Sinomenine derivative as well as synthesis method and application thereof

Abstract

The invention discloses a sinomenine derivative and a synthesis method and application thereof, and belongs to the technical field of biology and medicines.The sinomenine derivative is synthesized through the steps that sinomenine and acryloyl chloride are subjected to a room-temperature reaction under the catalysis of triethylamine, then a product in reaction liquid is separated and purified, and an intermediate is obtained; the intermediate and substituted aniline are subjected to a room temperature reaction for 11-13 h under the catalysis of organic alkali, then a product in reaction liquid is separated and purified, and the sinomenine derivative is obtained and can be used as an antifungal agent or applied to preparation of antifungal drugs, and the sinomenine derivative and fluconazole are combined to be used as an antifungal drug synergist. The sinomenine derivative not only provides a new candidate drug for fungal treatment, but also improves the antifungal effect of the existing drug.

Description

Technical Field

[0001] The invention belongs to the field of biology and medicine technology, and specifically relates to a sinomenine derivative and a synthesis method and application thereof. Background Art

[0002] Candida is the most common pathogen of invasive fungal infection. According to statistics, more than 95% of invasive fungal infections are caused by five pathogens, including Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis and Candida krusei. In recent years, the incidence of the above five fungi has increased to varying degrees, and there is a potential threat of outbreaks. Invasive fungal infections require long-term use of antifungal drugs for treatment. Commonly used antifungal drugs in clinical practice mainly include triazoles, polyenes and echinocandins. Among them, triazoles such as fluconazole are still the first-line antifungal drugs for the treatment of Candida.

[0003] With the long-term and repeated use of antifungal drugs in clinical practice, the drug resistance of Candida species has become increasingly serious. Currently, only a few antifungal drugs have been approved for clinical use, and the emergence of fungal resistance to existing drugs has made the treatment of fungal infections more difficult.

[0004] Sinomenine is an alkaloid extracted from the stems of Sinomenine or Sinomenine of the Menispermaceae family. It is widely present in natural plants and has a variety of biological activities, such as anti-inflammatory, analgesic, immunomodulatory, anti-arrhythmic, antioxidant, anti-tumor, and drug addiction treatment. Sinomenine derivatives are a series of new compounds obtained by introducing different functional groups or structures based on Sinomenine through chemical synthesis methods. Most Sinomenine derivatives retain the original pharmacological activity of Sinomenine and show higher efficacy and lower side effects. For example, some Sinomenine derivatives show better activity than Sinomenine in anti-inflammatory, analgesic, and immunosuppressive aspects. Therefore, the synthesis of Sinomenine derivatives has broad application prospects, and it is hoped that fungi will overcome the resistance to existing drugs and solve the problem of clinical fungal infection treatment. However, up to now, there is no safe and effective application of Sinomenine derivatives in antifungal drugs or synergists. Summary of the invention

[0005] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a sinomenine derivative and its synthesis method and application, the raw materials are easily available, the operation is simple and environmentally friendly, and the product is used in combination with fluconazole to treat fungal infections.

[0006] The present invention is implemented by the following technical solutions:

[0007] A sinomenine derivative, the structural formula of the sinomenine derivative is as shown in formula (I) or (II):

[0008]

[0009] In formula (I), R1 is hydrogen, methyl, hydroxy, halogen or methoxy;

[0010] In formula (II), R2 is an amino group.

[0011] Preferably, the specific structural formula of the sinomenine derivative is any one of the following formulas:

[0012]

[0013] A method for synthesizing a sinomenine derivative comprises the following steps:

[0014] S1, reacting sinomenine and acryloyl chloride at a molar ratio of 1:1.5 under the catalysis of triethylamine at room temperature, and then separating and purifying the product in the reaction solution to obtain an intermediate;

[0015] S2, reacting the intermediate and substituted aniline at a molar ratio of 1:4 under the catalysis of an organic base at room temperature for 11 to 13 hours, and then separating and purifying the product in the reaction solution to obtain a sinomenine derivative.

[0016] Preferably, the reactions described in S1 and S2 are both carried out in dichloromethane. When isolating and purifying the product, the product is first extracted into dichloromethane, then dried over anhydrous sodium sulfate, and then dichloromethane is removed by vacuum distillation. Finally, the product is purified by column chromatography.

[0017] Preferably, the molar ratio of triethylamine to sinomenine in S1 is 1:2, and the reaction is carried out for 2.5 to 3.5 hours.

[0018] Preferably, the substituted aniline structural formula described in S2 is:

[0019]

[0020] Among them, R 2-1 is hydrogen, hydroxy, bromine, methoxy, mercapto or methyl; R 2-2 is hydrogen or methoxy; R 2-3 is hydrogen, bromine, chlorine or methyl.

[0021] Preferably, the organic base in S2 is 1,8-diazabicyclo[5.4.0]undec-7-ene, and the molar ratio of 1,8-diazabicyclo[5.4.0]undec-7-ene to the substituted aniline is 10:1.

[0022] The invention discloses an application of a sinomenine derivative as an antifungal agent or in the preparation of an antifungal medicine.

[0023] Preferably, the sinomenine derivative is used in combination with fluconazole as an antifungal drug synergist.

[0024] Preferably, the fungi include resistant Candida albicans (103), Candida albicans SC5314, Candida parapsilosis, Candida krusei, Candida glabrata and Cryptococcus neoformans.

[0025] Compared with the prior art, the present invention has the following beneficial technical effects:

[0026] The sinomenine derivative of the present invention connects the modified aniline structure to sinomenine to form a new skeleton structure. The modified aniline can increase receptor selectivity, thereby reducing off-target effects and related toxicity, has better drugability, and is helpful for the development of different types of derivatives. The amino group is a highly active and easily oxidized group. The secondary amine structure retained after modification also provides convenience for the subsequent continued use of the derivative. The derivative can be used in combination with fluconazole to significantly enhance its anti-Candida albicans activity and can be used to develop drugs against drug-resistant fungi.

[0027] The present invention discloses a method for synthesizing sinomenine derivatives. Sinomenine is a plant extract. There are two relatively active reaction sites on its A ring, namely, active hydrogen at position 1 and phenolic hydroxyl at position 4. It can react with acryloyl chloride at room temperature under the catalysis of triethylamine, which not only ensures its thermal stability as a plant extract, but also provides a mild reaction method. An active intermediate with a terminal olefin structure is synthesized, which is helpful for reacting with substituted aniline at room temperature under the catalysis of an organic base to develop sinomenine derivatives. Traditional methods modify phenolic hydroxyl groups and mostly select etherification reactions. Synthesis is performed using flammable compounds, which needs to be controlled below zero degrees. The reaction conditions are relatively harsh, and reflux conditions are often used to derive the active hydrogen at position 1 on the A ring of sinomenine. The present invention has mild reaction conditions, is easy to operate and control, is simple to operate, has a wide range of applications, and is suitable for industrial production.

[0028] Sinomenine derivatives can be used as antifungal agents and can also be used in the preparation of antifungal drugs. Sinomenine derivatives not only provide new candidate drugs for fungal treatment, but also improve the antifungal effect of existing drugs. In the case of increasingly common and serious drug resistance of clinical fungi, antifungal drugs can restore their effects on drug-resistant fungi and reduce the dosage of antifungal drugs, thereby saving medical expenses for patients and reducing the toxic side effects of drugs.

[0029] Furthermore, the sinomenine derivatives can be used as synergists of antifungal drugs in combination with fluconazole. The sinomenine derivatives used in combination with the antifungal drug fluconazole can significantly reduce the dosage of the antifungal drug, enhance the inhibitory effect on drug-resistant fungi, and restore the effect of the antifungal drug fluconazole on drug-resistant fungi, with obvious antifungal effects, and the effect is better than the compound itself and fluconazole. As an antifungal drug synergist, this shows that the compound has certain advantages in antifungal effects and has certain development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This graph shows the effect of the combination of derivative 3ja and different concentrations of fluconazole on the growth of drug-resistant Candida albicans 103.

[0031] Figure 2 This graph shows the effect of different concentrations of 3ja combined with fluconazole on the growth of drug-resistant Candida albicans 103. DETAILED DESCRIPTION

[0032] The following will be combined with the embodiments to clearly and completely describe the concept, specific content and technical effects of the present invention, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work belong to the scope of protection of the present invention. The test methods used in the embodiments are conventional methods unless otherwise specified, and the materials and reagents used are all commercially available.

[0033] The present invention discloses a sinomenine derivative, the general structural formula of which is shown in formula (I) and (II):

[0034]

[0035] In formula (I), R1 includes any one of hydrogen, methyl, hydroxy, halogen or methoxy.

[0036] In formula (II), R2 is an amino group.

[0037] The specific structural formula is any one of the following:

[0038]

[0039] The present invention provides a method for synthesizing a sinomenine derivative, comprising the following steps:

[0040] S1, sinomenine hydrochloride is stirred in dichloromethane, and ammonia water is slowly added dropwise until sinomenine is dissolved in 2eq dichloromethane, the molar ratio of sinomenine hydrochloride to ammonia water is 1:1.5-2.5, and then reacted at room temperature for 0.5h, and then extracted three times (the organic phase is dichloromethane, the aqueous phase is saturated brine, and the volume ratio of the organic phase to the aqueous phase is 1:3), dried with anhydrous sodium sulfate, and finally distilled under reduced pressure to remove the solvent to obtain sinomenine.

[0041] S2, react the sinomenine obtained in S1 with acryloyl chloride (molar ratio of 1:1.5) in dichloromethane (10 mL for sinomenine 1 mmol) at room temperature for 2.5 to 3.5 h, the catalyst is triethylamine (molar ratio with sinomenine is 1:2), separate by extraction (organic phase is dichloromethane, aqueous phase is saturated saline, volume ratio of organic phase to aqueous phase is 1:3), dry with anhydrous sodium sulfate, and remove the solvent by reduced pressure distillation. Purify the compound by column chromatography, wherein dichloromethane and methanol with a volume ratio of 50:1 are used as the mobile phase, and the intermediate (4bR, 8aR, 9S)-3,7-dimethoxy-11-methyl-6-oxo-6,8a,9,10-tetrahydro-5H-9,4b-

[0042] (Epiaminoethyl)phenanthrene-4-acrylate.

[0043] S3, the intermediate obtained in S2 and different substituted anilines (molar ratio of 1:4) are placed in a test tube containing a stirrer, dissolved in dichloromethane (1 mL when the substituted aniline is 0.4 mmol), and then the catalyst 1,8-diazabicyclo[5.4.0]undec-7-ene (molar ratio of 10:1 to the substituted aniline) is added under stirring, and the reaction is carried out at room temperature for 11 to 13 hours. The reaction substances are observed over time by thin layer chromatography to determine the reaction process and the formation of products, and separated by extraction (the organic phase is dichloromethane, the aqueous phase is saturated salt water, and the volume ratio of the organic phase to the aqueous phase is 1:3), dried with anhydrous sodium sulfate, and the solvent is removed by distillation under reduced pressure. The compound is purified by column chromatography, with dichloromethane and methanol in a volume ratio of 25:1 as the mobile phase, and a derivative of the sinomenine structure is obtained.

[0044] Its structural formula is:

[0045]

[0046] The structural formula of substituted aniline is:

[0047] or or

[0048] Among them, R 2-1 is one of hydrogen, hydroxyl, bromine, methoxy, mercapto and methyl; R 2-2 is hydrogen or methoxy; R 2-3 is hydrogen, bromine, chlorine or methyl.

[0049] Example 1

[0050] In the first step, sinomenine hydrochloride (1 mol, 1.0 eq) was placed in a round-bottom flask with a stirrer, 2 eq of dichloromethane was added, and the molar ratio of dichloromethane to sinomenine hydrochloride was 1:2. Ammonia water was added dropwise at room temperature for 0.5 h until sinomenine was completely dissolved. The product was extracted three times (the organic phase was dichloromethane, the aqueous phase was saturated brine, and the volume ratio of the organic phase to the aqueous phase was 1:3). After the organic phase was dried with anhydrous sodium sulfate, the solvent was removed by reduced pressure distillation to obtain sinomenine (3.1 g, yield 95%).

[0051]

[0052] In the second step, sinomenine 1a (1 mol, 1.0 eq) was placed in a round-bottom flask with a stirrer, 10 mL of dichloromethane was added, triethylamine (2 mol, 2.0 eq) was added at room temperature, acryloyl chloride (1.5 mol, 1.5 eq) was slowly added dropwise, and the mixture was stirred at room temperature for three hours. The reaction was monitored by thin layer chromatography (TLC). The product was repeatedly extracted three times with dichloromethane and saturated brine, and the organic phase was dried with anhydrous sodium sulfate and the solvent was removed by reduced pressure distillation to obtain a crude product. After column chromatography (dichloromethane: methanol = 50: 1), (4bR, 8aR, 9S)-3,7-dimethoxy-11-methyl-6-oxo-6,8a, 9,10-tetrahydro-5H-9,4b-(epiaminoethyl)phenanthrene-4-acrylate (2.52 g, yield 66%) was obtained, and the reaction equation is as follows:

[0053]

[0054] In the third step, (4bR, 8aR, 9S)-3,7-dimethoxy-11-methyl-6-oxo-6,8a, 9,10-tetrahydro-5H-9,4b-(epiaminoethyl)phenanthrene-4-acrylate 2a (0.1mmol, 1.0eq) and substrate aniline (0.4mmol, 4.0eq) were placed in a test tube containing a stirrer, 1mL of dichloromethane was added, and (0.04mmol, 6μL) 1,8-diazabicyclo[5.4.0]undec-7-ene was added under stirring, and stirred at room temperature for twelve hours. The reaction was monitored by thin layer chromatography, and the product was repeatedly extracted three times with dichloromethane and saturated brine (volume ratio of 1:3), and the organic phase was dried with anhydrous sodium sulfate and the solvent was removed by reduced pressure distillation to obtain a crude product. After column chromatography (dichloromethane: methanol = 25:1), the target compound sinomenine derivative 3aa was obtained.

[0055]

[0056] The structural formula of the target compound is as follows:

[0057]

[0058] The compound1 H NMR and HRMS data are as follows:

[0059] 1 H NMR (600MHz, CDCl3) δ7.18(t,J=7.8Hz,2H),6.92(d,J=8.5Hz,1H),6.76(d,J=8.4 Hz,1H),6.69(d,J=8.0Hz,3H),5.48(s,1H),3.70(s,3H),3.49(s,3H),3.20(t,J= 4.5Hz,1H),3.09–3.01(m,1H),3.00(s,1H),2.88(ddd,J=16.3,6.6,4.7Hz,1H),2 .81–2.70(m,1H),2.56–2.47(m,2H),2.45(d,J=8.5Hz,4H).ESI-HRMS:calcd.for C 28 H 32 N2O5+H,477.2384found 477.2389.

[0060] It can be proved that the obtained substance is indeed derivative 3aa.

[0061] Example 2

[0062] The target compound 3ba was prepared according to the method and steps of Example 1, except that the raw material c in the third step was replaced by the compound c-1, and the rest of the method was the same as Example 1. The specific reaction formula is as follows:

[0063]

[0064] The structural formula is as follows:

[0065]

[0066] The compound 1 H NMR and HRMS data are as follows:

[0067] 1H NMR (400MHz, CDCl3) δ7.08(t,J=8.0Hz,1H),6.92(d,J=8.4Hz,1H),6.75(d,J=8.4Hz,1H),6 .33–6.20(m,3H),5.51–5.43(m,1H),3.77(s,3H),3.70(s,3H),3.49(s,3H),3.18(s,1H),3 .08(s,1H),3.03(s,1H),3.00–2.82(m,4H),2.76(d,J=5.8Hz,1H),2.71(d,J=5.7Hz,1H),2 .51(d,J=9.4Hz,2H),2.43(s,4H),1.83(d,J=4.7Hz,1H),1.25(s,2H).ESI-HRMS:calcd.for C 28 H 34 N2O6+Na,529.2309 found 529.2282.

[0068] It can be proved that the obtained substance is indeed the derivative 3ba.

[0069] Example 3

[0070] Prepared according to the method and steps of Example 1, the raw material c in the third step was changed to compound c-2, and the rest of the method was the same as in the example to obtain the target compound 3ca, the specific reaction formula is as follows:

[0071]

[0072] The structural formula is as follows:

[0073]

[0074] The compound 1 H NMR and HRMS data are as follows:

[0075] 1H NMR (600MHz, CDCl3) δ7.08(t,J=8.1Hz,1H),6.92(d,J=8.4Hz,1H),6.76(d,J=8.4Hz,1H),6.32–6. 26(m,2H),6.24(s,1H),5.48(s,1H),3.77(s,3H),3.71(s,3H),3.60(s,1H),3.49(s,3H),3.18(s,1 H),3.07(s,1H),3.04(s,1H),2.95(d,J=13.3Hz,2H),2.87(d,J=9.8Hz,1H),2.73(d,J=13.1Hz,1H ),2.50(d,J=8.0Hz,1H),2.46(s,1H),2.43(s,3H),1.43(s,1H),1.25(s,2H).ESI-HRMS:calcd.for C 28 H 34 N2O6+H,506.2417found506.2422.

[0076] It can be proved that the obtained substance is indeed the derivative 3ca.

[0077] Example 4

[0078] Prepared according to the method and steps of Example 1, the raw material c in the third step was changed to compound c-3, and the rest of the method was the same as in the example to obtain the target compound 3da, the specific reaction formula is as follows:

[0079]

[0080] The structure is as follows:

[0081]

[0082] The compound 1 H NMR and HRMS data are as follows:

[0083] 1H NMR (600MHz, CDCl3) δ7.16–7.12(m,1H),7.04(d,J=7.3Hz,1H),6.91(d,J=8.4Hz, 1H),6.79–6.60(m,3H),5.47(d,J=10.7Hz,1H),3.66(s,3H),3.47(s,3H),3.16(d, J=4.7Hz,1H),3.00–2.90(m,3H),2.74(dd,J=8.4,5.7Hz,1H),2.71(dd,J=8.5,5. 6Hz,1H),2.52–2.40(m,6H),2.11(s,4H),1.92–1.74(m,2H).ESI-HRMS:calcd.for C 29 H 34 N2O5+H,491.2540found 491.2546.

[0084] It can be proved that the obtained substance is indeed derivative 3da.

[0085] Example 5

[0086] Prepared according to the method and steps of Example 1, the raw material c in the third step was changed to compound c-4, and the rest of the method was the same as in the example to obtain the target compound 3ea, the specific reaction formula is as follows:

[0087]

[0088] The structure is as follows:

[0089]

[0090] The compound 1 H NMR and HRMS data are as follows:

[0091] 1H NMR (600MHz, CDCl3) δ7.00(d,J=8.4Hz,2H),6.91(d,J=8.4Hz,1H),6.75(d,J=8.5Hz,1H),6.62(d,J=8.4Hz, 2H),5.48(d,J=2.2Hz,1H),3.71(s,3H),3.49(s,3H),3.17(t,J=4.5Hz,1H),3.07(s,1H),3.04(s,1H),2.99– 2.92(m,2H),2.87(s,1H),2.74(d,J=6.7Hz,1H),2.71(d,J=6.4Hz,1H),2.49(dtd,J=12.0,6.0,3.6Hz,2H), 2.44(d,J=6.2Hz,1H),2.43(s,3H),2.24(s,3H),1.85–1.77(m,1H),1.32–1.18(m,2H).ESI-HRMS:calcd.for C 29 H 34 N2O5+H,491.2540found 491.2548.

[0092] It can be proved that the obtained substance is indeed the derivative 3ea.

[0093] Example 6

[0094] Prepared according to the method and steps of Example 1, the raw material c in the third step was changed to compound c-5, and the rest of the method was the same as in the example to obtain the target compound 3fa, the specific reaction formula is as follows:

[0095]

[0096] The structure is as follows:

[0097]

[0098] The compound 1 H NMR and HRMS data are as follows:

[0099] 1H NMR (600MHz, CDCl3) δ7.24(d,J=8.3Hz,2H),6.91(d,J=8.4Hz,1H),6.74(d,J=8.3 Hz,1H),6.57(d,J=8.3Hz,2H),5.49(s,1H),3.68(s,3H),3.48(s,3H),3.16(s,1H ),3.06(s,1H),3.03(s,1H),2.99–2.88(m,3H),2.83(dt,J=16.3,5.4Hz,1H),2.7 5-2.68(m,1H),2.41(s,6H),2.08(s,1H),1.87-1.72(m,2H).ESI-HRMS:calcd.for C 28 H 31 BrN2O5+H,555.1489found 555.1494.

[0100] It can be proved that the obtained substance is indeed the derivative 3fa.

[0101] Example 7

[0102] Prepared according to the method and steps of Example 1, the raw material c in the third step was changed to compound c-6, and the rest of the method was the same as in the example to obtain the target compound 3ga, the specific reaction formula is as follows:

[0103]

[0104] The structure is as follows:

[0105]

[0106] The compound 1 H NMR and HRMS data are as follows:

[0107] 1H NMR (600MHz, CDCl3) δ7.41(dd,J=7.9,1.5Hz,1H),7.20(t,J=7.7Hz,1H),6.91(d,J=8.4Hz,1H),6.75(d, J=9.8Hz,2H),6.59–6.56(m,1H),5.46(d,J=2.3Hz,1H),3.69(s,3H),3.47(s,3H),3.17(t,J=4.5Hz,1H), 3.06(s,1H),3.03(s,1H),3.01–2.91(m,3H),2.73(d,J=5.5Hz,1H),2.70(d,J=5.5Hz,1H),2.50(dd,J=4 .8,1.9Hz,1H),2.48(dd,J=4.7,2.0Hz,1H),2.42(s,4H),2.09(s,1H),1.25(s,2H).ESI-HRMS:calcd.for C 28 H 31 BrN2O5+H,556.4765found 556.4760.

[0108] It can be proved that the obtained substance is indeed the derivative 3ga.

[0109] Example 8

[0110] Prepared according to the method and steps of Example 1, the raw material c in the third step was changed to compound c-7, and the rest of the method was the same as in the example to obtain the target compound 3ha, the specific reaction formula is as follows:

[0111]

[0112] The structure is as follows:

[0113]

[0114] The compound 1 H NMR and HRMS data are as follows:

[0115] 1H NMR (600MHz, CDCl3) δ7.06(d,J=8.5Hz,2H),6.86(d,J=8.5Hz,1H),6.70(dd,J=8.4,6.7Hz,1H),6.56(d,J=8 .9Hz,2H),5.41(d,J=8.7Hz,1H),3.64(d,J=12.4Hz,3H),3.53–3.47(m,1H),3.42(d,J=3.6Hz,3H),3.11(s, 1H),3.01(s,1H),2.97(s,1H),2.89(s,1H),2.87–2.84(m,1H),2.77(d,J=11.1Hz,1H),2.67(s,1H),2.65(s ,1H),2.45–2.41(m,1H),2.40(d,J=6.2Hz,1H),2.36(s,3H),2.04(s,1H),1.76(s,2H).ESI-HRMS:calcd.for C 28 H 31 ClN2O5+H,511.1994found 511.1960

[0116] It can be proved that the obtained substance is indeed the derivative 3ha.

[0117] Example 9

[0118] Prepared according to the method and steps of Example 1, the raw material c in the third step was changed to compound c-9, and the rest of the method was the same as in the example to obtain the target compound 3ia, the specific reaction formula is as follows:

[0119]

[0120] The structure is as follows:

[0121]

[0122] The compound 1 H NMR and HRMS data are as follows:

[0123] 1H NMR (600MHz, CDCl3) δ7.37(d,J=9.4Hz,1H),7.07(t,J=7.6Hz,1H),6.84(d,J=8.5Hz,1H),6.70–6.65(m,2H ),6.63(t,J=7.5Hz,1H),5.40(d,J=2.4Hz,1H),4.41(s,2H),3.66(s,3H),3.41(s,3H),3.11(s,1H),3.09– 3.00(m,2H),2.91(s,1H),2.85(d,J=16.3Hz,1H),2.78–2.72(m,1H),2.68(d,J=5.4Hz,1H),2.65(d,J=5.4 Hz,1H),2.48–2.37(m,3H),2.36(s,3H),2.02(d,J=11.6Hz,1H),1.84–1.72(m,2H).ESI-HRMS:calcd.forC 28 H 32 N2O5S+H,509.2105found 509.2109.

[0124] It can be proved that the obtained substance is indeed derivative 3ia.

[0125] Example 10

[0126] Prepared according to the method and steps of Example 1, the raw material c in the third step was changed to compound c-10, and the rest of the method was the same as in the example to obtain the target compound 3ja, the specific reaction formula is as follows:

[0127]

[0128] The structure is as follows:

[0129]

[0130] The compound 1 H NMR and HRMS data are as follows:

[0131] 1H NMR (400MHz, CDCl3) δ6.92(s,1H),6.90(s,1H),6.82(d,J=8.0Hz,1H),6.76(d,J=8.4Hz,2H),6.64(d,J=8.2Hz,2H),5.43(s,1H),3.72(s,3H),3.4 6(s,3H),3.23(s,2H),3.12–2.88(m,5H),2.77(d,J=12.4Hz,2H),2.58–2.45(m,3H),2.43(s,3H),2.11(s,1H),1.25(s,2H).ESI-HRMS:calcd.for C 28 H 32 N2O6+H,493.5795found 493.5799.

[0132] It can be proved that the obtained substance is indeed derivative 3ja.

[0133] The antibacterial properties of some products were tested, and the description is continued with the previous example.

[0134] Embodiment 11

[0135] Effect of 3ja combined with fluconazole on fungal growth

[0136] 1. Drug testing

[0137] Drug 3ja: Laboratory synthesis.

[0138] Fluconazole: Sigma, batch number 036M4709V.

[0139] Dimethyl sulfoxide: Tianjin Tianli Chemical Reagent Co., Ltd.

[0140] Each test drug was stored at -20°C. Before the experiment, the drug was taken out and placed in a 35°C incubator to melt, mixed thoroughly, and then passed the pharmacodynamic test.

[0141] 2. Strains

[0142] The strains used include:

[0143] Resistant strains: Resistant Candida albicans (103), Candida albicans SC5314, Candida parapsilosis, Candida krusei, Candida glabrata, Cryptococcus neoformans

[0144] All of the above were provided by the Fungi Laboratory of Shanghai Changhai Hospital and were identified by morphology and biochemistry.

[0145] All experimental strains were activated by streaking on Sandcastle Dextrose Agar (SDA) medium, and single clones were picked out for re-streaking and activation. The single clone obtained for the second time was placed on an SDA slant, cultured using the above method, and stored at 4°C for future use.

[0146] 3. Culture medium / base

[0147] 1) RPMI 1640 liquid culture medium

[0148] RPMI 1640 (Gibco BRL) 10g, NaHCO3 2.0g, morpholinepropanesulfonic acid (Sigma) 34.5g (0.165mol), add triple distilled water 900mL to dissolve, adjust the pH to 7.0 (25℃) with 1mol / LNaOH, make up to 1000mL with triple distilled water, filter and sterilize with 0.22μM microporous filter membrane, and store at 4℃ for later use.

[0149] 2) Sandcastle Dextrose Agar (SDA)

[0150] Dissolve 10 g of peptone, 40 g of glucose, and 18 g of agar in 900 mL of triple-distilled water, add 50 mL of 2 mg / mL chloramphenicol aqueous solution, adjust the pH to 7.0, make up to 1000 mL with triple-distilled water, sterilize by high pressure (121°C, 15 min), and store at 4°C for later use.

[0151] 3) YEPD culture medium

[0152] Dissolve 10 g of yeast extract, 20 g of peptone, and 20 g of glucose in 900 mL of triple-distilled water, add 50 mL of 2 mg / mL chloramphenicol aqueous solution, make up to 1000 mL with triple-distilled water, sterilize by high pressure (121°C, 15 min), and store at 4°C for later use.

[0153] 4. Instruments and Equipment

[0154] Water-insulated electric constant temperature incubator (Shanghai Yuejin Medical Equipment Factory);

[0155] THZ-82A desktop constant temperature oscillator (Shanghai Yuejin Medical Equipment Factory);

[0156] SW-CT-IF ultra-clean workbench (Suzhou Antai Air Technology Co., Ltd.).

[0157] 5. 3ja and fluconazole stock solutions

[0158] 3ja and fluconazole were dissolved in DMSO respectively to prepare a drug solution with a concentration of 6.4 mg / mL, and stored at -20°C for later use.

[0159] 6. Preparation of bacterial solution

[0160] Before the experiment, a small amount of the above fungi was picked from the SDA medium stored at 4°C using an inoculation loop and inoculated into 1 mL of YEPD culture medium. The culture was shaken at 35°C and 200 rpm for 16 h to activate the fungi in the late exponential growth phase. Then, each bacterial solution was added to 1 mL of YEPD culture medium, activated again using the above method, cultured for 16 h, counted using a hemocytometer, and the concentration of each bacterial solution was adjusted to 1×10 3 ~5×10 3 CFU / mL.

[0161] 7. Preparation of drug sensitivity panels for multiple fungi using drug 3ja in combination with fluconazole:

[0162] Take a sterile 96-well plate for each strain, add 100 μL of RPMI 1640 liquid culture medium to well 1 in each row as a blank control; add 100 μL of the above freshly prepared bacterial solution to wells 3 to 12; add 198 μL of bacterial solution to well 2; well 12 does not contain any drug, and only 100 μL of bacterial solution is added as a positive growth control. The prepared bacterial solution and two tested drugs were added to wells 2 to 11, so that the final concentrations of fluconazole in each well in the horizontal direction (same in each row) were 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL and 0.125 μg / mL, respectively, and the concentrations of 3ja in each well in the vertical direction (a total of 6 rows, each column is the same) were 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL and 1 μg / mL, respectively. The DMSO content in each well was less than 1%. Each drug sensitivity plate was cultured in a 35°C incubator.

[0163] 8. MIC value determination:

[0164] In a 35°C incubator, the fungus was cultured for 24h or 72h, the experimental results were observed, and the MIC value was determined. When the MIC value of the drug exceeds the measured concentration range, it was counted as follows: when the MIC value is higher than the highest concentration of 64μg / mL, it is counted as >64μg / mL; when the MIC value is the lowest concentration or below the lowest concentration, no distinction is made, and it is counted as ≤0.125μg / mL. The above experiments were performed in parallel 3 times. When the MIC value can be accurately repeated or only differs by one concentration, it is accepted, and the higher concentration is used as the MIC value; when the MIC value differs by more than two concentrations, the experiment needs to be repeated until it meets the requirements.

[0165] Evaluation of the effect of drug combination: The main parameter for evaluating the interaction mode of the two drugs in combination is the fractional inhibitory concentration index (FICI). The fractional inhibitory concentration (FIC) is the ratio of the MIC required for each drug to inhibit bacteria in combination to the MIC when used alone, and the FICI is the sum of the FICs of the two drugs. When FICI ≤ 0.5, the interaction is a synergistic effect, and the smaller the FICI, the stronger the synergistic effect; when 0.5 < FICI ≤ 1, it is an additive effect; when 1 < FICI ≤ 4, it is an irrelevant effect; and when FICI > 4, the two drugs are antagonistic to each other.

[0166] The experimental results are shown in Table 1 below:

[0167] As can be seen from Table 1, the combination of 3ja and the antifungal drug fluconazole has an antibacterial synergistic effect. The MIC value of fluconazole alone against the drug-resistant Candida albicans 103 is >64 μg / mL, and the MIC value of 3ja is >64 μg / mL. After the two drugs are combined, the MIC value of fluconazole drops to 16 μg / mL, and 3ja drops to 8 μg / mL; this shows the synergistic effect of 3ja on fluconazole.

[0168] Table 1 MIC values (μg / mL) of the combination of 3ja and fluconazole against fungi

[0169]

[0170] Example 12

[0171] The drugs, strains and experimental materials used are the same as those shown in Example 11.

[0172] The clinical drug-resistant Candida albicans 103 was inoculated into 1 mL of YEPD culture medium at a ratio of 1:100, and cultured with shaking at 30 °C and 200 rpm for 16 h to activate it, so that the fungus was in the late exponential growth phase. Take this bacterial solution and add it to 1 mL of YEPD culture medium respectively. After activating it again for 16 h by the above method, dilute and adjust the bacterial solution concentration to 1×10 5 ~5×10 5 CFU / mL. Take 1 mL of freshly prepared bacterial solution and place it in a glass tube, and add 3ja or fluconazole (FLC) respectively. The drug grouping is as follows: blank group (drug-free) and the following different combined concentrations of FLC + 3ja:

[0173] (16+2)μg / mL, (16+4)μg / mL, (8+8)μg / mL, (16+16)μg / mL, (16+64)μg / mL, (2+8)μg / mL, (4+8)μg / mL, (16+8)μg / mL, (16+8)μg / mL, and (64+8)μg / mL were tested for the growth of resistant Candida albicans 103 for 24h and 48h respectively.

[0174] The experimental results are shown in Figure 1 and Figure 2 ,Depend on Figure 1 It can be seen that when 3ja (16μg / mL, 64μg / mL) and fluconazole (16μg / mL) were used alone, they had no inhibitory effect on the growth of drug-resistant Candida albicans 103, and the turbidity of the bacterial solution was equivalent to that of the blank group. When different concentrations of 3ja and fluconazole (16μg / mL) were used together for 24h and 48h, the culture fluid in the combination test tubes with 3ja+FLC combination concentrations of (16+16)μg / mL and (64+16)μg / mL remained clear, indicating that the anti-resistant Candida albicans effect was significantly enhanced after the two drugs were used together, and they had a good synergistic inhibitory effect on drug-resistant Candida albicans.

[0175] Depend on Figure 2 It can be seen that when 3ja (8 μg / mL) and different concentrations of fluconazole were used alone, the inhibitory effect on the growth of drug-resistant Candida albicans 103 was very small, and the bacterial solution became turbid. When 3ja was used in combination with different concentrations of fluconazole for 24h and 48h, the culture solution in the test tubes with the concentrations of (8+8) μg / mL, (8+16) μg / mL, and (8+64) μg / mL remained clear, which further demonstrated the synergistic effect of 3ja combined with fluconazole on antifungal drugs.

Claims

1. A sinomenine derivative, characterized in that: The structural formula of the sinomenine derivative is shown in formula (I) or (II): In formula (I), R1 is hydrogen, methyl, hydroxy, halogen or methoxy; In formula (II), R2 is an amino group.

2. The sinomenine derivative according to claim 1, characterized in that The specific structural formula of the sinomenine derivative is any one of the following formulas:

3. A method for synthesizing the sinomenine derivative according to claim 1, characterized in that: Includes steps: S1, reacting sinomenine and acryloyl chloride at a molar ratio of 1:1.5 under the catalysis of triethylamine at room temperature, and then separating and purifying the product in the reaction solution to obtain an intermediate; S2, reacting the intermediate and substituted aniline at a molar ratio of 1:4 under the catalysis of an organic base at room temperature for 11 to 13 hours, and then separating and purifying the product in the reaction solution to obtain a sinomenine derivative.

4. The method for synthesizing the sinomenine derivative according to claim 3, characterized in that: The reactions described in S1 and S2 are all carried out in dichloromethane. When isolating and purifying the product, the product is first extracted into dichloromethane, then dried over anhydrous sodium sulfate, and then dichloromethane is removed by vacuum distillation. Finally, the product is purified by column chromatography.

5. The method for synthesizing the sinomenine derivative according to claim 3, characterized in that: The molar ratio of triethylamine to sinomenine described in S1 is 1:2, and the reaction is carried out for 2.5 to 3.5 hours.

6. The method for synthesizing the sinomenine derivative according to claim 3, characterized in that: The substituted aniline structural formula described in S2 is: Among them, R 2-1 is hydrogen, hydroxy, bromine, methoxy, mercapto or methyl; R 2-2 is hydrogen or methoxy; R 2-3 is hydrogen, bromine, chlorine or methyl.

7. The method for synthesizing the sinomenine derivative according to claim 3, characterized in that: The organic base described in S2 is 1,8-diazabicyclo[5.4.0]undec-7-ene, and the molar ratio of 1,8-diazabicyclo[5.4.0]undec-7-ene to the substituted aniline is 10:

1.

8. Use of the sinomenine derivative as claimed in claim 1 as an antifungal agent or in the preparation of an antifungal drug.

9. The use according to claim 8, characterized in that: The sinomenine derivative is used in combination with fluconazole as an antifungal drug synergist.

10. The use according to claim 8, characterized in that: The fungi include resistant Candida albicans (103), Candida albicans SC5314, Candida parapsilosis, Candida krusei, Candida glabrata and Cryptococcus neoformans.

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