1.1) MeOH, Xylene, 0→135°C, 16h; (1.2) K2CO3, CH3CN, 80°C, 6h; (1.3) LiOH, H2O, MeOH, 40°C, 6h; (1.4) amino compound, PyBop, DIPEA, THF, rt, 4h; (1.5) H2, Pd / C, MeOH, rt, 6h.

[0034] In another aspect, the present invention provides the use of the above-mentioned 5,6-dihydroxypyrimidine compounds or their pharmaceutically acceptable salts, medically acceptable variants, racemates, racemates, stereoisomers, metabolites, metabolic precursors, prodrugs or solvates in the preparation of anti-biofilm drugs, anti-bacterial iron uptake drugs or antibiotic enhancers.

[0035] Preferably, the biofilm is a bacterial biofilm. More preferably, the bacteria is Pseudomonas aeruginosa.

[0036] Preferably, the antibiotic is used to inhibit Pseudomonas aeruginosa.

[0037] In another aspect, the use of the 5,6-dihydroxypyrimidine compound or its pharmaceutically acceptable salt, medically acceptable variant isomer, racemate, racemate, stereoisomer, metabolite, metabolic precursor, prodrug or solvate in the preparation of anti-biofilm drugs, anti-bacterial iron uptake drugs or antibiotic enhancers is characterized in that the drug also contains one or more pharmaceutically acceptable carriers or excipients.

[0038] Preferably, the bacteria is a bacterium, more preferably Pseudomonas aeruginosa.

[0039] In another aspect, the present invention provides a pharmaceutical composition comprising at least one of the 5,6-dihydroxypyrimidine compounds according to any one of claims 1 to 3 or a pharmaceutically acceptable salt, medically acceptable variant isomer, mesomorph, racemate, stereoisomer, metabolite, metabolic precursor, prodrug or solvate thereof.

[0040] Preferably, the drug further comprises one or more pharmaceutically acceptable carriers or excipients.

[0041] In another aspect, the present invention provides a combination drug comprising at least one of a 5,6-dihydroxypyrimidine compound or a pharmaceutically acceptable salt, medically acceptable variant isomer, mesomorph, racemate, stereoisomer, metabolite, metabolic precursor, prodrug or solvate thereof, and ciprofloxacin.

[0042] Preferably, the mass ratio of the 5,6-dihydroxypyrimidine compound or its pharmaceutically acceptable salt, medically acceptable variant isomer, mesomorph, racemate, stereoisomer, metabolite, metabolic precursor, prodrug or solvate to ciprofloxacin is 50:0.5-20, preferably 50:1-10.

[0043] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0044] (1) The present invention starts from the dihydroxypyrimidine structure with chelating properties and, through structural modification, invents a 5,6-dihydroxypyrimidine compound with stable chemical properties. The anti-biofilm inhibitory activity of the compound against Pseudomonas aeruginosa is detected by crystal violet staining. Compared with current clinical studies, this type of compound can significantly inhibit the formation of biofilm at a lower concentration, which is expected to reduce the dosage.

[0045] (2) The present invention identifies the total iron content in bacteria. 5,6-dihydroxypyrimidine compounds can effectively reduce the total iron content in bacteria, reduce the availability of bacterial iron, and disrupt bacterial iron homeostasis.

[0046] (3) The present invention uses an animal model to determine the antibiotic synergistic effect of the optimal compound, and determines the anti-biofilm effect and antibiotic synergistic effect of a 5,6-dihydroxypyrimidine compound described in the present invention on bacteria. The results show that the compound has good synergistic activity. It is expected to be further developed into an antibacterial synergist.

[0047] The present invention is further described below in conjunction with specific implementation modes. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Compound W13 interferes with the iron uptake system of Pseudomonas aeruginosa PAO1, leading to iron deficiency and a decrease in the total iron content in the bacteria.

[0049] Figure 2 Compound W13 enhances the activity of ciprofloxacin. (A) Bacterial survival rate in the infected area of ​​mouse wounds. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; (B) Monitoring of mouse wound area, and calculation of wound area using Image J. DETAILED DESCRIPTION

[0050] The present invention is further described below in conjunction with specific implementation modes.

[0051] Unless otherwise specified, the reagents used in the examples can be purchased from the market.

[0052] Example 1: Preparation of 5,6-bis(benzyloxy)-2-methylpyrimidine-4-carboxylic acid (Intermediate 3a-3c)

[0053] Step 1: Preparation of 5,6-dihydroxy-2-methylpyrimidine-4-carboxylic acid methyl ester (Intermediate 1)

[0054]

[0055] Dissolve N-hydroxyacetamidine (0.1 mol) in methanol, keep stirring in an ice-water bath, then dropwise add dimethyl butynedioate (0.11 mol), and continue the reaction for about 1 hour after the solvent is added. Terminate the reaction and remove the residual organic solvent in the reaction system. Then dissolve the obtained product in xylene and stir the reaction at 135°C for 16 hours. Terminate the reaction, then recrystallize with cold methyl tert-butyl ether / methanol solution (9:1), and filter under reduced pressure to obtain a light brown solid as intermediate 1 with a yield of 65%. 1 HNMR(400MHz,DMSO-d6)δ12.70(s,1H),10.12(s,1H),3.80(s,3H),2.20(s,3H). 13C NMR (101MHz, DMSO) δ166.75,159.42,148.06,145.29,128.94,52.57,20.88.

[0056] Step 2: Preparation of 5,6-bis(benzyloxy)-2-methylpyrimidine-4-carboxylic acid (Intermediate 3a)

[0057]

[0058] Place intermediate 1 (0.1 mol) in a 100 mL two-necked flask, dissolve it with an appropriate amount of anhydrous acetonitrile, and add benzyl bromide (0.24 mol) under stirring. Raise the temperature to 80°C and continue stirring the reaction for about 6 hours. Stop the reaction, then extract with 100 mL × 3 ethyl acetate and combine the organic phases, then wash with 100 mL × 3 saturated NH4Cl liquid and 100 mL × 3 saturated NaCl liquid until the outflowing water layer is neutral. After fully drying with MgSO4, filter and remove the solvent. The residue is purified by flash chromatography (petroleum ether-ethyl acetate = 80 / 20 v / v) to obtain a white solid as intermediate 2 with a yield of 60%. 1 H NMR (400MHz, Chloroform-d) δ7.45–7.40(m,2H),7.29(h,J=6.2Hz,6H),7.10–7.04(m,2H),5.29(s,2H),5.25(s,2H),3.84(s,3H),2.40(s,3H). 13 C NMR (101MHz,CDCl3)δ164.53,160.08,154.41,142.40,140.99,136.37,134.49,129.08,128.94,128.43,128.07,126.61,74.37,52.83,47.85,22.97.Intermediate 2 (0.1mol) was dissolved in methanol solution, LiOH (0.2mol) aqueous solution was added, stirred at 40℃ for 6h, and the reaction was stopped. The pH was adjusted to 5 with hydrochloric acid (6N), solid precipitated, and light brown solid intermediate 3a was obtained by suction filtration, with a yield of 70%. The preparation methods of intermediates 3b and 3c are the same as 3a.

[0059] Example 2: 5,6-dihydroxy-2-methyl-N-(4-phenylbutan-2-yl)pyrimidine-4-carboxamide (Compound W1)

[0060]

[0061] Intermediate 3a (1.0 mmol) was dissolved in tetrahydrofuran, and benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (PyBop, 1.5 mmol) and N,N-diisopropylethylamine (DIPEA, 2.0 mmol) were added, and then 4-phenyl-2-butylamine (1.2 mmol) was added to react for 4 h to terminate the reaction. It was extracted with 30 mL × 3 ethyl acetate, and washed with 30 mL × 3 saturated NaHCO3, 30 mL × 3 saturated NH4Cl and 100 mL × 3 saturated NaCl respectively until the outflowing water layer was neutral. The combined organic phases were fully dried with MgSO4 and then filtered to remove the solvent. The residue was purified by flash chromatography (petroleum ether-ethyl acetate = 60 / 40 v / v) to obtain a white solid as intermediate 4. Intermediate 4 was dissolved in methanol, 0.02 eq. of palladium carbon was added, and H2 was introduced. The reaction was stirred at room temperature for 6 h, the reaction was terminated, and the palladium carbon was removed by suction. After obtaining the filtrate, the solvent was removed and the product was recrystallized with petroleum ether-ethyl acetate. A white solid was obtained by suction filtration, namely compound W1. The yield was 58%. 1 HNMR (400MHz, DMSO-d6) δ12.55(d,J=20.2Hz,2H),8.49(d,J=8.8Hz,1H),7.21(dq,J=18.1,9.6,8 .6Hz,5H),4.01(s,1H),2.67–2.53(m,2H),2.26(s,3H),2.01–1.71(m,2H),1.19(d,J=6.6Hz,3H). 13 C NMR (101MHz, DMSO) δ168.37,158.57,148.22,147.70,142.05,128.72(2C),128.69(2C),127.28,126.17,44.98,37.44,32.47,20.98,20.72.

[0062] Example 3: Ethyl 2-(5,6-dihydroxy-2-methylpyrimidine-4-carboxamido)thiazole-4-carboxylate (Compound W2)

[0063] Using intermediate 3a (1.0 mmol) and ethyl 2-aminothiazole-4-carboxylate (1.2 mmol) as raw materials, the preparation method was similar to compound W1 to obtain a light brown solid as compound W2 with a yield of 61%. 1 H NMR (400MHz, DMSO-d6) δ13.66(d,J=541.8Hz,3H),8.10(s,1H),4.29(q,J=7.1Hz,2H),2.37(s,3H),1.30(q,J=5.7,4.4Hz,3H). 13C NMR (101MHz, DMSO) δ169.19,161.37,158.61,157.71,148.38,147.49,141.65,127.32,123.73,61.11,20.90,14.65.

[0064] Example 4: 5,6-dihydroxy-2-isopropyl-N-(2-(5-methoxy-1H-indol-3-yl)ethyl)pyrimidine-4-carboxamide (Compound W3)

[0065] Using intermediate 3b (1.0 mmol) and 5-methoxytryptamine (1.2 mmol) as raw materials, the preparation method was similar to compound W1 to obtain compound W3 as a white solid with a yield of 61%. 1 H NMR (400MHz, DMSO-d6) δ12.48(d,J=31.3Hz,2H),10.70(s,1H),8.67(s,1H),7.23(d,J=8.7Hz,1H),7.16(s,1H) ,7.08(s,1H),6.72(d,J=8.7Hz,1H),3.74(s,3H),3.56(s,2H),2.94(s,2H),2.75(s,1H),1.17(d,J=6.9Hz,6H). 13 CNMR(101MHz,DMSO)δ168.94,158.75,155.76,153.50,147.63,131.90,127.94 ,127.12,123.92,112.50,111.59,111.49,100.74,55.78,32.97,25.28,20.77.

[0066] Example 5: N-(cyclohexylmethyl)-5,6-dihydroxy-2-methylpyrimidine-4-carboxamide (Compound W4)

[0067] Using intermediate 3a (1.0 mmol) and cyclohexylmethylamine (1.2 mmol) as raw materials, the preparation method was similar to compound W1 to obtain compound W4 as a white solid with a yield of 69%. 1 H NMR (400MHz, DMSO-d6) δ12.54(d,J=22.1Hz,2H),8.71(t,J=6.4Hz,1H),3.11(t,J=6.7Hz,2H ),2.24(s,3H),1.63(q,J=15.4,12.7Hz,6H),1.16(t,J=11.4Hz,3H),0.90(q,J=11.3Hz,2H). 13C NMR (101MHz, DMSO) δ168.97,158.56,148.25,147.59,127.28,45.09,37.74,30.80(2C),26.44,25.77(2C),20.98.

[0068] Example 6: 5,6-dihydroxy-2-methyl-N-(pyridin-3-ylmethyl)pyrimidine-4-carboxamide (Compound W5)

[0069] Using intermediate 3a (1.0 mmol) and 3-aminomethylpyridine (1.2 mmol) as raw materials, the preparation method was similar to compound W1 to obtain compound W5 as a white solid with a yield of 63%. 1 H NMR (400MHz, DMSO-d6) δ12.58(d,2H),9.51(t,J=6.3Hz,1H),8.89–8.74(m,2H),8.43( dt,J=8.1,1.7Hz,1H),7.96(dd,J=8.1,5.5Hz,1H),4.64(d,J=6.3Hz,2H),2.26(s,3H). 13 C NMR (101MHz, DMSO) δ169.30,158.52,148.60,147.69,144.31,142.32,141.99,138.50,127.04,127.00,40.61,20.93.

[0070] Example 7: N-(4-fluorophenethyl)-5,6-dihydroxy-2-methylpyrimidine-4-carboxamide (Compound W6)

[0071] Using intermediate 3a (1.0 mmol) and 4-fluorophenethylamine (1.2 mmol) as raw materials, the preparation method was similar to compound W1 to obtain compound W6 as a white solid with a yield of 67%. 1 H NMR (400MHz, DMSO-d6) δ12.58(s,1H),12.40(s,J=3.4Hz,1H),8.89–8.72(m,1H),7.25(tq,J=7.8,4.8,3.6H z,2H),7.11(tp,J=10.0,3.5Hz,2H),3.53–3.42(m,2H),2.84(td,J=7.6,3.0Hz,2H),2.23(s,J=3.2Hz,3H). 13 C NMR(101MHz,DMSO)δ168.92,161.35(d, 1 J C-F=241.4Hz),158.52,148.29,147.60,135.60,130.90,130.82,127.21,115.65,115.44,40.59,34.35,20.98.

[0072] Example 8: N-(4-chlorophenethyl)-5,6-dihydroxy-2-phenylpyrimidine-4-carboxamide (Compound W7)

[0073] Using intermediate 3c (1.0 mmol) and 4-chlorophenethylamine (1.2 mmol) as raw materials, the preparation method was similar to compound W1 to obtain compound W7 as a white solid with a yield of 70%. 1 H NMR (400MHz, DMSO-d6) δ12.72(d,2H),9.08(s,1H),8.20(d,J=7.0Hz,2H),7.51(d,J=7.6Hz,3 H),7.37(d,J=7.9Hz,2H),7.30(d,J=8.1Hz,2H),3.55(q,J=7.1Hz,2H),2.90(t,J=7.4Hz,2H). 13 C NMR(101MHz,DMSO)δ168.94,159.17,148.42,146.54,138.52,131.37,131.02,12 9.82,128.91,128.83(2C),128.56(2C),127.88(2C),126.68(2C),40.61,34.60.

[0074] Example 9: 5,6-dihydroxy-N-(3-methoxybenzyl)-2-methylpyrimidine-4-carboxamide (Compound W8)

[0075] Using intermediate 3a (1.0 mmol) and 3-methoxybenzylamine (1.2 mmol) as raw materials, the preparation method was similar to compound W1 to obtain compound W8 as a white solid with a yield of 71%. 1 H NMR (400MHz, DMSO-d6) δ12.59(s,1H),12.32(s,1H),9.28(t,J=6.3Hz,1H),7.24(t ,J=8.0Hz,1H),6.93–6.78(m,3H),4.43(d,J=6.2Hz,2H),3.73(s,3H),2.25(s,3H). 13C NMR (101MHz, DMSO) δ169.01,159.74,158.53,148.38,147.68,140.61,129.91,127.30,120.10,113.85,112.71,55.46,42.57,20.99.

[0076] Example 10: 5,6-dihydroxy-N-(4-(2-hydroxyethyl)phenyl)-2-methylpyrimidine-4-carboxamide (Compound W9)

[0077] Using intermediate 3a (1.0 mmol) and 2-(4-aminophenyl)-1-ethanol (1.2 mmol) as raw materials, the preparation method was similar to compound W1 to obtain compound W9 as a white solid with a yield of 62%. 1 H NMR (400MHz, DMSO-d6) δ12.78(d,2H),10.39(s,1H),7.64(dd,J=8.8,2.4Hz,2H),7.21(dd,J=8. 7,2.4Hz,2H),5.31(s,1H),3.59(t,J=7.0Hz,2H),2.70(t,J=7.0Hz,2H),2.33(d,J=2.3Hz,3H). 13 C NMR (101MHz, DMSO) δ166.59,158.78,148.59,148.09,136.70,135.33,129.60(2C),126.92,121.46(2C),62.56,38.94,20.67.

[0078] Example 11: 5,6-dihydroxy-2-methylpyrimidine-4-carboxylic acid) tyrosine methyl ester (Compound W10)

[0079] Using intermediate 3a (1.0 mmol) and L-tyrosine methyl ester (1.2 mmol) as raw materials, the preparation method was similar to compound W1 to obtain compound W10 as a white solid with a yield of 55%. 1 H NMR (400MHz, DMSO-d6) δ12.20(d,2H),9.25(d,J=8.1Hz,1H),7.28(s,1H),6.99(d,J=8.0Hz,2H) ,6.66(d,J=7.9Hz,2H),4.69(q,J=7.3Hz,1H),3.66(s,3H),3.09(d,J=6.8Hz,2H),2.26(s,3H). 13C NMR (101MHz, DMSO) δ171.49,158.58,156.59,149.15,147.55,131.37,130.50(2C),127.08,126.06,115.69(2C),53.83,52.68,35.74,20.70.

[0080] Example 12: N-(2-(Benzo[d][1,3]dioxol-5-yl)ethyl)-5,6-dihydroxy-2-methylpyrimidine-4-carboxamide (Compound W11)

[0081] Using intermediate 3a (1.0 mmol) and piperonyl ethylamine (1.2 mmol) as raw materials, the preparation method was similar to compound W1 to obtain compound W11 as a white solid with a yield of 63%. 1 H NMR (400MHz, DMSO-d6) δ12.57(s,1H),12.37(s,1H),8.83–8.69(m,1H),6.93–6.74(m,2H),6.67(dt,J=7. 8,2.0Hz,1H),5.96(d,J=2.0Hz,2H),3.45(q,J=8.2,7.3Hz,2H),2.82–2.69(m,2H),2.23(d,J=2.2Hz,3H). 13 C NMR (101MHz, DMSO) δ168.88,158.53,148.30,147.71,147.59,146.08,133.21,127.22,121.97,109.42,108.64,101.16,40.75,34.89,20.99.

[0082] Example 13: N-(4-benzoylphenyl)-5,6-dihydroxy-2-isopropylpyrimidine-4-carboxamide (Compound W12)

[0083] Using intermediate 3b (1.0 mmol) and 4-aminobenzophenone (1.2 mmol) as raw materials, the preparation method was similar to compound W1 to obtain compound W12 as a white solid with a yield of 65%. 1 H NMR(400MHz,DMSO-d6)δ12.63(s,1H),11.77(s,1H),10.10(s,1H),7.64(d, J=8.4Hz,2H),7.31–7.17(m,7H),2.83(p,J=6.9Hz,1H),1.30–1.20(m,6H). 13C NMR (101MHz, DMSO) δ167.17,158.79,156.00,147.68,141.72,138.59,135. 12,129.45,129.11,128.90,127.52,126.45,122.07,33.20,21.07,20.74.

[0084] Example 14: N-(3-fluorobenzyl)-5,6-dihydroxy-2-methylpyrimidine-4-carboxamide (Compound W13)

[0085] Using intermediate 3a (1.0 mmol) and 3-fluorobenzylamine (1.2 mmol) as raw materials, the preparation method was similar to compound W1 to obtain a light brown solid as compound W13 with a yield of 71%. 1 H NMR (400MHz, DMSO-d6) δ12.60 (s, 1H), 12.23 (s, 1H), 9.38 (t, J = 6.4Hz, 1H), 7.3 8(td,J=7.9,6.2Hz,1H),7.19–7.04(m,3H),4.47(d,J=6.5Hz,2H),2.25(s,3H). 13 C NMR(101MHz,DMSO)δ169.15,162.61(d, 1 J C-F =243.6Hz),158.52,148.41,147.69,142.04(d, 3 J C-F =7.2Hz),130.79(d, 5 J C-F =8.4Hz),127.26,123.95(d, 4 J C-F =2.7Hz),114.65(d, 6 J C-F =21.7Hz),114.26(d, 2 J C-F =21.0Hz),42.17,21.00.

[0086] Example 15: 5,6-dihydroxy-2-methyl-N-(3-(trifluoromethyl)benzyl)pyrimidine-4-carboxamide (Compound W14)

[0087] Using intermediate 3a (1.0 mmol) and 3-trifluoromethylbenzylamine (1.2 mmol) as raw materials, the preparation method was similar to compound W1 to obtain compound W14 as a white solid with a yield of 77%. 1H NMR (400MHz, DMSO-d6) δ12.60(s,1H),12.20(s,1H),9.45(t,J=6.4Hz,1H),7.68(s,1 H),7.63(d,J=7.9Hz,2H),7.58(d,J=7.4Hz,1H),4.54(d,J=6.3Hz,2H),2.25(s,3H). 13 C NMR (101MHz, DMSO) δ169.23,158.51,148.45,147.71,140.60,132.20,129.93,129. 64,129.33,127.22,126.05,124.62,124.58,124.28,124.24,123.34,42.28,21.00.

[0088] Example 16: N-(2-fluorobenzyl)-5,6-dihydroxy-2-methylpyrimidine-4-carboxamide (Compound W15)

[0089] Using intermediate 3a (1.0 mmol) and 2-fluorobenzylamine (1.2 mmol) as raw materials, the preparation method was similar to compound W1 to obtain compound W15 as a white solid with a yield of 67%. 1 H NMR (400MHz, DMSO-d6) δ12.61 (s, 1H), 12.20 (s, 1H), 9.27 (t, J = 6.4Hz, 1H), 7.33 (dt,J=11.5,6.4Hz,2H),7.22–7.14(m,2H),4.52(d,J=6.2Hz,2H),2.25(s,3H). 13 C NMR(101MHz,DMSO)δ169.16,160.41(d, 1 J C-F =244.7Hz),158.52,148.45,147.66,129.95(d, 3 J C-F =4.2Hz),129.56(d, 5 J C-F =8.2Hz),127.22,125.61(d, 2 J C-F =14.6Hz),124.85(d, 4 J C-F =3.4Hz),115.60(d, 6 J C-F =21.2Hz),36.46(d,J=4.8Hz),21.00.

[0090] Example 17: N-(4-chlorobenzyl)-5,6-dihydroxy-2-methylpyrimidine-4-carboxamide (Compound W16)

[0091] Using intermediate 3a (1.0 mmol) and 4-chlorobenzylamine (1.2 mmol) as raw materials, the preparation method was similar to compound W1 to obtain compound W16 as a white solid with a yield of 69%. 1 H NMR (400MHz, DMSO-d6) δ12.41(d,J=90.6Hz,2H),9.37(t,J=6.4Hz,1H),7.45–7.27(m,4H),4.44(d,J=6.5Hz,2H),2.25(s,3H). 13 C NMR (101MHz, DMSO) δ169.10,158.52,148.39,147.72,138.14,132.05,129.87(2C),128.76(2C),127.23,42.01,20.98.

[0092] Example 18: N-(4-bromobenzyl)-5,6-dihydroxy-2-methylpyrimidine-4-carboxamide (Compound W17)

[0093] Using intermediate 3a (1.0 mmol) and 4-bromobenzylamine (1.2 mmol) as raw materials, the preparation method was similar to compound W1 to obtain compound W17 as a white solid with a yield of 68%. 1 H NMR (400MHz, DMSO-d6) δ12.60 (s, 1H), 12.26 (s, 1H), 9.37 (t, J = 6.5Hz, 1H), 7. 52(d,J=8.0Hz,2H),7.28(d,J=8.0Hz,2H),4.42(d,J=6.4Hz,2H),2.25(s,3H). 13 C NMR (101MHz, DMSO) δ169.12,158.51,148.41,147.71,138.56,131.68(2C),130.23(2C),127.24,120.53,42.07,21.00.

[0094] Example 19: N-(4-cyanobenzyl)-5,6-dihydroxy-2-methylpyrimidine-4-carboxamide (Compound W18)

[0095] Using intermediate 3a (1.0 mmol) and 4-cyanobenzylamine (1.2 mmol) as raw materials, the preparation method was similar to compound W1 to obtain compound W18 as a white solid with a yield of 58%. 1H NMR (400MHz, DMSO-d6) δ12.62(s,1H),12.16(s,1H),9.45(t,J=6.4Hz,1H),7. 80(d,J=7.9Hz,2H),7.50(d,J=7.9Hz,2H),4.53(d,J=6.3Hz,2H),2.26(s,3H). 13 C NMR (101MHz, DMSO) δ169.31,158.51,148.47,147.70,144.91,132.80(2C),128.72(2C),127.21,119.31,110.22,42.45,21.01.

[0096] Example 20: N-(3-chlorobenzyl)-5,6-dihydroxy-2-methylpyrimidine-4-carboxamide (Compound W19)

[0097] Using intermediate 3a (1.0 mmol) and 3-chlorobenzylamine (1.2 mmol) as raw materials, the preparation method was similar to compound W1 to obtain compound W20 as a white solid with a yield of 63%. 1 H NMR (400MHz, DMSO-d6) δ12.60 (s, 1H), 12.22 (d, J = 5.7Hz, 1H), 9.39 (t, J = 6.3Hz, 1H) ,7.33(ddd,J=20.9,10.4,5.0Hz,4H),4.46(d,J=6.0Hz,2H),2.25(d,J=5.4Hz,3H). 13 C NMR (101MHz, DMSO) δ169.16,158.51,148.42,147.71,141.66,133.41,130.72,127.82,127.45,127.24,126.69,42.15,21.00.

[0098] Example 21: 5,6-dihydroxy-N-(4-isopropylbenzyl)-2-methylpyrimidine-4-carboxamide (Compound W20)

[0099] Using intermediate 3a (1.0 mmol) and 4-isopropylbenzylamine (1.2 mmol) as raw materials, the preparation method was similar to compound W1 to obtain compound W22 as a white solid with a yield of 75%. 1H NMR (400MHz, DMSO-d6) δ12.59(s,1H),12.35(s,1H),9.26(t,J=6.4Hz,1H),7.27–7.15( m, 4H), 4.41 (d, J = 6.4Hz, 2H), 2.85 (p, J = 6.9Hz, 1H), 2.24 (s, 3H), 1.18 (d, J = 6.9Hz, 6H). 13 C NMR (101 MHz, DMSO) δ 168.92, 13 C NMR (101MHz, DMSO) δ168.92,167.17,158.53,148.35,147.69,136.45,128.09(2C),127.31,126.71(2C),42.37,33.59,24.38(2C),20.98.

[0100] Example 22: N-([1,1'-biphenyl]-4-ylmethyl)-5,6-dihydroxy-2-methylpyrimidine-4-carboxamide (Compound W21)

[0101] Using intermediate 3a (1.0 mmol) and 4-phenylbenzylamine (1.2 mmol) as raw materials, the preparation method was similar to compound W1 to obtain compound W23 as a white solid with a yield of 73%. 1 H NMR (400MHz, DMSO-d6) δ12.48(d,J=99.0Hz,2H),9.37(t,J=6.6Hz,1H),7.63(t,J=6.6 Hz,4H),7.48–7.40(m,4H),7.35(t,J=7.3Hz,1H),4.51(d,J=6.2Hz,2H),2.26(s,3H). 13 C NMR(101MHz,DMSO)δ169.07,158.54,148.40,147.72,140.40,139.47,138.30,12 9.37(2C),128.61(2C),127.82,127.60,127.15(2C),127.06(2C),42.37,20.99.

[0102] Example 23: N-(3,5-dichlorobenzyl)-5,6-dihydroxy-2-methylpyrimidine-4-carboxamide (Compound W22)

[0103] Using intermediate 3a (1.0 mmol) and 3,5-dichlorobenzylamine (1.2 mmol) as raw materials, the preparation method was similar to compound W1 to obtain a light brown solid as compound W24 with a yield of 69%. 1H NMR(400MHz,DMSO-d6)δ12.61(s,1H),12.24–12.00(m,1H),9.41(t,J=6.5Hz,1H) ,7.49(t,J=1.9Hz,1H),7.37(d,J=1.9Hz,2H),4.45(d,J=6.2Hz,2H),2.26(s,3H). 13 C NMR (101MHz, DMSO) δ169.26,158.50,148.46,147.70,143.46,134.40(2C),127.21,127.15,126.80(2C),41.81,21.01.

[0104] Example 24: Inhibitory activity of compounds against Pseudomonas aeruginosa biofilm

[0105] Experimental method: First, prepare the compound premix with cell-grade DMSO, and then dilute it with ABTGC medium to a certain concentration for use. Pseudomonas aeruginosa PAO1 strain was diluted with ABTGC medium to OD 600 =0.05, and added to a 96-well plate, each well containing 75 μL of medium containing the compound and 75 μL of diluted bacterial solution. At the same time, a blank group (medium only) and a control group (bacteria only and positive control group) were set up, with azithromycin as the positive control drug, and 6 replicate wells were set up for each group of data. The plate was placed in an incubator at 37°C for 24 hours, and then each well was washed 3 times with PBS to remove planktonic bacteria. Next, the 96-well plate was fixed with 160 μL of methanol at room temperature for 30 minutes, then evacuated and dried. Then it was stained with 150 μL of 0.1% crystal violet for 30 minutes, blotted dry, washed with PBS buffer, and dried. Then 150 μL of 33% glacial acetic acid was added to each well to dissolve the crystal violet, and the plate was placed on a microshaker and shaken thoroughly, and the absorbance was measured at a wavelength of 570 nm using an enzyme reader. All experiments were performed in triplicate to screen out compounds with biofilm inhibitory activity. The results of the biofilm inhibitory activity experiment are shown in Table 1.

[0106] Calculation method of biofilm inhibition rate:

[0107] Biofilm inhibition rate = [(control group OD值 -Blank Group OD值 )-(Drug sensitivity group OD值 -blank OD值 )] / (control group OD值 -Blank Group OD值 )×100%. IC of the compound 50 Greater than 10 μM was marked as +; between 10 μM and 1 μM was marked as ++; between 1 μM and 0.1 μM was marked as +++.

[0108] Table 1 Inhibitory effects of all compounds on Pseudomonas aeruginosa biofilm

[0109]

[0110] a IC 50 = inhibitor concentration required to inhibit 50% biofilm formation. b IC of most compounds 50 The screening concentration is 0.001~10μM.

[0111] Experimental results: Most of the 5,6-dihydroxypyrimidine compounds described in the present invention can inhibit the formation of biofilm at a relatively low concentration. The IC 50 Less than 1μM, better than the IC of the positive control drug azithromycin 50 Among them, compound W13 had the best activity, inhibiting the biofilm IC 50 0.1μM.

[0112] Example 25: Inhibitory activity of compound W13 on total iron content in Pseudomonas aeruginosa

[0113] Experimental method: In this example, a microplate reader was used to detect the total iron content of bacteria. PAO1 was incubated overnight and then diluted to OD 600 The experimental group and the control group (bacteria only and positive control group) were set up. In the experimental group, the compound premix prepared with cell-grade DMSO was added to the diluted bacterial solution, and the samples were incubated at 37°C with shaking (200 rpm) for 24 hours. Commercially available deferiprone (DFP) was used as a positive control drug. Deferiprone is an orally absorbed iron chelator that has been marketed and has a good iron chelation effect. Subsequently, each group of bacterial cultures was centrifuged at 4000 rpm for 10 min, the precipitate was washed twice with 5 mL of HEPES buffer solution (Biosharp, China), and then centrifuged at 4000 rpm for 10 min. 5 mL (50 mM) of HEPES buffer solution was added to the precipitate, shaken well, 800 μL of bacterial suspension was aspirated, 200 μL of lysozyme was added to it, vortexed to mix, placed on an ice box for lysis for 10 min, and then centrifuged at 15000 rpm for 10 min. Take 80 μL of supernatant into the corresponding well of the ELISA plate, and then add 80 μL of reagent III reaction solution. Incubate at 37°C for 40 min, and measure the OD value of each well at 593 nm on an ELISA reader.

[0114] Experimental results: Figure 1 As shown, compared with the commercially available iron chelator DFP, compound W13 can significantly reduce the total iron content in bacteria at low concentrations, indicating that the compound can effectively inhibit bacterial iron uptake and reduce the availability of iron in bacteria, and is expected to be used as an anti-bacterial iron uptake drug.

[0115] Example 26: Study on the synergistic effect of compound W13 on ciprofloxacin (CIP) in a mouse wound infection model

[0116] Experimental method: 3-5 week old healthy female balb / c mice were purchased from SPF (Beijing) Biotechnology Co., Ltd. (SPFBeijing Biotechnology.co.Ltd.). This animal experiment was carried out in accordance with the national animal experiment management regulations. Compound W13 was mixed with 0.1% Tween 80 and saline to the required concentration, and antibiotics (ciprofloxacin) were prepared in saline. First, 30 mice (5 per group) were anesthetized with 4% chloral hydrate (intraperitoneal injection, 0.15 mL), and the back hair was shaved. A wound of about 5 mm was made on the back of the mouse, and then 30 μL PAO1 (5×10 8 CFU, prepared with 0.9% saline solution) was inoculated on the wound surface. After 24 hours of establishing the wound infection model, the wound was treated with 30 μL of normal saline, antibiotics alone, or antibiotics combined with compound W13, and the medication was given once every 24 hours. After 3 consecutive days of administration, the mice were killed with isoflurane. Next, the skin with a diameter of about 7 mm at the wound was taken, placed in a centrifuge tube containing 4 mm grinding beads and 1 mL of normal saline, homogenized with a grinder, and then CFU counted on an agar plate. The wounds of the mice were monitored by photography every day, and the wound area was calculated by ImageJ.

[0117] Experimental results: Figure 2 As shown in the figure, after three days of continuous administration of drugs to mice, the bacteria in the wounds of mice in the combined drug administration group were significantly reduced compared with those in the saline group (Control). By calculating the bacterial survival rate in the wounds, the bacterial survival rate in the saline group was 100%, and after three days of treatment with 1mg / ml CIP, the bacteria were basically completely eliminated. After the combined drug treatment of 0.002mg / ml CIP+0.5μM W13, very few bacteria survived, indicating that the synergistic effect of W13 on CIP reached 500 times. In the combined drug group of 0.001mg / ml CIP+0.5μM W13, the bacterial survival rate was relatively low compared with the Control group, only about 1%. Subsequently, the monitoring of the wound area of ​​mice also obtained consistent experimental results. After three days of combined administration of W13 and diluted CIP, the wound area was basically the same as that of the CIP group alone, and both were much smaller than the wound area of ​​the Control group. On the 9th day, the degree of wound healing reached about 80%.

[0118] The above embodiments are only used to help understand the method and core idea of ​​the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A 5,6-dihydroxypyrimidine compound or a pharmaceutically acceptable salt, medically acceptable variant isomer, mesomorph, racemate, stereoisomer, metabolite, metabolic precursor, prodrug or solvate thereof, characterized in that: It has a chemical structure as shown in Formula I: Wherein R1 is selected from one of C1-C4 straight chain or branched saturated or unsaturated hydrocarbon groups, aryl groups, benzyl groups, and 5-6 membered heteroaryl groups; R2 is selected from C3-C6 straight chain or branched saturated or unsaturated hydrocarbon group, C3-C6 cyclic hydrocarbon group, 3-6 membered heterocyclic group, aryl group, One of arylethyl, arylsulfonyl, 5- to 6-membered heteroaryl, and 5- to 6-membered heteroarylmethyl; R3 is selected from halogen, hydroxyl, amino, cyano, C1-C3 straight chain or branched saturated or unsaturated hydrocarbon group, C1-C3 straight chain or branched saturated or unsaturated hydrocarbonoxy group, trifluoromethyl, trifluoromethoxy, aryl, aryloxy group.

2. The 5,6-dihydroxypyrimidine compound or a pharmaceutically acceptable salt, medically acceptable variant isomer, mesomorph, racemate, stereoisomer, metabolite, metabolic precursor, prodrug or solvate thereof according to claim 1, characterized in that: The compound is selected from:

3. The 5,6-dihydroxypyrimidine compound or a pharmaceutically acceptable salt, medically acceptable variant isomer, meso-body, racemate, stereoisomer, metabolite, metabolic precursor, prodrug or solvate thereof according to claim 1 or 2, characterized in that: The pharmaceutically acceptable salt of the 5,6-dihydroxypyrimidine compound is an acid addition salt or a base addition salt of the 5,6-dihydroxypyrimidine compound, including but not limited to: hydrochloride, hydrobromide, hydroiodide, phosphate, sulfate, nitrate, ethanesulfonate, toluenesulfonate, benzenesulfonate, acetate, maleate, tartrate, succinate, citrate, benzoate, ascorbate and salicylate, malonate, adipate, caproate, arginine salt, fumarate, nicotinate, phthalate, oxalate, lithium salt, sodium salt, potassium salt, barium salt, calcium salt, magnesium salt, aluminum salt, iron salt, ferrous salt, copper salt, zinc salt, or any one or a combination of at least two of the salts of the 5,6-dihydroxypyrimidine compound and morpholine, diethylamine, triethylamine, isopropylamine, trimethylamine, lysine or histidine.

4. A method for preparing the 5,6-dihydroxypyrimidine compound according to claim 1 or 2, characterized in that The following steps are involved: Step (1.1) dissolving N-hydroxyacetamidine or N-hydroxyisobutylamidine or N-hydroxybenzamidine and dimethyl butynedioate in methanol, reacting at 0°C, then removing methanol and refluxing in xylene for reaction, and purifying the reaction solution obtained after the reaction to obtain compound 1; Step (1.2) Compound 1, benzyl bromide and potassium carbonate are placed in an acetonitrile solvent and reacted at 80° C. The obtained reaction solution is purified to obtain Compound 2; Step (1.3) Compound 2 and lithium hydroxide aqueous solution are placed in a methanol solvent for reaction at 50° C. The obtained reaction solution is purified to obtain Compound 3; Step (1.4) Compound 3, an amine compound, benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (PyBop) and N,N-diisopropylethylamine (DIPEA) are reacted in tetrahydrofuran (THF) solvent at room temperature, and the obtained reaction solution is purified to obtain Compound 4; Step (1.5) Compound 4 is dissolved in methanol, a palladium-carbon catalyst is added, and then hydrogen is introduced to react at room temperature. The obtained reaction solution is purified to obtain compound 5; The synthetic route is:

5. Use of the 5,6-dihydroxypyrimidine compound or its pharmaceutically acceptable salt, medically acceptable variant isomer, mesomorph, racemate, stereoisomer, metabolite, metabolic precursor, prodrug or solvate according to any one of claims 1 to 3 in the preparation of anti-biofilm drugs, antibacterial iron homeostasis drugs or antibiotic synergists.

6. The use according to claim 5, characterized in that: The medicament further comprises one or more pharmaceutically acceptable carriers or excipients.

7. The use according to claim 5, wherein the bacterium is Pseudomonas aeruginosa.

8. A pharmaceutical composition comprising at least one of the 5,6-dihydroxypyrimidine compounds according to any one of claims 1 to 3 or a pharmaceutically acceptable salt, medically acceptable variant isomer, mesomorph, racemate, stereoisomer, metabolite, metabolic precursor, prodrug or solvate thereof, and further comprising one or more pharmaceutically acceptable carriers or excipients.

9. A combination drug comprising at least one of the 5,6-dihydroxypyrimidine compound or a pharmaceutically acceptable salt, medically acceptable variant isomer, mesomorph, racemate, stereoisomer, metabolite, metabolic precursor, prodrug or solvate thereof according to any one of claims 1 to 3, and ciprofloxacin.

10. The combination drug according to claim 9, wherein the mass ratio of the 5,6-dihydroxypyrimidine compound or its pharmaceutically acceptable salt, medically acceptable variant, mesomorph, racemate, stereoisomer, metabolite, metabolic precursor, prodrug or solvate to ciprofloxacin is 50:0.5-20, preferably 50:1-10.

Images