A jatrorrhizine derivative with antibacterial activity, synthesis method and application

Abstract

The present invention relates to the technical field of medicinal chemistry, and discloses a jatrorrhizine derivative with antibacterial activity, a synthesis method and an application. Starting from isovanillin, the present invention simply and efficiently synthesizes a series of novel 3-alkyl / acyl jatrorrhizine derivatives through Henry reaction, reduction reaction, Pictet-Spengler reaction, reductive amination, Friedel-Crafts cyclization and nucleophilic substitution reaction. R in the structural formula of this type of derivative is an aliphatic or aromatic hydrocarbon group, acyl group, cinnamoyl group or triazole with C2- 12 The antibacterial activity of 3-decyloxy jatrorrhizine is excellent, the cytotoxicity is small, and the relative safety is large, and it can be further developed into a potential drug for anti-Gram-positive bacterial infection. 3-Aromatic acyloxy jatrorrhizine shows good inhibitory activity against Helicobacter pylori.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical chemistry, and particularly to a jatrorrhizine derivative with antibacterial activity, a synthesis method and an application thereof. Background Art

[0002] Antibiotics are the most widely used antibacterial drugs at present. However, due to continuous overuse and misuse, the emergence of various multi-drug resistant strains and superbugs poses a potential threat to human health and survival. Therefore, the research and development of new antibacterial drugs with good antibacterial activity and low drug resistance is not only urgent, but also of great scientific significance, which helps to solve the bacterial drug resistance crisis.

[0003] Since the Eastern Han Dynasty, Coptis chinensis has been widely used in clinical practice, with the effects of clearing heat and purging fire, purging fire and detoxifying, killing insects and stopping vomiting. The main active ingredients of Coptis chinensis are isoquinoline-type berberine alkaloids with quaternary ammonium ion fragments, including berberine, coptisine, epiberberine, palmatine, jatrorrhizine and columbamine. Among them, the content of berberine is the highest (not less than 5.5%), and the content of jatrorrhizine is relatively low (only 0.26%). Among them, jatrorrhizine has good pharmacological effects such as detoxification, bactericidal and hypoglycemic effects. However, due to the relative scarcity of natural resources and the disadvantages of traditional extraction and separation, the extraction rate of jatrorrhizine is not optimistic. Therefore, it is very necessary to prepare jatrorrhizine in large quantities by chemical synthesis methods and carry out reasonable structural modification and transformation in the field of antibacterial drug research and development.

[0004]

[0005] At present, although there are some reports on the synthetic routes of the key intermediate of 3-hydroxy-4-methoxyphenethylamine, there is still no overall study on its reaction conditions, reaction time, and yield. Therefore, by exploring the synthetic process of phenethylamine derivatives, reducing raw material costs, and optimizing the synthetic route of alkaloids, and finally synthesizing jatrorrhizine with high yield, it will undoubtedly provide a very important material basis for the industrial production of jatrorrhizine and its related research. Since the 3-position of the jatrorrhizine skeleton has a hydroxyl group that is easily modified, therefore, the jatrorrhizine derivative 1 with a phenyl group introduced at the 3-position can improve the lipophilicity of jatrorrhizine. Its MIC against Staphylococcus aureus is 7.8 μg / mL, and its MIC against Shigella dysenteriae is 7.8 μg / mL. The antibacterial activity is 30 times higher than that of the unmodified jatrorrhizine. When a triazole-modified compound 2 is introduced at the 3-position, its antibacterial activity is significantly improved against both Gram-positive and Gram-negative bacteria. Therefore, structural modification of jatrorrhizine and preliminary evaluation of its in vitro antibacterial activity and cytotoxicity, exploring the effects of introducing alkoxy groups, ester groups, and antibacterial groups at the C-3 position of jatrorrhizine on antibacterial activity, and constructing a structure-activity relationship model will provide some experimental data support for obtaining jatrorrhizine antibacterial agents with strong antibacterial activity and low toxicity. This research will greatly enrich the research on the antibacterial active substance basis of Coptis chinensis and is of great significance for the development of traditional Chinese medicine. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides the following technical solutions:

[0007] The present invention provides a jatrorrhizine derivative with antibacterial activity, which is characterized in that it is an isoquinoline-type berberine alkaloid with a quaternary imine ion fragment, and the general chemical structure formula I of the derivative is as follows:

[0008]

[0009] In general formula I, R is H, ethyl, butyl, hexyl, octyl, decyl, dodecyl, tetradecyl, ethylbenzene, propylbenzene, 1,2,4-triazol-1-ylbutyl, benzimidazolylbutyl, acetyl, butyryl, hexanoyl, octanoyl, decanoyl, dodecanoyl, tetradecanoyl, benzoyl, p-methylbenzoyl, p-chlorobenzoyl, p-methoxybenzoyl, cinnamoyl; Y is chloride, bromide ion.

[0010] The synthetic route of the jatrorrhizine derivative includes the following steps:

[0011] 1) Synthesis of (E)-2-methoxy-5-(2-nitrovinyl)phenol: Under a nitrogen atmosphere, 3-hydroxy-4-methoxybenzaldehyde, NH4OAc, The molecular sieve was mixed with CH3NO2 and refluxed with stirring at 110 °C for 2 h to obtain a reaction solution; the reaction solution was cooled to room temperature and filtered through diatomaceous earth to remove the 5A molecular sieve, obtaining a filtrate; the filtrate was subjected to vacuum distillation using a rotary evaporator at 55 rpm to obtain a mixed organic matter from which nitromethane was removed; methanol was added to the mixed organic matter and dissolved at 60 °C, and then recrystallized in a -20 °C refrigerator to obtain a yellowish-brown crystal (E)-2-methoxy-5-(2-nitrovinyl)phenol;

[0012] 2) Synthesis of 2-methoxy-5-(2-nitroethyl)phenol: THF and MeOH were mixed to obtain a mixed solvent; (E)-2-methoxy-5-(2-nitrovinyl)phenol was dissolved in the mixed solvent, and then sodium borohydride was added in three batches at 0 °C with an interval of 10 min for a total of 12 h to obtain a mixed solution; the mixed solution was transferred to room temperature and reacted for 12 h, and then the pH value was adjusted to 7 with 1 M hydrochloric acid, THF was removed by reduced pressure concentration, extracted 3 times with ether, the ether layers were combined, washed 2 times with saturated NaCl, dried with anhydrous MgSO4, the filtrate was concentrated to obtain a residue, and the residue was purified by silica gel column chromatography to obtain a light yellow oily liquid 2-methoxy-5-(2-nitroethyl)phenol; among them, an eluent with a volume ratio of petroleum ether∶ethyl acetate = 8∶1 was used for elution;

[0013] 3) Synthesis of 3-hydroxy-4-methoxyphenethylamine hydrochloride: 2-methoxy-5-(2-nitroethyl)phenol was dissolved in ethyl acetate, 37 wt% concentrated hydrochloric acid was added dropwise, and stirred at 100 r / min for 5 min to obtain a mixture; zinc powder was added to the mixture at 70 °C, stirred at 100 r / min for 10 min, then the temperature was adjusted to 50 °C and refluxed with stirring to obtain a mixed solution, the mixed solution was filtered to remove zinc powder to obtain a filtrate; the filtrate was dried with anhydrous sodium sulfate, filtered, and ethyl acetate was removed by rotary evaporation under reduced pressure to obtain a remaining oily liquid; concentrated hydrochloric acid was added to the remaining oily liquid, frozen overnight at -20 °C, and filtered to obtain a white solid 3-hydroxy-4-methoxyphenethylamine hydrochloride;

[0014] 4) Synthesis of 1-(dimethoxymethyl)-7-methoxy-1,2,3,4-tetrahydroisoquinolin-6-ol: 3-Hydroxy-4-methoxyphenethylamine hydrochloride, triethylamine and dichloromethane were mixed in a reaction vessel. 60 wt% of 2,2-dimethoxyacetaldehyde was added to the above mixture after being extracted with dichloromethane to obtain a reaction mixture. The reaction mixture was stirred at room temperature for 30 min and then trifluoroacetic acid was added dropwise under ice bath conditions and reacted for 1 h. After the reaction, the pH was adjusted to 7 with 3 M sodium hydroxide solution, and then extracted 3 times with DCM. The organic phases were combined, dried over anhydrous Na2SO4, filtered and concentrated to obtain a residue. The residue was purified by silica gel column chromatography to obtain 1-(dimethoxymethyl)-7-methoxy-1,2,3,4-tetrahydroisoquinolin-6-ol as a yellow oily liquid. Among them, an eluent with a volume ratio of dichloromethane:methanol = 10:1 was used for elution;

[0015] 5) Synthesis of 2-(2,3-dimethoxybenzyl)-7-methoxy-1-(dimethoxymethyl)-1,2,3,4-tetrahydroisoquinolin-6-ol: 1-(Dimethoxymethyl)-7-methoxy-1,2,3,4-tetrahydroisoquinolin-6-ol was dissolved in 1,2-dichloroethane, and then 2,3-dimethoxybenzaldehyde was added. After thorough mixing, sodium triacetoxyborohydride was added at room temperature. The mixture was stirred and reacted at 100 r / min for 4 h, and then saturated NaHCO3 was added to obtain a mixed solution. The mixed solution was extracted 3 times with DCM, the organic layers were combined, anhydrous MgSO4 was added for drying, filtered and concentrated to obtain a residue. The residue was purified by silica gel column chromatography to obtain 2-(2,3-dimethoxybenzyl)-7-methoxy-1-(dimethoxymethyl)-1,2,3,4-tetrahydroisoquinolin-6-ol as a dark yellow oily liquid. Among them, an eluent with a volume ratio of petroleum ether:ethyl acetate = 1:1 was used for elution;

[0016] 6) Synthesis of jatrorrhizine: 2-(2,3-Dimethoxybenzyl)-7-methoxy-1-(dimethoxymethyl)-1,2,3,4-tetrahydroisoquinolin-6-ol and dichloromethane were mixed and added to a round-bottom flask. Trifluoromethanesulfonic acid was added dropwise at 0 °C under argon protection. After reacting for 5 min, the ice bath was removed and the mixture was transferred to room temperature at 25 °C and stirred for 1 h to obtain a mixture;

[0017] The mixture was diluted with dichloromethane and washed with sodium bicarbonate solution to remove acid, obtaining an organic layer; the organic layer was dried with anhydrous Na2SO4, filtered and concentrated to obtain a concentrated filtrate; potassium acetate and ethanol were added to the concentrated filtrate, and an iodine solution of ethanol was added dropwise at 1000 r / min at a rate of 1 mL / s. After reacting for 1 h, a 10 wt% sodium thiosulfate solution was added, and after filtration through diatomaceous earth and washing the filter cake with methanol, a concentrated filtrate was obtained; the concentrated filtrate was purified by silica gel column chromatography to obtain a light yellow solid product; among them, an eluent with a volume ratio of dichloromethane∶methanol = 15∶1 was used for elution;

[0018] 7) Synthesis of jatrorrhizine derivatives: At room temperature, jatrorrhizine, an organic acid-binding agent containing a lone pair of electrons, and a solvent N,N-dimethylformamide / acetonitrile were added to a sealed tube, stirred at room temperature for 30 min, and then bromoalkane / alkyl acyl chloride was added. The mixed solution was stirred at room temperature for 16 - 18 h, and the reaction was monitored by TLC; after the reaction was completed, separation was carried out to obtain jatrorrhizine derivatives.

[0019] During the above operation process, during the synthesis of the intermediate 3-hydroxy-4-methoxyphenethylamine hydrochloride, inorganic salt ammonium acetate was used instead of methylamine, thereby increasing the yield of 2-methoxy-5-(2-nitroethyl)phenol; in step two, the solvent was replaced from methanol with a mixed solvent of THF and MeOH, and the yield was increased to 91%; in step three, the hydrochloride could be directly prepared without column separation, improving the yield of 3-hydroxy-4-methoxyphenethylamine hydrochloride.

[0020] Preferably, the inorganic and organic acid-binding agents containing a lone pair of electrons in step 7) are sodium carbonate, potassium carbonate, cesium carbonate, triethylamine, trimethylamine or pyridine;

[0021] Preferably, the separation is carried out by silica gel column chromatography, and the eluent is a mixed solution of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 15∶1;

[0022] Preferably, the general structural formulas of the haloalkane and acyl halide are as follows:

[0023]

[0024] Among them, R is one of H, ethyl, butyl, hexyl, octyl, decyl, dodecyl, tetradecyl, ethylbenzene, propylbenzene, 1,2,4-triazol-1-ylbutyl, benzimidazolylbutyl, acetyl, butyryl, hexanoyl, octanoyl, decanoyl, dodecanoyl, tetradecanoyl, benzoyl, p-methylbenzoyl, p-chlorobenzoyl, p-methoxybenzoyl, cinnamoyl; Y is one of chlorine and bromide ions;

[0025] Application of jatrorrhizine derivatives with antibacterial activity in preparing drugs against Gram-positive and Gram-negative bacterial infections.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] 1. When synthesizing jatrorrhizine, inexpensive and readily available isovanillin is used as the starting material; when preparing the intermediate 3-hydroxy-4-methoxyphenethylamine hydrochloride of jatrorrhizine, methylamine is replaced with inorganic salt ammonium acetate to obtain (E)-2-methoxy-5-(2-nitrovinyl)phenol with a higher yield; during the preparation of 2-methoxy-5-(2-nitroethyl)phenol, THF and MeOH are used as a mixed solvent to dissolve the raw materials, thereby improving the yield of the double bond reduction product; zinc powder is used as a catalyst, and 2-methoxy-5-(2-nitroethyl)phenol reacts with concentrated hydrochloric acid directly to obtain the hydrochloride, omitting the column chromatography separation step, thereby increasing the yield of the hydrochloride. Other routes adopt the method of Bn protection, adding two steps of protection and deprotection, and deprotection requires Pd / C and H2, which has potential safety hazards.

[0028] 2. A series of 3-alkyl / acyl jatrorrhizine derivatives are synthesized using the prepared jatrorrhizine as an intermediate. The R in this derivative is an aliphatic or aromatic hydrocarbon group, triazole, imidazole group, acyl group, and cinnamoyl group of C 2-12 This type of derivative has good inhibitory activity against the measured Gram-positive bacteria, even increasing by 128 times compared with unmodified jatrorrhizine, higher than the positive control drug erythromycin, and comparable to vancomycin. In addition, some derivatives have good inhibitory effects on Helicobacter pylori. Among them, 3-decyloxy jatrorrhizine has excellent antibacterial activity, low cytotoxicity, and relatively high safety, and can be further developed into a potential drug against Gram-positive bacterial infections; 3-aromatic acyloxy jatrorrhizine shows good inhibitory activity against Helicobacter pylori, and its MIC value is 16 μg / mL, which is 4 times higher than that of jatrorrhizine. Description of the Drawings

[0029] Figure 1 is the structure of the jatrorrhizine derivatives prepared in the present invention;

[0030] Figure 2 is the test comparison chart of the inhibition rate of some alkaloid derivatives on HepG2 cells;

[0031] Figure 3 is the synthetic route diagram of jatrorrhizine derivatives. Detailed Embodiments

[0032] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0033] Next, please refer to Figures 1 - 3 。

[0034] Example 1: Preparation method of jatrorrhizine, including the following steps

[0035] 1. Synthesis of (E)-2-methoxy-5-(2-nitrovinyl)phenol: Under a nitrogen atmosphere, 7.6 g of 3-hydroxy-4-methoxybenzaldehyde, 3.25 g of NH4OAc, 10 g of molecular sieve and 150 mL of CH3NO2 were mixed, and refluxed and stirred at 110 °C for 2 h to obtain a reaction solution; the reaction solution was cooled to room temperature and filtered through diatomaceous earth to remove the molecular sieve to obtain a filtrate; the filtrate was distilled under reduced pressure using a rotary evaporator at 55 rpm to obtain a mixed organic matter with nitromethane removed; 10 mL of methanol was added to the mixed organic matter and dissolved at 60 °C, and then recrystallized in a -20 °C refrigerator to obtain 7.69 g of yellowish-brown crystals of (E)-2-methoxy-5-(2-nitrovinyl)phenol.

[0036] 2. Synthesis of 2-methoxy-5-(2-nitroethyl)phenol: 37 mL of THF and 6 mL of MeOH were mixed to obtain a mixed solvent; 3.32 g of (E)-2-methoxy-5-(2-nitrovinyl)phenol was dissolved in the mixed solvent, and then 1.93 g of sodium borohydride was added in three batches at 0 °C with an interval of 10 min to obtain a mixed solution; the mixed solution was transferred to room temperature and reacted for 12 h, and then the pH value was adjusted to 7 with 1 M hydrochloric acid, concentrated under reduced pressure to remove THF, extracted 3 times with 30 mL of ether, the ether layers were combined, washed 2 times with 30 mL of saturated NaCl solution, dried with anhydrous MgSO4, the filtrate was concentrated to obtain a residue, and the residue was purified by silica gel column chromatography to obtain 2.9 g of light yellow oily liquid of 2-methoxy-5-(2-nitroethyl)phenol; among them, an eluent with a volume ratio of petroleum ether:ethyl acetate = 8:1 was used for elution.

[0037] 3. Synthesis of 3-hydroxy-4-methoxyphenethylamine hydrochloride: Dissolve 715.2 mg of 2-methoxy-5-(2-nitroethyl)phenol in 12 mL of ethyl acetate, dropwise add 5 mL of 37 wt% concentrated hydrochloric acid at a rate of 1 mL / s, and stir at 100 r / min for 5 min to obtain a mixture. Add 3.1 g of zinc powder to the mixture at 70 °C, stir at 100 r / min for 10 min, then adjust the temperature to 50 °C and reflux and stir for 4 h to obtain a mixed solution. Filter the mixed solution to remove the excess zinc powder to obtain a filtrate; dry the filtrate with anhydrous sodium sulfate, filter, and rotary evaporate the ethyl acetate under reduced pressure to obtain a remaining oily liquid; add 2 mL of concentrated hydrochloric acid to the remaining oily liquid, freeze overnight at -20 °C, and filter to obtain the white solid 3-hydroxy-4-methoxyphenethylamine hydrochloride.

[0038] 4. Synthesis of 1-(dimethoxymethyl)-7-methoxy-1,2,3,4-tetrahydroisoquinolin-6-ol: Mix 1.02 g of 3-hydroxy-4-methoxyphenethylamine hydrochloride, 700 μL of triethylamine, and 6.5 mL of dichloromethane in a reaction vessel; add 2.25 mL of 60 wt% 2,2-dimethoxyacetaldehyde after extraction with dichloromethane to the above mixture to obtain a reaction mixture; stir the reaction mixture at room temperature for 30 min, then dropwise add 5.75 mL of TFA dropwise under ice bath conditions and react for 1 h; after the reaction is completed, adjust the pH to 7 with 3 M sodium hydroxide solution, then extract 3 times with 10 mL of DCM, combine the organic phases, dry with anhydrous Na2SO4, filter and concentrate to obtain a residue; purify the residue by silica gel column chromatography to obtain 558.1 mg of the yellow oily liquid 1-(dimethoxymethyl)-7-methoxy-1,2,3,4-tetrahydroisoquinolin-6-ol; among them, an eluent with a volume ratio of dichloromethane:methanol = 10:1 is used for elution.

[0039] 5. Synthesis of 2-(2,3-dimethoxybenzyl)-7-methoxy-1-(dimethoxymethyl)-1,2,3,4-tetrahydroisoquinolin-6-ol: Dissolve 62.7 mg of 1-(dimethoxymethyl)-7-methoxy-1,2,3,4-tetrahydroisoquinolin-6-ol in 1.2 mL of 1,2-dichloroethane, then add 79.8 mg of 2,3-dimethoxybenzaldehyde. After thorough mixing, add 152.6 mg of sodium triacetoxyborohydride at room temperature. Stir and react at 100 r / min for 4 h, then add saturated NaHCO3 to obtain a mixed solution. Extract the mixed solution with 10 mL of DCM three times, combine the organic layers, add anhydrous MgSO4 for drying, filter and concentrate to obtain a residue. Purify the residue by silica gel column chromatography to obtain 88.8 mg of 2-(2,3-dimethoxybenzyl)-7-methoxy-1-(dimethoxymethyl)-1,2,3,4-tetrahydroisoquinolin-6-ol as a dark yellow oily liquid; among them, elute with an eluent with a volume ratio of petroleum ether:ethyl acetate = 1:1.

[0040] 6. Synthesis of jatrorrhizine: Mix 65.3 mg of 2-(2,3-dimethoxybenzyl)-7-methoxy-1-(dimethoxymethyl)-1,2,3,4-tetrahydroisoquinolin-6-ol and 3.2 mL of dichloromethane and add them to a 50 mL round-bottom flask. Dropwise add 86 μL of trifluoromethanesulfonic acid at 0 °C under argon protection at a rate of 1 mL / s. After reacting for 5 min, remove the ice bath and transfer to room temperature at 25 °C and continue stirring for 1 h to obtain a mixture. 2) Dilute the mixture with dichloromethane and wash with sodium bicarbonate solution to remove acid to obtain an organic layer; dry the organic layer with anhydrous Na2SO4, filter and concentrate to obtain a concentrated filtrate; add 19.7 mg of potassium acetate and 5 mL of ethanol to the concentrated filtrate, dropwise add an iodine solution in ethanol at 1000 r / min at a rate of 1 mL / s. After reacting for 1 h, add a 10 wt% sodium thiosulfate solution, filter through diatomaceous earth and wash the filter cake with methanol to obtain a concentrated filtrate; purify the concentrated filtrate by silica gel column chromatography to obtain 39.3 mg of a light yellow solid product; among them, elute with an eluent with a volume ratio of dichloromethane:methanol = 15:1.

[0041] Example 2: Preparation of 3-butoxy jatrorrhizine I-2

[0042] Under argon atmosphere, 0.1 mmol of jatrorrhizine and 1.5 mL of N,N-dimethylformamide were added into a 15 mL sealed tube and stirred thoroughly. After jatrorrhizine was completely dissolved in the solvent, 0.2 mmol of cesium carbonate was added, and the reaction was carried out at room temperature for 30 min. Then 0.13 mmol of butyl bromide was added to obtain a mixed solution. The mixed solution was stirred at room temperature for 16 h, and the reaction progress was monitored by TLC. After the reaction was completed, it was washed with saturated brine and extracted 5 times with 10 mL of dichloromethane. The organic layers were combined and dried over anhydrous Na2SO4. After filtration and concentration, it was purified by silica gel column chromatography to obtain 23.7 mg of the yellow solid compound 3-butoxy jatrorrhizine I-2; among them, the eluent with a volume ratio of dichloromethane:methanol = 15:1 was used for elution; with dichloromethane-methanol with a volume ratio of 15:1 as the developing agent, the R f value of the prepared 3-butoxy jatrorrhizine I-2 was 0.26, and the chemical structural formula was as follows:

[0043]

[0044] 1 H NMR(400MHz,CDCl3:δ=10.03 - 9.92(m,1H),8.75(s,1H),8.09(d,J=9.1Hz,1H),7.61(d,J=9.1Hz,1H),7.43(s,1H),6.70(s,1H),5.05(t,J=6.4Hz,2H),4.27(s,3H),4.08(s,5H),3.99(s,3H),3.25(t,J=6.4Hz,2H),1.97 - 1.82(m,2H),1.53(dt,J=14.2,7.5Hz,2H),1.02(t,J=7.4Hz,3H).

[0045] 13 C NMR(100MHz,CDCl3):δ=151.87,150.21,149.78,145.66,144.62,137.80,133.65,127.80,125.96,123.39,121.81,119.81,118.50,111.83,108.91,69.12,62.68,57.50,56.99,56.55,31.12,27.00,19.22,13.86.

[0046] Example 3: Preparation of 3-ethoxy jatrorrhizine I-3

[0047] Under argon atmosphere, 0.1 mmol of jatrorrhizine and 1.5 mL of N,N-dimethylformamide were added into a 15 mL sealed tube and stirred well. After jatrorrhizine was completely dissolved in the solvent, 0.2 mmol of cesium carbonate was added and the reaction was carried out at room temperature for 30 min. Then 0.13 mmol of 1-bromooctane was added to obtain a mixed solution. The mixed solution was stirred at room temperature for 16 h, and the reaction progress was monitored by TLC. After the reaction was completed, it was washed with saturated brine and extracted 5 times with 10 mL of dichloromethane. The organic layers were combined and dried over anhydrous Na2SO4. After filtration and concentration, it was purified by silica gel column chromatography to obtain 18.6 mg of yellow solid compound 3-ethoxy jatrorrhizine I-3. Among them, the eluent with a volume ratio of dichloromethane:methanol = 15:1 was used for elution; with dichloromethane-methanol with a volume ratio of 15:1 as the developing agent, the R f value of the prepared 3-ethoxy jatrorrhizine I-3 was 0.26, and the chemical structural formula was as follows:

[0048]

[0049] Example 4: Preparation of 3-ethoxy jatrorrhizine I-4

[0050] Under argon atmosphere, 0.1 mmol of jatrorrhizine and 1.5 mL of N,N-dimethylformamide were added into a 15 mL sealed tube and stirred well. After jatrorrhizine was completely dissolved in the solvent, 0.2 mmol of cesium carbonate was added and the reaction was carried out at room temperature for 30 min. Then 0.13 mmol of 1-bromooctane was added to obtain a mixed solution. The mixed solution was stirred at room temperature for 16 h, and the reaction progress was monitored by TLC. After the reaction was completed, it was washed with saturated brine and extracted 5 times with 10 mL of dichloromethane. The organic layers were combined and dried over anhydrous Na2SO4. After filtration and concentration, it was purified by silica gel column chromatography to obtain 20.2 mg of yellow solid compound 3-ethoxy jatrorrhizine I-4. Among them, the eluent with a volume ratio of dichloromethane:methanol = 15:1 was used for elution; with dichloromethane-methanol with a volume ratio of 15:1 as the developing agent, the R f value of the prepared 3-ethoxy jatrorrhizine I-4 was 0.22, and the chemical structural formula was as follows:

[0051]

[0052] 1HNMR(400MHz,CD3OD):δ=9.94(s,1H),8.60(s,1H),8.03(d,J=9.0Hz,1H),7.63(d,J=8.9Hz,1H),7.39(s,1H),6.70(s,1H),5.05(t,J=6.4Hz,2H),4.24(s,3H),4.07(d,J=9.1Hz,5H),3.97(s,3H),3.26(t,J=6.3Hz,2H),1.98-1.82(m,2H),1.48(q,J=7.4Hz,2H),1.34(dd,J=18.6,7.1Hz,7H),1.26(s,1H),0.90(t,J=6.4Hz,3H).

[0053] 13 C NMR(100MHz,CDCl3):δ=151.77,150.15,149.68,145.66,144.53,137.76,133.53,127.91,125.93,123.48,121.77,119.77,118.48,111.74,108.78,69.37,62.57,57.25,56.89,56.45,31.80,29.34,29.20,29.05,27.04,25.94,22.64,14.07.

[0054] Example 5: Preparation of 3-ethoxy jatrorrhizine I-5

[0055] Under argon, 0.1 mmol of jatrorrhizine and 1.5 mL of N,N-dimethylformamide were added to a 15 mL sealed tube and stirred thoroughly. After the jatrorrhizine was completely dissolved in the solvent, 0.2 mmol of cesium carbonate was added, and the reaction was carried out at room temperature for 30 min. Then, 0.13 mmol of 1-bromodecane was added to obtain a mixed solution; the mixed solution was stirred at room temperature for 16 h, and the reaction was monitored by TLC. After the reaction was complete, it was washed with saturated brine and extracted 5 times with 10 mL of dichloromethane. The organic layers were combined and dried over anhydrous Na2SO4. After filtration and concentration, it was purified by silica gel column chromatography to obtain 26.3 mg of the yellow solid compound 3-decyloxy jatrorrhizine I-5; among them, an eluent with a volume ratio of dichloromethane:methanol = 15:1 was used for elution; with a dichloromethane-methanol with a volume ratio of 15:1 as the developing agent, the R f value of the prepared 3-ethoxy jatrorrhizine I-5 was 0.22, and the chemical structural formula was as follows:

[0056]

[0057] Example 6: Preparation of 3-dodecyloxy jatrorrhizine I-6

[0058] Under argon, 0.1 mmol of jatrorrhizine and 1.5 mL of N,N-dimethylformamide were added to a 15 mL sealed tube and stirred well. After jatrorrhizine was completely dissolved in the solvent, 0.2 mmol of cesium carbonate was added, and the reaction was carried out at room temperature for 30 min. Then 0.13 mmol of dodecyl bromide was added to obtain a mixed solution. The mixed solution was stirred at room temperature for 16 h, and the reaction was monitored by TLC. After the reaction was complete, it was washed with saturated brine and extracted 5 times with 10 mL of dichloromethane. The organic layers were combined and dried over anhydrous Na2SO4. After filtration and concentration, it was purified by silica gel column chromatography to obtain 26.4 mg of the yellow solid compound 3-dodecyloxy jatrorrhizine I-6; among them, an eluent with a volume ratio of dichloromethane:methanol = 15:1 was used for elution; with dichloromethane-methanol with a volume ratio of 15:1 as the developing agent, the R f value was 0.25, and the chemical structural formula is as follows:

[0059]

[0060] Example 7: Preparation of 3-ethoxy jatrorrhizine I-8

[0061] Under argon, 0.1 mmol of jatrorrhizine and 1.5 mL of N,N-dimethylformamide were added to a 15 mL sealed tube and stirred well. After jatrorrhizine was completely dissolved in the solvent, 0.2 mmol of cesium carbonate was added, and the reaction was carried out at room temperature for 30 min. Then 0.13 mmol of 2-bromoethylbenzene was added to obtain a mixed solution. The mixed solution was stirred at room temperature for 16 h, and the reaction was monitored by TLC. After the reaction was complete, it was washed with saturated brine and extracted 5 times with 10 mL of dichloromethane. The organic layers were combined and dried over anhydrous Na2SO4. After filtration and concentration, it was purified by silica gel column chromatography to obtain 36.6 mg of the yellow solid compound 3-ethoxy jatrorrhizine I-8; among them, an eluent with a volume ratio of dichloromethane:methanol = 15:1 was used for elution; with dichloromethane-methanol with a volume ratio of 15:1 as the developing agent, the R f value was 0.23, and the chemical structural formula is as follows:

[0062]

[0063] Example 8: Preparation of 3-ethoxy jatrorrhizine I-9

[0064] Under argon, 0.1 mmol of jatrorrhizine and 1.5 mL of N,N-dimethylformamide were added to a 15 mL sealed tube and stirred thoroughly. After the jatrorrhizine was completely dissolved in the solvent, 0.2 mmol of cesium carbonate was added, and the reaction was carried out at room temperature for 30 min. Then, 0.13 mmol of 1-bromo-3-phenylpropane was added to obtain a mixed solution. The mixed solution was stirred at room temperature for 16 h, and the reaction progress was monitored by TLC. After the reaction was complete, it was washed with saturated brine and extracted 5 times with 10 mL of dichloromethane. The organic layers were combined and dried over anhydrous Na2SO4. After filtration and concentration, it was purified by silica gel column chromatography to obtain 20.2 mg of the yellow solid compound 3-ethoxy jatrorrhizine I-9; among them, an eluent with a volume ratio of dichloromethane:methanol = 15:1 was used for elution; with dichloromethane-methanol with a volume ratio of 15:1 as the developing agent, the R f value of the prepared 3-ethoxy jatrorrhizine I-9 was 0.22, and the chemical structural formula is as follows:

[0065]

[0066] 1 H NMR(400MHz,CDCl3):δ=10.30(s,1H),8.61(s,2H),7.98(d,J=8.3Hz,1H),7.70(d,J=7.4Hz,1H),7.44(s,1H),7.33(d,J=6.8Hz,5H),6.72(s,1H),5.21(s,2H),4.32-4.21(m,5H),4.04(s,3H),3.99(s,3H),3.20(t,J=7.2Hz,2H),2.10(s,1H),1.25(t,J=9.0Hz,2H).

[0067] 13 C NMR(100MHz,CDCl3):δ=151.56,150.32,149.84,146.07,144.79,137.76,137.72,133.72,129.20,128.75,128.02,126.90,126.00,123.74,121.95,120.33,119.10,112.17,109.47,77.48,77.16,76.84,70.13,62.64,57.33,57.04,56.23,53.52,35.83,27.22.

[0068] HRMS(ESI):C 29 H 30 NO4 + [M] +The calculated exact molecular weight is 456.2169, and the measured value is 456.2159.

[0069] Example 9: Preparation of 3-(4-(1,2,4-triazole))butoxy jatrorrhizine I-10

[0070] Under argon atmosphere, 0.1 mmol of jatrorrhizine and 1.5 mL of N,N-dimethylformamide were added to a 15 mL sealed tube and stirred well. After the jatrorrhizine was completely dissolved in the solvent, 0.2 mmol of cesium carbonate was added, and the reaction was carried out at room temperature for 30 min. Then 0.13 mmol of 1-(4-bromobutyl)-1,2,4-triazole was added to obtain a mixed solution. The mixed solution was stirred at room temperature for 16 h, and the reaction was monitored by TLC. After the reaction was complete, it was washed with saturated brine and extracted 5 times with 10 mL of dichloromethane. The organic layers were combined and dried over anhydrous Na2SO4. After filtration and concentration, it was purified by silica gel column chromatography to obtain 24.9 mg of the yellow solid compound 3-(4-(1,2,4-triazole))butoxy jatrorrhizine I-10; among them, the eluent with a volume ratio of dichloromethane:methanol = 15:1 was used for elution; with dichloromethane-methanol with a volume ratio of 15:1 as the developing agent, the R f value of the prepared 3-(4-(1,2,4-triazole))butoxy jatrorrhizine I-10 is 0.22, and the chemical structural formula is as follows:

[0071]

[0072] 1 1H NMR (400 MHz, CD3OD): δ = 9.76 (s, 1H), 8.81 (s, 1H), 8.57 (s, 1H), 8.11 (d, J = 9.0 Hz, 1H), 8.04 - 7.99 (m, 2H), 7.67 (s, 1H), 7.02 (s, 1H), 4.97 - 4.90 (m, 2H), 4.39 (t, J = 6.8 Hz, 2H), 4.21 (s, 3H), 4.15 (t, J = 5.9 Hz, 2H), 4.10 (s, 3H), 4.01 (s, 3H), 3.29 - 3.25 (m, 2H), 2.16 - 2.08 (m, 2H), 1.86 - 1.77 (m, 2H).

[0073] 1313C NMR (100 MHz, CD3OD): δ = 151.70, 150.80, 150.53, 149.76, 144.96, 144.43, 138.44, 133.96, 128.64, 126.83, 123.08, 121.93, 119.94, 119.22, 112.05, 108.88, 68.56, 61.16, 56.33, 56.00, 55.68, 53.37, 48.24, 48.02, 47.81, 47.60, 47.38, 47.17, 46.96, 46.59, 26.58, 26.41, 25.36.

[0074] HRMS (ESI): C 26 H 29 N4O4 + [M] + Calculated exact molecular weight 461.2183, found 461.2182.

[0075] Example 10: Preparation of 3-butyryloxy jatrorrhizine I-11

[0076] Under argon atmosphere, 0.1 mmol of jatrorrhizine and 1.5 mL of acetonitrile were added to a 15 mL sealed tube and stirred thoroughly. After the jatrorrhizine was completely dissolved in the solvent, 0.44 mmol of triethylamine was added, and the reaction was carried out at room temperature for 30 min. Then 0.12 mmol of butyryl chloride was added to obtain a mixed solution. The mixed solution was stirred at room temperature for 18 h, and the reaction was monitored by TLC. After the reaction was complete, it was washed with saturated brine and extracted 5 times with 10 mL of dichloromethane. The organic layers were combined and dried over anhydrous Na2SO4. After filtration and concentration, it was purified by silica gel column chromatography to obtain 16 mg of the yellow solid compound 3-butyryloxy jatrorrhizine I-11; among them, the eluent with a volume ratio of dichloromethane:methanol = 15:1 was used for elution; with dichloromethane-methanol with a volume ratio of 15:1 as the developing agent, the R f value was 0.27, and the chemical structural formula is as follows:

[0077]

[0078] 11H NMR (400 MHz, CDCl3): δ = 10.09 (s, 1H), 9.23 (s, 1H), 8.12 (d, J = 8.9 Hz, 1H), 7.74 (s, 1H), 7.62 (d, J = 9.0 Hz, 1H), 6.92 (s, 1H), 5.08 (t, J = 6.4 Hz, 2H), 4.21 (s, 3H), 4.05 (s, 3H), 3.99 (s, 3H), 3.18 (t, J = 6.2 Hz, 2H), 2.60 (t, J = 7.3 Hz, 2H), 1.82 (h, J = 7.3 Hz, 2H), 1.09 (t, J = 7.4 Hz, 3H).

[0079] 13 13C NMR (100 MHz, CDCl3): δ = 171.60, 151.79, 151.00, 146.14, 145.01, 142.77, 137.22, 133.33, 127.38, 126.50, 125.08, 123.67, 123.04, 122.41, 121.49, 109.92, 62.53, 57.06, 56.84, 56.31, 35.90, 26.66, 18.53, 13.58.

[0080] HRMS (ESI): C 24 H 26 NO5 + [M] + Calculated exact molecular weight 408.1805, found 408.1803.

[0081] Example 11: Preparation of 3-octanoyloxy jatrorrhizine I-12

[0082] Under argon atmosphere, 0.1 mmol of jatrorrhizine and 1.5 mL of acetonitrile were added to a 15 mL sealed tube and stirred well. After the jatrorrhizine was completely dissolved in the solvent, 0.44 mmol of triethylamine was added, and the reaction was carried out at room temperature for 30 min. Then 0.12 mmol of butyryl chloride was added to obtain a mixed solution. The mixed solution was stirred at room temperature for 18 h, and the reaction progress was monitored by TLC. After the reaction was complete, it was washed with saturated brine and extracted 5 times with 10 mL of dichloromethane. The organic layers were combined and dried over anhydrous Na2SO4. After filtration and concentration, it was purified by silica gel column chromatography to obtain 27.5 mg of the yellow solid compound 3-octanoyloxy jatrorrhizine I-12; among them, the eluent with a volume ratio of dichloromethane:methanol = 15:1 was used for elution; with dichloromethane-methanol with a volume ratio of 15:1 as the developing agent, the R f value of the prepared 3-octanoyloxy jatrorrhizine I-12 was 0.23, and the chemical structural formula was as follows:

[0083]

[0084] 1 1H NMR (400 MHz, CD3OD): δ = 9.83 (s, 1H), 8.96 (s, 1H), 8.16 (d, J = 9.1 Hz, 1H), 8.09 (d, J = 9.1 Hz, 1H), 7.83 (s, 1H), 7.18 (s, 1H), 5.01 - 4.93 (m, 2H), 4.23 (s, 3H), 4.12 (s, 3H), 4.00 (s, 3H), 3.28 (d, J = 6.0 Hz, 2H), 2.62 (t, J = 7.2 Hz, 2H), 1.76 (dt, J = 14.8, 7.2 Hz, 2H), 1.44 - 1.26 (m, 8H), 0.93 (t, J = 6.9 Hz, 3H).

[0085] 13 13C NMR (100 MHz, CD3OD): δ = 173.10, 153.13, 152.48, 146.91, 146.04, 134.97, 129.16, 128.20, 126.74, 124.77, 124.74, 124.02, 123.69, 122.70, 122.70, 111.10, 62.64, 57.73, 57.43, 54.77, 34.68, 32.87, 30.04, 30.03, 27.38, 26.03, 23.64, 14.37.

[0086] Example 12: Preparation of 3-benzoyloxy jatrorrhizine I-14

[0087] Under argon atmosphere, 0.1 mmol of jatrorrhizine and 1.5 mL of acetonitrile were added to a 15 mL sealed tube and stirred well. After the jatrorrhizine was completely dissolved in the solvent, 0.44 mmol of triethylamine was added, and the reaction was carried out at room temperature for 30 min. Then 0.12 mmol of benzoyl chloride was added to obtain a mixed solution. The mixed solution was stirred at room temperature for 18 h, and the reaction was monitored by TLC. After the reaction was complete, it was washed with saturated brine and extracted 5 times with 10 mL of dichloromethane. The organic layers were combined and dried over anhydrous Na2SO4. After filtration and concentration, it was purified by silica gel column chromatography to obtain 11.5 mg of the yellow solid compound 3-benzoyloxy jatrorrhizine I-14; among them, the eluent with a volume ratio of dichloromethane:methanol = 15:1 was used for elution; with dichloromethane-methanol with a volume ratio of 15:1 as the developing agent, the Rf value of the prepared 3-benzoyloxy jatrorrhizine I-14 was 0.22, and the chemical structural formula is as follows: f The value is 0.22, and the chemical structural formula is as follows:

[0088]

[0089] 1 1H NMR (400 MHz, CD3OD): δ = 9.76 (s, 1H), 8.90 (s, 1H), 8.08 (dd, J = 8.3, 5.1 Hz, 3H), 8.00 (d, J = 9.0 Hz, 1H), 7.79 (s, 1H), 7.63 (t, J = 7.5 Hz, 1H), 7.49 (t, J = 7.7 Hz, 2H), 7.24 (s, 1H), 4.90 (t, J = 6.2 Hz, 2H), 4.14 (s, 3H), 4.04 (s, 3H), 3.90 (s, 3H), 3.25 (s, 2H).

[0090] 13 13C NMR (100 MHz, DMSO): δ = 164.22, 151.38, 151.23, 146.38, 144.31, 142.04, 137.18, 134.71, 133.13, 130.28, 129.52, 128.70, 128.46, 127.29, 126.04, 124.09, 123.46, 122.20, 121.79, 110.62, 62.39, 57.54, 57.16, 55.85, 25.94.

[0091] HRMS (ESI): C 27 H 24 NO5 + [M] + Calculated exact molecular weight 442.1649, found 442.1647.

[0092] Example 13: Preparation of 3-p-methylbenzoyloxy jatrorrhizine I-15

[0093] Under argon, 0.1 mmol of jatrorrhizine and 1.5 mL of acetonitrile were added to a 15 mL sealed tube and stirred well. After the jatrorrhizine was completely dissolved in the solvent, 0.44 mmol of triethylamine was added, and the reaction was carried out at room temperature for 30 min. Then 0.12 mmol of p-methylbenzoyl chloride was added to obtain a mixed solution; the mixed solution was stirred at room temperature for 18 h, and the reaction was monitored by TLC. After the reaction was complete, it was washed with saturated brine and extracted 5 times with 10 mL of dichloromethane. The organic layers were combined and dried over anhydrous Na2SO4. After filtration and concentration, it was purified by silica gel column chromatography to obtain 32.0 mg of the yellow solid compound 3-p-methylbenzoyloxy jatrorrhizine I-15; among them, the eluent with a volume ratio of dichloromethane:methanol = 15:1 was used for elution; with a dichloromethane-methanol with a volume ratio of 15:1 as the developing agent, the R f value was 0.22, and the chemical structural formula is as follows:

[0094]

[0095] Example 14: Preparation of 3-cinnamoyloxy jatrorrhizine I-16

[0096] Under argon atmosphere, 0.1 mmol of jatrorrhizine and 1.5 mL of acetonitrile were added into a 15 mL sealed tube and stirred well. After jatrorrhizine was completely dissolved in the solvent, 0.44 mmol of triethylamine was added, and the reaction was carried out at room temperature for 30 min. Then 0.12 mmol of cinnamoyl chloride was added to obtain a mixed solution. The mixed solution was stirred at room temperature for 18 h, and the reaction was monitored by TLC. After the reaction was complete, it was washed with saturated brine and extracted 5 times with 10 mL of dichloromethane. The organic layers were combined and dried over anhydrous Na2SO4. After filtration and concentration, it was purified by silica gel column chromatography to obtain 43.8 mg of yellow solid compound 3-cinnamoyloxy jatrorrhizine I-16; among them, the eluent with a volume ratio of dichloromethane:methanol = 15:1 was used for elution; with dichloromethane-methanol with a volume ratio of 15:1 as the developing agent, the R f value was 0.23, and the chemical structural formula was as follows:

[0097]

[0098] Test 1: Preliminary in vitro antibacterial activity detection

[0099] Experimental strains: Staphylococcus aureus (Staphylococcus aureus ATCC 29213), Bacillus subtilis (Bacillus subtilis ATCC 66330), Bacillus cereus (Bacillus cereus ATCC 14579), Staphylococcus epidermidis (Staphylococcus epidermidis ATCC 14990); Gram-negative bacteria: Escherichia coli (Escherichia coli DHB4), Pseudomonas aeruginosa (P. Aeruginosa PA14), Helicobacter pylori (Helicobacter pylori ATCC 43504) were all inoculated in the Microbiology Laboratory of the School of Pharmacy, Southwest University.

[0100] Positive control drugs: vancomycin hydrochloride, erythromycin.

[0101] Configuration of the culture medium:

[0102] LB liquid medium: Weigh 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl powder, mix and dissolve them in 1000 mL of deionized water, and stir well to dissolve. Then add 3 M NaOH dropwise to adjust the pH to 7.2. Use an autoclave, set the temperature to 121 °C, and perform moist heat sterilization for 20 min under the condition of 1×105 Pa. Store it in a 4 °C refrigerator for later use.

[0103] LB solid medium: Weigh 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl powder, dissolve them in 1000 mL of deionized water, and stir well to dissolve. Adjust the pH value to 7.2 with 3 M NaOH, and finally add 15 g of agar powder. Use an autoclave, set the temperature to 121 °C, and perform moist heat sterilization for 20 min under the condition of 1×105 Pa. When it cools to 50 - 60 °C, pour the medium into plates for later use. After the medium solidifies and cools to room temperature, invert the plates and store them in a 4 °C refrigerator for later use.

[0104] Columbia liquid medium: Weigh 29 g of Columbia medium powder and dissolve it in 1000 mL of deionized water, heat to boiling and stir until completely dissolved. Use an autoclave, set the temperature to 121 °C, and perform moist heat sterilization for 20 min under the condition of 1×105 Pa. Store it in a 4 °C refrigerator for later use.

[0105] Helicobacter pylori solid medium: Take 5.2 g of Columbia solid medium, heat and stir to completely dissolve it in deionized water (93 mL). Use an autoclave, set the temperature to 121 °C, and perform moist heat sterilization for 15 min under the condition of 1×105 Pa. When it cools to 50 - 55 °C, add defibrinated sheep blood (7 mL) and 1 HP inhibitor, mix well, pour it into a sterile petri dish. After the medium solidifies and cools to room temperature, invert the plates and store them in a 4 °C refrigerator for later use.

[0106] Cultivation of bacteria: Use a disposable sterile inoculation loop in a laminar flow hood to pick the test strains Staphylococcus aureus, Bacillus cereus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and inoculate them into the sterilized LB liquid medium, and culture overnight in a 37 °C constant temperature shaker. Then dilute the cultured bacterial solution with the corresponding liquid medium according to the ratio of bacteria: liquid medium = 1:1000. Among them, Helicobacter pylori is inoculated into the sterilized Columbia liquid medium, placed in a 37 °C triple-gas incubator and cultured overnight with shaking. Use a turbidimeter to test and adjust the concentration of the bacterial suspension to about 1.0, and then dilute the bacterial solution with Columbia liquid medium to 6×107 CFU / mL.

[0107] Determination of MIC value: Dissolve the sample to be tested and the positive control drug in DMSO solution to prepare a stock solution with a concentration of 2.56 mg / mL, ensuring that the DMSO concentration in each well is <5%. Then add 100 μL of LB liquid medium to each row of the 96-well plate, and then add 80 μL of LB liquid medium to the wells in the first row of the 96-well plate. Subsequently, take 20 μL of the sample to be tested and the positive control drug and add them to the first well in each row of the 96-well plate to ensure that the total volume of the solvent in the wells of the first row is 200 μL. Use a pipette to continuously blow and mix the solvent in the wells of the first row to make it homogeneous, and then perform two-fold dilutions on wells 1-9 in each column in sequence, skipping well 10 without adding. Take 100 μL of the solvent from well 9 and directly add it to well 11 to obtain a series of sample solutions with concentrations of 128-1 μg / mL. Then shake the prepared bacterial suspension well and take 100 μL and add it to wells 1-10, without adding the bacterial suspension to well 11, finally keeping the volume of each well at 200 μL. Then place the 96-well plate in a constant temperature incubator at 37 °C and incubate it at a constant temperature for 24 h. Then use a microplate reader to measure the absorbance of each well at 600 nm, and then judge the MIC value of the drug based on the absorbance value of each well. If well 10 is clear, the sample in that row needs to be further diluted to determine its accurate MIC value. If the wells in the first row are turbid, the sample in that row needs to be increased in concentration to determine its accurate MIC value. Set up 3 parallel experiments for each sample and the positive control drug.

[0108] For the determination of the MIC value of Helicobacter pylori, the two-fold dilution method is also used for drug dilution. That is, first add 180 μL and 100 μL of the prepared bacterial suspension to well 1 and wells 2-11 respectively, then take 20 μL of the drug and add it to the first well, mix it well and take 100 μL and add it to well 2, and so on until well 10. After mixing, take 100 μL and discard it to ensure that the final total volume of each well is 100 μL; the blank control group is 100 μL of the medium to obtain a series of sample solutions with concentrations of 128-1 μg / mL. Place the 96-well plate in a triple gas incubator at 37 °C and incubate it with constant shaking for 24 h. Set up 3 parallel experiments for each sample and the positive control drug.

[0109] Determination of MBC value: Take 10 μL of the solution in the well with the minimum MIC value in the MIC experiment, dilute it according to the ratio of solution: liquid medium = 1:100, mix it well and take 20 μL of the diluted solution and add it to the corresponding solid medium. Use a disposable sterile spreader to evenly spread the solution on the solid medium, and incubate the agar plate in a constant temperature incubator at 37 °C for 24 h. Observe the growth of bacteria. The concentration at which the number of bacterial colonies is less than 5 is regarded as the MBC value of the corresponding compound. The unit of MBC is μg / mL.

[0110] Table 1 Analysis of the anti-G+ bacteria activity of jatrorrhizine and its derivatives

[0111] Compd. S.aureus B.subtilis B.cereus S.epidermidis Jatrorrhizine >128 >128 >128 >128 I-2 >128 128 >128 64 I-3 <![CDATA[8 a > 8 <![CDATA[16 a > 4 I-4 4 4 2 1 I-5 1 2 2 0.5 I-6 8 8 2 1 I-8 8 16 8 4 I-9 <![CDATA[16 a > 16 <![CDATA[16 a > 8 I-10 >128 >128 >128 >128 I-11 >128 >128 >128 >128 I-12 32 64 <![CDATA[8 a > 16 I-14 64 128 <![CDATA[32 a > 32 I-15 64 64 64 <![CDATA[16 a > I-16 64 64 128 32 Erythromycin 4 1 1 2 Vancomycin hydrochloride 1 2 1 0.5

[0112] As can be seen from Table 1, the antibacterial activities of the 3-substituted jatrorrhizine derivatives in the examples are significantly better than those of jatrorrhizine. Among them, the antibacterial activity of Compound I-5 is better than that of the positive control drug erythromycin and is comparable to that of vancomycin hydrochloride. Judging from the experimental results, for most 3-substituted jatrorrhizine derivatives, the MIC values of their antibacterial activities against Gram-positive bacteria show an obvious trend of first increasing, then decreasing, and then increasing again as the length of the substituent chain increases. When the length of the introduced alkyl carbon chain is 6-12, the compounds show good antibacterial activities against all the tested G+ bacteria (Table 1, I-3, I-4, I-5, I-6). Among them, 3-decyloxy jatrorrhizine I-5 shows the best inhibitory activities against Staphylococcus aureus, Bacillus subtilis, Bacillus cereus and Staphylococcus epidermidis. In particular, its MIC value against Staphylococcus epidermidis can reach 0.5 μg / mL; in addition, when a phenylalkyloxy group is introduced at the 3-position, the derivatives also show good inhibitory activities against the tested G+ bacteria (Table 1, I-8, I-9). When an acyloxy group is introduced at the 3-position of jatrorrhizine and the number of carbon atoms in the acyloxy chain is less than 6, the inhibitory effects on Staphylococcus aureus and Bacillus subtilis are basically not improved. When the length of the introduced acyloxy carbon chain is 8-12, the inhibitory activities of the compounds against Staphylococcus aureus, Bacillus subtilis, Bacillus cereus and Staphylococcus epidermidis are significantly improved (Table 1, I-12). When an aromatic group is introduced at the 3-position, the compounds show general inhibitory activities against the tested G+ bacteria (Table 1, I-14, I-15, I-16), but there are varying degrees of improvement compared with jatrorrhizine.

[0113] Table 2 Analysis of the anti-G- bacterial activities of jatrorrhizine and its derivatives

[0114]

[0115]

[0116] As can be seen from Table 2, jatrorrhizine and a few derivatives have good inhibitory effects on Helicobacter pylori. Among them, 3-butoxy jatrorrhizine I-2 shows good antibacterial activity against Helicobacter pylori, and its MIC value is 8 μg / mL. Based on this compound, further structural modification and optimization can be carried out to obtain excellent Helicobacter pylori inhibitors. When an aromatic group is introduced at the 3-position (Table 2, I-14, I-15 and I-16), this type of derivative also shows good inhibitory activity against Helicobacter pylori, and its MIC values are all 16 μg / mL, which is 4 times higher than that of jatrorrhizine. In addition, the antibacterial effects of this type of jatrorrhizine on Escherichia coli and Pseudomonas aeruginosa are poor. The reason may be that the structures of these two Gram-negative bacteria are relatively complex, and it is difficult for drug molecules to enter, resulting in insignificant antibacterial effects. The antibacterial activity of the jatrorrhizine derivatives obtained in this invention is significantly better than that of the unmodified original alkaloid. Compounds I-4, I-5, and I-6 showed good antibacterial activity in the previous antibacterial activity study. To further explore their drug-likeness, their cytotoxicity against human tumor cell HepG2 was further studied.

[0117] Test 2: Cytotoxicity detection

[0118] Configuration of MTT: Weigh 250 mg of MTT, dissolve it in 50 mL of PBS, and sonicate it to fully dissolve it to prepare a 5 mg / mL MTT solution. Filter and dispense it through a 0.22 μm sterile filter membrane and store it at -20 °C for later use.

[0119] Configuration of DMEM medium: Prepared with 1% double antibodies (50 mg / L penicillin, 50 mg / L streptomycin) and 10% fetal bovine serum. After preparation, place it in a -4 °C refrigerator for later use.

[0120] Cell culture: Human hepatocellular carcinoma HepG2 cells are cultured in DMEM culture medium containing 10% fetal bovine serum in a cell culture incubator at 5% CO2 and 37 °C. When the cell growth reaches a confluence of 90%, digest the cells and plate them for experiments.

[0121] MTT method: Collect HepG2 cells in the logarithmic growth phase, discard the original medium, wash them 3 times repeatedly with PBS, add 2 mL of trypsin, place them in an incubator for digestion, quickly pour out the trypsin and add 5 mL of complete medium to cover the cell culture flask, continuously pipette and pat, collect the cell suspension, and centrifuge at 1000 rpm for 5 min. Add PBS (200 μL / well) to the outer wells of the 96-well plate, and add 100 μL of cell suspension to each of the remaining wells. After mixing, incubate in an incubator for 24 h. Observe the cell state under a microscope, discard the supernatant. For the 0 group, add 100 μL of medium to each well as a blank control group. For the 1 group, add 100 μL of the prepared solution to each well, perform gradient dilution, and incubate in an incubator for 24 h. Take out the 96-well plate, add 100 μL of PBS to each well and then aspirate and discard it. Add 100 μL of MTT (5 mg / mL) solution to each well, and incubate in an incubator for 4 h. Take out the 96-well plate, discard the supernatant, add 100 μL of DMSO, mix well on a micro oscillator, and after 5 min, measure the absorbance value of each well at a wavelength of 490 nm using a microplate reader.

[0122] Calculate the inhibition rate of the compound on HepG2 cells according to the following formula:

[0123]

[0124] From Figure 2 the results, it can be concluded that compounds I-4 and I-5 have a certain inhibitory effect on the proliferation of HepG2 cells. Among them, the IC 50 value of I-4 is 7.56 μg / mL, showing a certain cytotoxicity to HepG2 cells; the IC 50 value of I-5 is 35.54 μg / mL, and its therapeutic index for Staphylococcus aureus is 35.54 (35.54 / 1 = 35.54), and its therapeutic index for Staphylococcus epidermidis is 71.08 (35.54 / 0.5 = 71.08). Therefore, 3-decyloxy jatrorrhizine I-5 has relatively low cytotoxicity and relatively high safety, and can be further developed into a potential anti-Gram-positive bacterial infection drug. In addition, the inhibition rate of compound I-6 on HepG2 cells is relatively low, and its IC 50 values are all far greater than 400 μg / mL, and it has almost no cytotoxicity.

[0125] The above content is only an example and explanation of the present invention. Those skilled in the art of this technology can make various modifications or supplements to the described specific embodiments or use similar methods for substitution, as long as they do not deviate from the invention or exceed the scope defined by this claim book, they should all fall within the protection scope of the present invention.

Claims

1. A method for synthesizing jatrorrhizine derivatives with antibacterial activity, characterized in that, It includes the following steps: 1) Synthesis of (E)-2-methoxy-5-(2-nitrovinyl)phenol: Under a nitrogen atmosphere, 3-hydroxy-4-methoxybenzaldehyde, NH4OAc, molecular sieve and CH3NO2 were mixed and refluxed with stirring at 110 °C for 2 h to obtain a reaction solution; the reaction solution was cooled to room temperature and filtered through diatomaceous earth to remove the 5A molecular sieve to obtain a filtrate; the filtrate was distilled under reduced pressure using a rotary evaporator at 55 rpm to obtain a mixed organic matter from which nitromethane was removed; the mixed organic matter was dissolved in methanol at 60 °C and then recrystallized in a -20 °C refrigerator to obtain a yellowish-brown crystal of (E)-2-methoxy-5-(2-nitrovinyl)phenol; 2) Synthesize 2-methoxy-5-(2-nitroethyl)phenol: Mix THF and MeOH to obtain a mixed solvent; dissolve (E)-2-methoxy-5-(2-nitrovinyl)phenol in the mixed solvent, and then add sodium borohydride in three batches at 0 °C with an interval of 10 min for a total of 12 h to obtain a mixed solution; transfer the mixed solution to room temperature and react for 12 h, then adjust the pH value to 7 with 1 M hydrochloric acid, concentrate under reduced pressure to remove THF, extract with ether 3 times, combine the ether layers, wash with saturated NaCl 2 times, add anhydrous MgSO4 for drying, concentrate the filtrate to obtain a residue, and purify the residue by silica gel column chromatography to obtain 2-methoxy-5-(2-nitroethyl)phenol as a light yellow oily liquid; among them, an eluent with a volume ratio of petroleum ether∶ethyl acetate = 8∶1 is used for elution; 3) Synthesize 3-hydroxy-4-methoxyphenethylamine hydrochloride: Dissolve 2-methoxy-5-(2-nitroethyl)phenol in ethyl acetate, add 37 wt% concentrated hydrochloric acid, and stir at 100 r / min for 5 min to obtain a mixture; add zinc powder to the mixture at 70 °C, stir at 100 r / min for 10 min, then adjust the temperature to 50 °C and reflux and stir to obtain a mixed solution, filter the mixed solution to remove zinc powder to obtain a filtrate; dry the filtrate with anhydrous sodium sulfate, filter, and concentrate under reduced pressure to evaporate ethyl acetate to obtain a remaining oily liquid; add concentrated hydrochloric acid to the remaining oily liquid, freeze overnight at -20 °C, and filter to obtain white solid 3-hydroxy-4-methoxyphenethylamine hydrochloride; 4) Synthesize 1-(dimethoxymethyl)-7-methoxy-1,2,3,4-tetrahydroisoquinolin-6-ol: Mix 3-hydroxy-4-methoxyphenethylamine hydrochloride, triethylamine, and dichloromethane in a reaction vessel, and add 60 wt% of 2,2-dimethoxyacetaldehyde after extraction with dichloromethane to the above mixture to obtain a reaction mixture; stir the reaction mixture at room temperature for 30 min, and then dropwise add TFA under ice bath conditions and react for 1 h; after the reaction is completed, adjust the pH to 7 with 3 M sodium hydroxide solution, extract with DCM 3 times, combine the organic phases, dry with anhydrous Na2SO4, filter and concentrate to obtain a residue; purify the residue by silica gel column chromatography to obtain 1-(dimethoxymethyl)-7-methoxy-1,2,3,4-tetrahydroisoquinolin-6-ol as a yellow oily liquid; among them, an eluent with a volume ratio of dichloromethane∶methanol = 10∶1 is used for elution; 5) Synthesis of 2-(2,3-dimethoxybenzyl)-7-methoxy-1-(dimethoxymethyl)-1,2,3,4-tetrahydroisoquinolin-6-ol: Dissolve 1-(dimethoxymethyl)-7-methoxy-1,2,3,4-tetrahydroisoquinolin-6-ol in 1,2-dichloroethane, add 2,3-dimethoxybenzaldehyde, mix well, then add sodium triacetoxyborohydride at room temperature. Stir and react at 100 r / min for 4 h, and then add saturated NaHCO₃ to obtain a mixed solution; Extract the mixed solution with DCM three times, combine the organic layers, add anhydrous MgSO₄ for drying, filter and concentrate to obtain a residue; Purify the residue by silica gel column chromatography to obtain 2-(2,3-dimethoxybenzyl)-7-methoxy-1-(dimethoxymethyl)-1,2,3,4-tetrahydroisoquinolin-6-ol as a dark yellow oily liquid; Among them, elute with an eluent with a volume ratio of petroleum ether∶ethyl acetate = 1∶1; 6) Synthesis of jatrorrhizine: Mix 2-(2,3-dimethoxybenzyl)-7-methoxy-1-(dimethoxymethyl)-1,2,3,4-tetrahydroisoquinolin-6-ol and dichloromethane and add them to a round-bottom flask. Dropwise add trifluoromethanesulfonic acid at 0 °C under argon protection. After reacting for 5 min, remove the ice bath and transfer it to room temperature at 25 °C and continue stirring for 1 h to obtain a mixture; Dilute the mixture with dichloromethane and wash it with sodium bicarbonate solution to remove acid to obtain an organic layer; Dry the organic layer with anhydrous Na₂SO₄, filter and concentrate to obtain a concentrated filtrate; Add potassium acetate and ethanol to the concentrated filtrate, dropwise add an iodine solution of ethanol at 1 mL / s at 1000 r / min. After reacting for 1 h, add a 10 wt% sodium thiosulfate solution, filter through diatomaceous earth and wash the filter cake with methanol to obtain a concentrated filtrate; Purify the concentrated filtrate by silica gel column chromatography to obtain a light yellow solid product; Among them, elute with an eluent with a volume ratio of dichloromethane∶methanol = 15∶1; 7) Synthesis of jatrorrhizine derivatives: Add jatrorrhizine, a base scavenger, and the solvent N,N-dimethylformamide / acetonitrile to a sealed tube at room temperature. Stir at room temperature for 30 min, then add an alkyl halide / alkyl acyl chloride. Stir the mixed solution at room temperature for 16 - 18 h, and detect the reaction by TLC; After the reaction is completed, separate to obtain jatrorrhizine derivatives; The said jatrorrhizine derivatives are isoquinoline-type berberine alkaloids with a quaternary iminium ion fragment, and the chemical structure general formula I of the said jatrorrhizine derivatives is as follows: Among them, in general formula I, R is phenylpropyl or benzoyl; Y is one of chlorine and bromide ions.

2. The synthesis method of jatrorrhizine derivatives according to claim 1, characterized in that, The base scavenger in step 7) is sodium carbonate, potassium carbonate, cesium carbonate, triethylamine, trimethylamine or pyridine.

3. The synthesis method of jatrorrhizine derivatives according to claim 1, characterized in that, The said separation adopts silica gel column chromatography separation method, and the eluent is a mixed solution of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 15:

1.

4. The synthesis method of jatrorrhizine derivatives according to claim 1, characterized in that, The structural general formulas of the said alkyl halide and acyl halide are as follows: Among them, R and Y are defined as in claim 1.

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