CN112679456B - Kdo-based glycolipid derivatives, their preparation and their antibacterial applications - Google Patents
Kdo-based glycolipid derivatives, their preparation and their antibacterial applications Download PDFInfo
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Abstract
The invention discloses a Kdo-based glycolipid derivative, and preparation and antibacterial application thereof, wherein the structural formula of the derivative is as follows:
in the formula R 1 Is an aliphatic amino group (i.e. CH) 3 (CH 2 ) n NH‑，n＝1～17)，R 2 ＝‑NH 3 + Cl ‑ (ii) a Or R 1 is-OH or-O ‑ NH 4 + ，R 2 Is RCONH- (R is C) 1～18 Saturated or unsaturated alkyl groups of (ii). Compared with the reported glycolipid, the glycolipid disclosed by the invention is an unnatural glycolipid compound prepared based on an important monosaccharide Kdo (3-deoxy-D-manno-2-octulosonic acid) from gram-negative bacteria, and the compound can be used as a novel antibacterial substance.
Description
Technical Field
The invention belongs to the technical field of synthesis of glycolipid compounds, and particularly relates to a glycolipid derivative taking a scarce monosaccharide Kdo (3-deoxy-D-mannose-2-octulosonic acid) in organisms such as gram-negative bacteria, plants, algae and the like as a framework. The compounds can be used as potential novel antibacterial compounds, so that the compounds are used for inhibiting the growth of gram-positive bacteria and gram-negative bacteria.
Background
The natural glycolipid has the advantages of low toxicity, degradability, reproducibility, environmental friendliness and the like, and the natural glycolipid represented by the sophorolipid, the trehalose glycolipid and the rhamnolipid can reduce the surface and interface tension, promote the formation of detergent foam and the wetting capacity, and is widely applied to cosmetics, medicines, environmental protection and energy-saving technologies. Importantly, glycolipids also induce the formation of channels or ion channels in cell membranes, destroying membrane integrity and permeability. Thus, glycolipids also have antibacterial, antifungal, viral and mycoplasma action. In addition, glycolipids can also be used to modulate the activity of enzymes and to increase or inhibit the activity of enzymes (carbohydrate res.2015,416, 59.).
Nevertheless, the natural glycolipids have microscopic heterogeneity, and the research on physicochemical properties and functions of natural glycolipids with definite structures has great limitation, and the research is mainly focused on the research on some common glycolipids such as sophorolipids. Studies based on chemically synthesized glycolipids have also been performed only on the basis of simple glucose, mannose, lactose, etc. (chem. Rev.2016,116, 1693.).
Kdo, 3-deoxy-D-manno-2-octulosonic acid, is an important component of the outermost lipopolysaccharide layer of gram-negative bacteria. Introduction of Kdo is a critical step in lipopolysaccharide synthesis during lipopolysaccharide synthesis. Meanwhile, a large number of studies have shown that two derivatives of Kdo (2-deoxy- β -Kdo and 8-amino-2, 8-dideoxy- β -Kdo) have very good inhibitory activity on enzymes critical for lipopolysaccharide synthesis in vitro tests. However, due to the strong water solubility, the compounds are difficult to penetrate cell membranes to reach the cells for inhibitory activity. To solve this problem, there are several groups that hopefully screen compounds with good inhibitory effect on gram-negative bacteria by derivatization for Kdo (nat. Prod. Rep.2010,27, 1618-1629). For example, claesson et al introduced amino acids and dipeptides at position 8 of 8-amino-2, 8-dideoxy-. Beta. -Kdo, wherein dipeptide derivatives introduced through the N-terminus showed better inhibitory effects against several gram-negative bacteria (J.Med.chem.1987, 30, 2309-2313). In addition, other terminal Kdo derivatives (Carbohydr. Res.1987,166, 233-251) or 8-position Kdo derivatives have been used for inhibition assays of key enzymes in lipopolysaccharide synthesis. However, even compounds having inhibitory activity against enzymes do not necessarily achieve growth inhibition of bacteria (J.Med.chem.1989, 32, 1069-1074.).
Disclosure of Invention
The invention aims to provide a kind of glycolipid derivative based on Kdo, and a preparation method and application of the glycolipid derivative.
For the above purpose, the structural formula of the Kdo-based glycolipid derivative employed in the present invention is shown below:
in the formula, R 1 Is an aliphatic amino group, R 2 is-NH 3 + Cl - The aliphatic amino group is CH 3 (CH 2 ) n NH-, n is an integer of 1 to 17; or R 1 is-OHor-O - NH 4 + ，R 2 Is RCONH-, wherein R is C 1～18 Saturated or unsaturated alkyl groups.
When R in the above formula is 1 Is an aliphatic amino group, R 2 is-NH 3 + Cl - The method for preparing the glycolipid derivative comprises the following steps:
1. taking methanol or water as a reaction medium, reacting the compound 1 and alkali according to a molar ratio of 1-10 at 0-50 ℃ for 1-24 hours, and separating and purifying a product to obtain a compound 2, wherein the reaction equation is as follows:
2. reacting compound 2 with an aliphatic amine (CH) 3 (CH 2 ) n NH 2 Wherein N is an integer of 1 to 17), a condensing agent and N, N-Diisopropylethylamine (DIPEA) are added into N, N-Dimethylformamide (DMF) according to the molar ratio of 1 to 2, 1.5 to 2.5, and the mixture is reacted at 0 to 40 ℃ for 3 to 24 hours, and a product is separated and purified to obtain a compound 3, wherein the reaction equation is as follows:
3. dissolving a compound 3 in a polar solvent, adding palladium carbon, introducing hydrogen, stirring at room temperature for 1-12 hours to obtain a reduction product, filtering to remove the palladium carbon, and acidifying the obtained solution with an aqueous solution of hydrogen chloride to obtain a target compound I, namely the glycolipid derivative, wherein the reaction equation is as follows:
when R in the above structural formula 1 is-OH or-O - NH 4 + ，R 2 Is RCONH-, wherein R is C 1～18 Saturated or unsaturated alkyl groups, the glycolipid derivatives being prepared by a process such asThe following:
1. dissolving the compound 1 and organic phosphine in a molar ratio of 1-5 in a mixed solution of tetrahydrofuran and water, reacting at 40-90 ℃ for 3-24 hours, cooling the reaction solution to room temperature, concentrating to remove the solvent, and performing column chromatography to obtain a compound 4, wherein the reaction equation is as follows:
2. adding the compound 4, aliphatic carboxylic acid, a condensing agent and N, N-Diisopropylethylamine (DIPEA) into DMF according to the molar ratio of 1-2; or adding the compound 4, pentafluorophenol ester and N, N-Diisopropylethylamine (DIPEA) into DMF according to the mol ratio of 1 to 2. The reaction equation is as follows:
the structural formulas RCOOH and pentafluorophenol ester of the above aliphatic carboxylic acid
In which R is C 1～18 Saturated or unsaturated alkyl groups.
3. Dissolving a compound 5 in a mixed solution of dichloromethane and methanol, adding an aqueous alkali solution into the mixed solution under stirring at room temperature, wherein the molar ratio of the compound 5 to the alkali is 1-8, reacting for 3-6 hours under stirring at room temperature, neutralizing a product with acidic ion exchange resin after the reaction is finished, and filtering and concentrating to obtain a target compound II. The target compound II can be further treated by ammonia water and concentrated to obtain the ammonium salt thereof, namely the target compound III, and the specific reaction equation is as follows:
in the above preparation method, the alkali is specifically sodium hydroxide, lithium hydroxide, etc.; the condensing agent is benzotriazole-1-yl-oxy tripyrrolidinyl phosphorus hexafluorophosphate (PyBOP) or a mixture of 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride (EDCI) and 1-hydroxybenzotriazole (HOBt) in a molar ratio of 1-2; the organic phosphine is specifically trimethyl phosphine or triphenyl phosphine; the polar solvent is specifically alcohol solvent such as ethanol and methanol.
The glycolipid derivative can be prepared into an antibacterial material for inhibiting the growth of gram-positive bacteria and gram-negative bacteria.
The invention has the following beneficial effects:
the invention is based on that the glycolipid compound can realize antibacterial effect by physically damaging the structure of a bacterial membrane, the Kdo derivative has inhibitory activity on key enzyme of gram-negative bacteria, the membrane penetrating capacity of the Kdo derivative is improved by introducing an alkyl chain on an 8-amino-2, 8-dideoxy-beta-Kdo skeleton, and the obtained glycolipid derivative, particularly a cationic compound, has good to medium inhibitory effect on gram-positive bacteria or gram-negative bacteria.
Drawings
FIG. 1 is a nuclear magnetic hydrogen spectrum of the final product in example 1.
FIG. 2 is a nuclear magnetic hydrogen spectrum of the final product in example 2.
FIG. 3 is a nuclear magnetic hydrogen spectrum of the final product in example 3.
FIG. 4 is a nuclear magnetic hydrogen spectrum of the final product in example 4.
FIG. 5 is a nuclear magnetic hydrogen spectrum of the final product in example 5.
FIG. 6 is a graph comparing the bacteriostatic effects of the final product of example 3 on E.coli and P.aeruginosa.
Detailed Description
The invention will be further described in detail with reference to the following figures and examples, but the scope of the invention is not limited to these examples.
Example 1
1. 100mg (0.36 mmol) of compound 1 was dissolved in 1mL of methanol, 3.6mL of 0.5mol/L aqueous lithium hydroxide solution was added dropwise thereto with stirring at room temperature, the resulting mixture was stirred at room temperature for 2 hours, neutralized to neutrality with an acidic ion exchange resin, filtered and concentrated to obtain compound 2 (87mg, 0.36mmol, yield 100%), and the resulting crude product was used directly in the subsequent reaction.
2. 87mg (0.36 mmol) of Compound 2, 116mg (0.43 mmol) of stearyl amine were dissolved in 4mL of DMF, 89mg (0.46 mmol) of EDCI, 63mg (0.47 mmol) of HOBt and 108. Mu.L (0.62 mmol) of DIPEA were added to the solution successively with stirring at room temperature, and after stirring at room temperature for 8.5 hours, the solvent was removed by concentration, and the resulting syrup was taken up in CH 2 Cl 2 Dissolving and then adding H 2 O-wash, collect the organic phase and add Na 2 SO 4 Drying, filtering, concentrating, and performing column Chromatography (CH) 2 Cl 2 MeOH =10, 1,v/v) yielded compound 3-1 (80mg, 0.16mmol, yield 44%).
The structural characterization data of the obtained compound 3-1 are: 1 H NMR(400MHz,DMSO-d 6 )δ7.41(t,J＝5.8Hz,1H),5.28(d,J＝6.2Hz,1H),4.69(d,J＝5.4Hz,1H),4.34(d,J＝4.7Hz,1H),4.25(d,J＝6.2Hz,1H),3.87(d,J＝7.0Hz,1H),3.71(d,J＝4.3Hz,1H),3.57(dd,J＝12.8,2.3Hz,1H),3.28–3.14(m,3H),3.00(dd,J＝12.8,6.4Hz,1H),2.01(dd,J＝12.4,4.6Hz,1H),1.81(td,J＝12.1,6.5Hz,1H),1.42(s,2H),1.23(s,30H),0.85(t,J＝6.6Hz,3H)； 13 C NMR(100MHz,DMSO)δ170.01,73.08,67.97,65.89,65.80,54.05,38.49,31.27,29.08,29.00,28.93,28.74,28.68,27.03,26.39,22.08,13.94；MALDI-TOF MS:[M+Na] + theoretical value 521.3679, calculated value 521.3854.
3. 80mg (0.16 mmol) of the compound 3-1 was dissolved in 5mL of methanol, 17mg 10% of Pd/C was added to the solution with stirring, oxygen was removed under reduced pressure, hydrogen was introduced into the reaction flask and the reaction was stirred at room temperature for 12 hours. After completion of the reaction, pd/C was removed by filtration, and 0.5mL of 1mol/L HCl aqueous solution was added to the filtrate, followed by concentration to obtain Compound I-1 (31mg, 0.06mmol, yield 32%) as a white solid.
The structural characterization data of the obtained compound I-1 are: 1 H NMR(400MHz,DMSO-d 6 ) δ 8.05 (t, J =5.8hz, 2h), 7.88 (t, J =5.8hz, 1h), 4.30 (dd, J =6.0,1.5hz, 1h), 3.86 (td, J =7.8,3.0hz, 1h), 3.72 (d, J =2.8hz, 1h), 3.44 (ddd, J =11.7,4.8,2.8hz, 1h), 3.23 (dd, J =7.7,1.0hz, 1h), 3.15-3.06 (m, 1H), 3.06-2.97 (m, 1H), 2.82-2.69 (m, 1H), 2.03-1.81 (m, 2H), 1.39 (t, J =6.9hz, 2h), 1.22 (s, 30H), 0.87 (m, 0.87H), see fig. 1.3.8 (m, 1H); 13 C NMR(100MHz,DMSO-d 6 )δ170.84,76.40,72.61,65.97,65.81,65.59,42.81,38.62,31.33,29.15,29.10,29.08,29.05,28.81,28.75,28.15,26.47,22.12,13.95；MALDI-TOF MS:[M+H] + the theoretical value is 473.3949, and the actual value is 473.3700.
Example 2
1. Dissolving 200mg (0.73 mmol) of compound 1 in 7mL of a mixed solution of tetrahydrofuran and deionized water in a volume ratio of 6, adding 526mg (1.78 mmol) of triphenylphosphine, and heating the mixture to 60 ℃ for reaction for 7h; after the reaction mixture was cooled to room temperature, the solvent was removed by concentration, the resulting syrup was dissolved in deionized water and washed three times with ethyl acetate, and the aqueous phase was concentrated to give compound 4 as a white solid (172mg, 0.68mmol, yield 96%).
The structural characterization data for compound 4 obtained are: 1 H NMR(400MHz,Methanol-d 4 )δ4.61–4.53(m,1H),4.30–4.18(m,2H),3.94(d,J＝2.5Hz,1H),3.93–3.87(m,1H),3.64–3.54(m,1H),3.49(dd,J＝8.5,0.8Hz,1H),3.01(dd,J＝13.4,3.4Hz,1H),2.93(dd,J＝13.4,5.7Hz,1H),2.20–2.09(m,2H),1.30(t,J＝7.1Hz,3H).
2. 132mg (0.53 mmol) of Compound 4, 305mg (0.68 mmol) of pentafluorophenol octadecanecarboxylic acid ester, and 111. Mu.L (0.64 mmol) of DIPEA were added to 3mL of DMF, and reacted at 50 ℃ for 8 hours; cooling the solution to room temperature, concentrating under reduced pressure to remove the solvent, and separating by column Chromatography (CH) 2 Cl 2 MeOH =10, 1,v/v) yielded compound 5-1 (113mg, 0.22mmol, yield 32%).
The structural characterization data of the obtained compound 5-1 are: 1 H NMR(400MHz,Chloroform-d)δ7.09(dd,J＝7.8,4.5Hz,1H),4.53(d,J＝6.24Hz,1H),4.22(q,J＝7.1Hz,2H),4.05(s,1H),4.00–3.85(m,2H),3.62(ddt,J＝12.2,5.4,2.5Hz,1H),3.44(d,J＝8.9Hz,1H),3.26(dt,J＝14.5,4.2Hz,1H),2.36–2.12(m,4H),1.69–1.54(m,2H),1.36–1.12(m,33H),0.93–0.83(m,3H)； 13 C NMR(100MHz,CDCl 3 )δ176.55,172.11,74.81,72.42,68.83,66.62,66.20,61.57,42.92,36.49,31.90,29.70,29.69,29.68,29.64,29.55,29.36,29.34,29.32,29.01,25.70,22.67,14.18,14.10；MALDI-TOF MS:[M+Na] + the theoretical value is 538.3720, and the actual value is 538.3184.
3. 75mg (0.14 mmol) of compound 5-1 was dissolved in 3mL of a mixed solution of dichloromethane and methanol in a volume ratio of 1.
The structural characterization data of the obtained compound II-1 are: 1 h NMR (400mhz, dmso-d 6) δ 4.36 (d, J =5.3hz, 1h), 3.72 (d, J =2.8hz, 1h), 3.68-3.60 (m, 3H), 3.38 (ddd, J =11.2,5.8,2.7hz, 1h), 3.26 (d, J =8.7hz, 1h), 2.88-2.78 (m, 1H), 2.08 (t, J =7.4hz, 2h), 2.00-1.84 (m, 2H), 1.46 (p, J =7.2hz, 2h), 1.21 (s, 26H), 0.89-0.78 (m, 3H), see fig. 12； 13 C NMR(100MHz,DMSO)δ172.50,75.62,72.01,67.26,66.61,65.93,42.77,35.53,31.35,29.11,29.06,28.95,28.77,25.40,22.15,14.01；MALDI-TOF MS:[M-H] - Theoretical value is 486.3436, and actual value is 486.8383.
And adding excessive ammonia water/MeOH solution into the compound II-1, stirring for 10min, and spin-drying to obtain ammonium carboxylate, namely the target compound III-1.
Example 3
1. 396mg (1.44 mmol) of the compound 1 was dissolved in 6mL of methanol, 13.34mL of a 0.5mol/L aqueous solution of lithium hydroxide was added dropwise thereto with stirring at room temperature, the resulting mixture was stirred at room temperature for 4 hours, neutralized to neutrality with an acidic ion exchange resin, filtered and concentrated to obtain a compound 2 (340mg, 1.38mmol, yield 96%) and the resulting crude product was used directly in the subsequent reaction.
2. 184mg (0.73 mmol) of Compound 2, 173mg (0.81 mmol) of saturated decatetramine were dissolved in 4mL of DMF, 172mg (0.90 mmol) of EDCI, 118mg (0.56 mmol) of HOBt and 300. Mu.L (1.82 mmol) of DIPEA were added to the solution in this order with stirring at room temperature, and after stirring at room temperature for 12 hours, the solvent was removed by concentration, and the resulting syrup was taken up in CH 2 Cl 2 Dissolving and then adding H 2 O-wash, collect the organic phase and add Na 2 SO 4 Drying, filtering, concentrating, and performing column Chromatography (CH) 2 Cl 2 MeOH =10, 1,v/v) yielded compound 3-2 (206mg, 0.47mmol, yield 64%).
The structural characterization data of the obtained compound 3-2 are: 1 H NMR(400MHz,DMSO-d 6 )δ7.41(t,J＝5.7Hz,1H),5.29(d,J＝6.1Hz,1H),4.70(d,J＝5.4Hz,1H),4.35(d,J＝4.6Hz,1H),4.25(d,J＝6.1Hz,1H),3.93–3.83(m,1H),3.71(s,1H),3.56(dd,J＝12.8,2.2Hz,1H),3.46–3.37(m,1H),3.30–3.14(m,2H),3.00(dq,J＝12.5,6.7Hz,1H),2.02(dt,J＝12.4,4.4Hz,1H),1.81(td,J＝12.3,6.5Hz,1H),1.48–1.34(m,2H),1.23(s,22H),0.89–0.81(m,3H)； 13 C NMR(100MHz,DMSO-d 6 )δ170.05,75.12,73.12,67.99,65.92,65.83,54.06,38.53,31.32,29.11,29.08,29.05,29.02,28.97,28.78,28.73,27.04,26.43,22.11,13.95；MALDI-TOF MS:[M+Na] + the theoretical value is 465.3053, and the actual value is 465.2897.
3. 192mg (0.38 mmol) of Compound 3-2 was dissolved in 10mL of methanol, and 158mg 10% of Pd/C was added to the solution under stirring, and after removing oxygen under reduced pressure, hydrogen gas was introduced and the solution was stirred at room temperature for 12 hours. After completion of the reaction, pd/C was removed by filtration, and 1mL of 1mol/L aqueous HCl solution was added to the filtrate to concentrate the mixture to obtain Compound I-2 (170mg, 0.038mmol, yield 100%) as a white solid.
The structural characterization data of the obtained compound I-2 are: 1 H NMR(400MHz,DMSO-d 6 ) δ 7.99 (s, 2H), 7.87 (t, J =5.7hz, 1h), 4.32 (d, J =5.3hz, 1h), 4.18-4.04 (m, 1H), 3.87 (td, J =7.7,2.9hz, 1h), 3.73 (s, 1H), 3.45 (ddd, J =11.8,4.8,2.8hz, 1h), 3.23 (d, J =7.9hz, 1h), 3.19-2.97 (m, 2H), 2.83-2.72 (m, 1H), 2.06-1.83 (m, 2H), 1.45-1.33 (m, 2H), 1.23 (s, 22H), 0.87-0.83 (t, J =6.9, 3h), see fig. 3; 13 C NMR(100MHz,DMSO-d 6 )δ170.87,76.45,72.62,65.97,65.83,65.60,42.81,38.62,31.33,29.15,29.10,29.06,28.80,28.75,28.18,26.47,22.13,13.98；MALDI-TOF MS:[M+H] + the theoretical value is 417.3323, and the actual value is 417.3350.
Example 4
1. 200mg (0.73 mmol) of compound 1 was dissolved in 14mL of a mixed solution of tetrahydrofuran and deionized water in a volume ratio of 6, 300mg (1.14 mmol) of triphenylphosphine was added, and the resulting mixture was heated to 60 ℃ for reaction for 12 hours; after the reaction mixture was cooled to room temperature, the solvent was removed by concentration, the resulting syrup was dissolved in deionized water and washed three times with ethyl acetate, and the aqueous phase was concentrated to give compound 4 as a white solid (152mg, 0.61mmol, yield 84%).
2. 100mg (0.40 mmol) of Compound 4, 271mg (0.69 mmol) of pentafluorophenol tetradecylcarboxylate, and 111. Mu.L (0.64 mmol) of DIPEA were added to 3mL of DMF and reacted at 50 ℃ for 8 hours; cooling the solution to room temperature, concentrating under reduced pressure to remove the solvent, and separating by column Chromatography (CH) 2 Cl 2 MeOH = 10) to give compound 5-2 (120mg, 0.26mmol, yield 49%).
The structural characterization data of the obtained compound 5-2 are: 1 H NMR(400MHz,Chloroform-d)δ6.91(t,J＝6.1Hz,1H),4.52(d,J＝6.7Hz,1H),4.23(q,J＝7.2Hz,2H),4.08(s,1H),4.00–3.86(m,2H),3.71–3.57(m,2H),3.46(d,J＝9.0Hz,2H),3.37–3.25(m,1H),2.36–2.22(m,2H),2.15(td,J＝12.9,6.7Hz,1H),1.70–1.58(m,2H),1.38–1.18(m,25H),0.88(t,J＝6.7Hz,3H). 13 C NMR(100MHz,CDCl 3 )δ176.94,171.98,77.32,77.00,76.68,74.61,72.36,69.44,66.56,66.30,61.58,43.05,36.40,31.90,30.12,29.68,29.65,29.52,29.34,29.28,25.66,22.67,14.20,14.10；MALDI-TOF MS:[M+Na] + the theoretical value is 482.3094, and the actual value is 482.2968.
3. 80mg (0.17 mmol) of compound 5-2 was dissolved in 3mL of a mixed solution of dichloromethane and methanol in a volume ratio of 1.
The structural characterization data of the obtained compound II-2 are: 1 H NMR(400MHz,DMSO-d 6 ) δ 4.71 (s, 1H), 4.64 (s, 1H), 4.33 (s, 1H), 4.23 (s, 1H), 3.73 (s, 1H), 3.70-3.62 (m, 2H), 3.43-3.26 (m, 2H), 2.83 (ddd, J =13.7,6.7,3.4hz, 1h), 2.09 (t, J =7.5hz, 2h), 1.97-1.89 (m, 2H), 1.52 (t, J =7.1hz, 3h), 1.24 (s, 22H), 0.90-0.82 (t, 3H), see fig. 4; 13 C NMR(100MHz,DMSO)δ172.52,75.51,72.11,67.23,66.60,65.95,42.74,35.51,31.30,29.08,29.03,29.00,28.95,28.90,28.72,25.36,22.10,13.95；MALDI-TOF MS:[M-H] - theoretical value of 430.2810 and measured value of 430.6582.
Example 5
1. This step is the same as step 1 of example 2.
2. 62.5mg (0.25 mmol) of Compound 4 and 90mg (0.32 mmol) of linoleic acid were dissolved in 2mL of DMF, and 198mg (0.38 mmol) of PyBOP and 66. Mu.L (0.40 mmol) of DIPEA were added to the mixture solution with stirring at room temperature, and after stirring at room temperature for 16 hours, the reaction mixture was concentrated and subjected to silica gel column chromatography to obtain Compound 5-3 (71mg, 0.14mmol, 56%).
3. 71mg (0.14 mmol) of compound 5-3 was dissolved in 1.8mL of methanol, 0.45mL of 0.5mol/L aqueous sodium hydroxide solution was added thereto with stirring at room temperature, and after stirring at room temperature for 1.5h, the mixture was neutralized with an acidic ion exchange resin Dowex 50WX8, and concentrated by filtration to obtain compound II-3 (44mg, 0.091mmol, yield 65%).
The structural characterization data of the obtained compound II-3 are: 1 h NMR (400mhz, meod) δ 5.41-5.31 (m, 2H), 4.47 (d, J =6.1hz, 1h), 4.00-3.91 (m, 2H), 3.85 (dd, J =14.0,2.7hz, 1h), 3.66 (ddd, J =11.8,4.6,2.9hz, 1h), 3.55 (d, J =8.7hz, 1h), 3.24 (dd, J =13.9,4.7hz, 1h), 2.35-2.09 (m, 4H), 2.09-1.97 (m, 4H), 1.74-1.55 (m, 2H), 1.47-1.21 (m, 20H), 0.92-0.88 (t, J =6.7hz, 3h), see fig. 5.
Example 6
In vitro antibacterial Activity test of glycolipid derivatives prepared in examples 1 and 3
The testing process comprises the following steps: two gram-positive bacteria (enterococcus faecalis E.faecium and staphylococcus aureus S.aureus) and two gram-negative bacteria (escherichia coli E.coli and pseudomonas aeruginosa) are respectively selected, and the inhibition effect of the glycolipid derivative on the bacterial growth (24 h) is determined by adopting a multiple dilution method. Two commercially available antibiotics vancomycin and gentamicin were used as positive controls. The MIC value was measured by the Constant broth dilution method (Constant broth dilution method) in the following specific procedures: 384. Mu.L of 4mg/mL sample solution was added to 1.808mL of medium, after mixing, 1mL was aspirated into tube 2, after mixing, 1mL was aspirated into tube 3, and this was repeated, and 1mL was discarded from the last 10 th tube, tube 11 being a drug-free growth control. The sample concentrations were 768, 384, 192, 96, 48, 24, 12, 6, 3, 1.5. Mu.g/mL. 0.2mL of the new shake strain is taken, 100 times of the shake strain is diluted to 20mL, 1mL of the new shake strain is respectively taken and put into the sample, and the concentration of the bacterial liquid is about 5X 105CFU/mL. The final concentration of the sample is 384, 192, 96, 48, 24, 12, 6, 3, 1.5 and 0.75 mu g/mL, and after incubation in an incubator at 37 ℃ for 24 hours, the solution is visually inspected to have no turbidity and OD 600 The measured value is equivalent to the negative control and is the MIC value, and the final MIC value is determined by repeating the steps for three times.
And (3) testing results: experimental results show that the compound I-1 has good inhibition effect on gram-positive bacteria, and the inhibition concentrations of the compound I-1 on staphylococcus aureus and enterococcus faecalis are 12 mu g/mL and 6 mu g/mL respectively. And example 3 contains C 14 The compound I-2 of the alkyl chain shows good inhibitory activity to gram-positive bacteria and gram-negative bacteria, and the inhibitory concentration to escherichia coli, pseudomonas aeruginosa, staphylococcus aureus and enterococcus faecalis is as follows in sequence: 24. 24, 24 and 12. Mu.g/mL. The transmission electron microscope can observe that after the bacteria and the glycolipid I-2 are incubated for 24h, the membrane structure of the bacteria is destroyed, thereby causing the bacteria to die, and the bacterial state before and after partial antibiosis is shown in figure 6.
Claims (6)
1. A Kdo-based glycolipid derivative characterized in that it has the following structural formula:
in the formula, R 1 Is an aliphatic amino group, R 2 is-NH 3 + Cl - Wherein said aliphatic amino group is CH 3 (CH 2 ) n NH-, and n is an integer of 13 to 17.
2. A method of producing a Kdo-based glycolipid derivative according to claim 1, characterized in that:
(1) Taking methanol and water as reaction media, reacting the compound 1 and alkali according to a molar ratio of 1-10 at 0-50 ℃ for 1-24 hours, and separating and purifying a product to obtain a compound 2;
(2) Adding the compound 2, aliphatic amine, a condensing agent and N, N-diisopropylethylamine into N, N-dimethylformamide according to a molar ratio of 1-2; wherein, the structural formula of the aliphatic amine is CH 3 (CH 2 ) n NH 2 In the formula, n is an integer of 13 to 17;
(3) Dissolving a compound 3 in a polar solvent, adding palladium carbon, introducing hydrogen, stirring at room temperature for 1-12 hours to obtain a reduction product, filtering to remove the palladium carbon, and acidifying the obtained solution with a hydrogen chloride aqueous solution to obtain a target compound I;
3. the method of preparing Kdo-based glycolipid derivatives according to claim 2, characterized in that: in the step (1), the alkali is sodium hydroxide or lithium hydroxide.
4. The method of preparing Kdo-based glycolipid derivatives according to claim 2, characterized in that: in the step (2), the condensing agent is benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate or a mixture of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole in a molar ratio of 1-2.
5. The method of preparing Kdo-based glycolipid derivatives according to claim 2, characterized in that: in the step (3), the polar solvent is ethanol or methanol.
6. Use of a Kdo-based glycolipid derivative according to claim 1, in the preparation of an antibacterial material, said bacterium being a gram-positive or gram-negative bacterium.
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