A sinomenine derivative, and a synthesis method and application thereof

Abstract

The application discloses a sinomenine derivative, a synthetic method and application thereof, and belongs to the technical field of biology and medicine. In the synthesis, sinomenine and acryloyl chloride are subjected to a room temperature reaction under the catalysis of triethylamine, and then the product in the reaction solution is separated and purified to obtain an intermediate; the intermediate and substituted aniline are subjected to a room temperature reaction under the catalysis of an organic base for 11-13 hours, and then the product in the reaction solution is separated and purified to obtain the sinomenine derivative, which can be used as an antifungal agent or for preparing an antifungal medicine and used in combination with fluconazole as an antifungal medicine synergist. The sinomenine derivative not only provides a new candidate medicine for fungal treatment, but also improves the antifungal effect of the existing medicine.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biology and medicine, and particularly relates to a sinomenine derivative, a synthesis method and application thereof. BACKGROUND

[0002] Candida is the main pathogenic fungus of invasive fungal infection. According to statistics, more than 95% of invasive fungal infections are caused by five pathogenic fungi 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 outbreak. Invasive fungal infection requires long-term treatment with antifungal drugs. The commonly used antifungal drugs in clinical practice mainly include triazoles, polyenes and echinocandins. Among them, fluconazole is still the first-line antifungal drug for Candida treatment.

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

[0004] Sinomenine is an alkaloid extracted from the stems of Sinomenium acutum or S. maculatum, and widely exists in natural plants. It has various biological activities such as anti-inflammatory, analgesic, immunomodulatory, anti-arrhythmic, antioxidant, anti-tumor, and drug withdrawal. Sinomenine derivatives are a series of new compounds obtained by introducing different functional groups or structures into sinomenine through chemical synthesis. Most sinomenine derivatives not only retain the original pharmacological activity of sinomenine, but also 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 is expected to overcome the drug resistance of fungi to existing drugs and solve the problem of clinical fungal infection treatment. However, as of now, there is no safe and effective application of sinomenine derivatives in antifungal drugs or synergists. SUMMARY

[0005] In view of the deficiencies in the prior art, the purpose of the present application is to provide a sinomenine derivative, a synthesis method and application thereof. The raw materials are easy to obtain, the operation is simple, and the product is environmentally friendly. The product is used in combination with fluconazole for treating fungal infection drugs.

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

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

[0008]

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

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

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

[0012]

[0013] A method for synthesizing a sinomenine derivative, comprising the steps of:

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

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

[0016] Preferably, the reactions in S1 and S2 are both carried out in dichloromethane, and when separating and purifying the product, the product is first extracted into dichloromethane, then dried with anhydrous sodium sulfate, then dichloromethane is removed by reduced pressure distillation, and 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 2.5-3.5 hours.

[0018] Preferably, the structure of the substituted aniline in S2 is:

[0019]

[0020] wherein, R 2-1 is hydrogen, hydroxyl, bromine, methoxy, sulfydryl or methyl; R 2-2 is hydrogen or methoxy; and 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 substituted aniline is 10:1.

[0022] Use of a sinomenine derivative as an antifungal agent or for preparing an antifungal drug.

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

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

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

[0026] The sinomenine derivative of the present application connects the modified aniline structure to sinomenine to form a new skeleton structure, and the modified aniline can increase the receptor selectivity, thereby reducing the off-target effect and the related toxicity, having better drugability, and being helpful for developing different types of derivatives. The amino group is an active group that is easily oxidized, and the remaining secondary amine structure after modification also provides convenience for subsequent utilization of the derivative. The derivative can be used in combination with fluconazole, which can significantly improve the anti-Candida albicans activity, and can be used for developing anti-drug-resistant fungi drugs

[0027] The present application relates to a synthesis method of a sinomenine derivative, and sinomenine is a plant extract. There are two relatively active reaction sites on the A ring of sinomenine, i.e., a reactive hydrogen at 1 position and a phenolic hydroxyl group at 4 position. The reactive hydrogen and the phenolic hydroxyl group can be reacted at room temperature under the catalysis of triethylamine, which not only ensures the thermal stability of the plant extract, but also provides a mild reaction method. A reactive intermediate with a terminal olefin structure is synthesized, which is helpful for the room temperature reaction of the sinomenine derivative and a substituted aniline under the catalysis of an organic base. In the traditional method, the phenolic hydroxyl group is usually modified by etherification reaction, flammable compounds are used for synthesis, the reaction needs to be controlled below zero degrees, the reaction conditions are relatively harsh, and the reactive hydrogen at 1 position on the A ring of sinomenine is usually derivatized under reflux conditions. The reaction conditions of the present application are mild, easy to operate and control, simple in operation, wide in application range, and suitable for industrial production.

[0028] The sinomenine derivative can be used as an antifungal agent and can also be applied to the preparation of an antifungal drug. The sinomenine derivative not only provides a new candidate drug for fungal treatment, but also improves the antifungal effect of the existing drug. In the case that fungal drug resistance is becoming more and more common and the degree of drug resistance is becoming more and more serious, the sinomenine derivative can restore the effect of the antifungal drug on drug-resistant fungi, reduce the dosage of the antifungal drug, save the medical expenses of patients, and reduce the toxic and side effects of the drug.

[0029] Further, the sinomenine derivative can be used as a synergist of the antifungal drug. The sinomenine derivative and the antifungal drug fluconazole can significantly reduce the dosage of the antifungal drug, enhance the inhibitory effect on drug-resistant fungi, restore the effect of the antifungal drug fluconazole on drug-resistant fungi, have obvious antifungal effect, and the effect is better than that of the compound itself and fluconazole. As an antifungal drug synergist, it shows that the compound has a certain advantage in antifungal effect and has a certain development prospect. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Figure for the influence of the combination of derivative 3ja and fluconazole with different concentrations on the growth of drug-resistant Candida albicans 103.

[0031] Figure 2 Figure for the influence of the combination of derivative 3ja and fluconazole with different concentrations on the growth of drug-resistant Candida albicans 103. DETAILED DESCRIPTION

[0032] The concept, specific content and technical effects of the present application will be described in detail below in combination with examples, so as to fully understand the purpose features and effects of the present application. Obviously, the described examples are only a part of the examples of the present application, but not all the examples. Based on the examples of the present application, other examples obtained by those skilled in the art without creative labor belong to the protection scope of the present application. In the examples, the test methods used are conventional methods, and the materials and reagents used are commercially available reagents and reagents, unless otherwise specified.

[0033] The sinomenine derivative of the present application has a structure general formula as shown in formula (I) and (II):

[0034]

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

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

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

[0038]

[0039] The synthesis method of the sinomenine derivative of the present application comprises the following steps:

[0040] S1, the sinomenine hydrochloride is stirred in dichloromethane, ammonia water is slowly added dropwise until the sinomenine is dissolved in 2 eq dichloromethane, the molar ratio of sinomenine hydrochloride to ammonia water is 1:1.5-2.5, then the reaction is carried out at room temperature for 0.5 h, then extraction is carried out 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 the solvent is removed by reduced pressure distillation to obtain sinomenine.

[0041] S2, the intermediate obtained in S1 was reacted with acryloyl chloride (mole ratio 1:1.5) in dichloromethane (10 mL for 1 mmol of sinomenine) at room temperature for 2.5-3.5 h, with triethylamine as catalyst (mole ratio 1:2 with sinomenine), and the product was separated by extraction (dichloromethane as organic phase, saturated brine as aqueous phase, volume ratio of organic phase to aqueous phase 1:3), dried with anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. The compound was purified by column chromatography, with dichloromethane and methanol (volume ratio 50:1) as mobile phase, to obtain the intermediate (4bR, 8aR, 9S)-3,7-dimethoxy-11-methyl-6-oxo-6,8a,9,10-tetrahydro-5H-9,4b-

[0042] (4aR, 8aR, 9S)-3,7-dimethoxy-11-methyl-6-oxo-6,8a,9,10-tetrahydro-5H-9,4b-

[0043] S3, the intermediate obtained in S2 was dissolved in dichloromethane (1 mL for 0.4 mmol of substituted aniline) in a test tube with a stirrer, and then 1,8-diazabicyclo[5.4.0]undec-7-ene (mole ratio 10:1 with substituted aniline) was added under stirring at room temperature, and the reaction was carried out for 11-13 h. The reaction progress and product formation were determined by observing the change of reaction substances with time by thin layer chromatography. The product was separated by extraction (dichloromethane as organic phase, saturated brine as aqueous phase, volume ratio of organic phase to aqueous phase 1:3), dried with anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. The compound was purified by column chromatography, with dichloromethane and methanol (volume ratio 25:1) as mobile phase, to obtain the product, a sinomenine-structured derivative.

[0044] The structural formula is:

[0045]

[0046] The structural formula of the substituted aniline is:

[0047] or or

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

[0049] Example 1

[0050] First step, put sinomenine hydrochloride (1 mol, 1.0 eq) into a round bottom flask with a stirrer, add 2 eq dichloromethane, with the molar ratio of sinomenine hydrochloride to dichloromethane being 1:2, add ammonia water drop by drop during stirring at room temperature for 0.5 h until sinomenine is completely dissolved, extract the product three times (the organic phase is dichloromethane and the aqueous phase is saturated brine, the volume ratio of organic phase to aqueous phase is 1:3), dry the organic phase with anhydrous sodium sulfate and then remove the solvent by distillation under reduced pressure to obtain sinomenine (3.1 g, yield 95%).

[0051]

[0052] Second step, put sinomenine 1a (1 mol, 1.0 eq) into a round bottom flask with a stirrer, add 10 mL dichloromethane, add triethylamine (2 mol, 2.0 eq) at room temperature, slowly add acryloyl chloride (1.5 mol, 1.5 eq), stir for three hours at room temperature, monitor the reaction by thin layer chromatography (TLC), extract the product repeatedly three times with dichloromethane and saturated brine, dry the organic phase with anhydrous sodium sulfate and then remove the solvent by distillation under reduced pressure to obtain the 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%) is obtained, and the reaction equation is as follows:

[0053]

[0054] Third step, put (4bR,8aR,9S)-3,7-dimethoxy-11-methyl-6-oxo-6,8a,9,10-tetrahydro-5H-9,4b-(epiaminoethyl)phenanthrene-4-acrylate 2a (0.1 mmol, 1.0 eq) and the substrate aniline (0.4 mmol, 4.0 eq) into a test tube with a stirrer, add 1 mL dichloromethane, add (0.04 mmol, 6 μL) 1,8-diazabicyclo[5.4.0]undec-7-ene under stirring, stir for twelve hours at room temperature, monitor the reaction by thin layer chromatography (TLC), extract the product repeatedly three times with dichloromethane and saturated brine (volume ratio 1:3), dry the organic phase with anhydrous sodium sulfate and then remove the solvent by distillation under reduced pressure to obtain the crude product. After column chromatography (dichloromethane:methanol=25:1), the target compound sinomenine derivative 3aa is obtained.

[0055]

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

[0057]

[0058] The compound has1 H NMR and HRMS data are as follows:

[0059] 1 H NMR (600 MHz, CDC13) δ 7.18 (t, J = 7.8 Hz, 2H), 6.92 (d, J = 8.5 Hz, 1H), 6.76 (d, J = 8.4 Hz, 1H), 6.69 (d, J = 8.0 Hz, 3H), 5.48 (s, 1H), 3.70 (s, 3H), 3.49 (s, 3H), 3.20 (t, J = 4.5 Hz, 1H), 3.09 - 3.01 (m, 1H), 3.00 (s, 1H), 2.88 (ddd, J = 16.3, 6.6, 4.7 Hz, 1H), 2.81 - 2.70 (m, 1H), 2.56 - 2.47 (m, 2H), 2.45 (d, J = 8.5 Hz, 4H). ESI-HRMS: calcd. for C 28 H 32 N2O5+H, 477.2384 found 477.2389.

[0060] The resulting material proved to be indeed the derivative 3aa.

[0061] Example 2

[0062] Prepared according to the method and procedure of Example 1, with the exception that the starting material c in the third step was replaced by the following compound c-1, and the rest of the procedure was identical to Example 1. The target compound 3ba was obtained, and the specific reaction scheme is as follows:

[0063]

[0064] The structure is as follows:

[0065]

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

[0067] 1H NMR (400 MHz, CDC13) δ 7.08 (t, J = 8.0 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 6.75 (d, J = 8.4 Hz, 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 = 5.8 Hz, 1H), 2.71 (d, J = 5.7 Hz, 1H), 2.51 (d, J = 9.4 Hz, 2H), 2.43 (s, 4H), 1.83 (d, J = 4.7 Hz, 1H), 1.25 (s, 2H). ESI-HRMS: calcd. for C 28 H 34 N2O6+ Na, 529.2309 found 529.2282.

[0068] The resulting material proved to be indeed the derivative 3ba.

[0069] Example 3

[0070] Prepared according to the method and procedure of Example 1, with compound c-2 as the starting material in the third step, and otherwise identical to Example 1. The target compound 3ca was obtained, with the following specific reaction scheme:

[0071]

[0072] The structure is as follows:

[0073]

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

[0075] 1H NMR (600 MHz, CDC13) δ 7.08 (t, J = 8.1 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 6.76 (d, J = 8.4 Hz, 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, 1H), 3.07 (s, 1H), 3.04 (s, 1H), 2.95 (d, J = 13.3 Hz, 2H), 2.87 (d, J = 9.8 Hz, 1H), 2.73 (d, J = 13.1 Hz, 1H), 2.50 (d, J = 8.0 Hz, 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, 506.2417 found 506.2422.

[0076] The resulting material proved to be indeed the derivative 3ca.

[0077] Example 4

[0078] Prepared according to the method and procedure of Example 1, with the exception that in the third step the starting material c was replaced by compound c-3. The remaining procedure was identical to Example 1. The target compound 3da was obtained. The specific reaction scheme is as follows:

[0079]

[0080] The structure is as follows:

[0081]

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

[0083] 1H NMR (600 MHz, CDC13) δ 7.16 - 7.12 (m, 1H), 7.04 (d, J = 7.3 Hz, 1H), 6.91 (d, J = 8.4 Hz, 1H), 6.79 - 6.60 (m, 3H), 5.47 (d, J = 10.7 Hz, 1H), 3.66 (s, 3H), 3.47 (s, 3H), 3.16 (d, J = 4.7 Hz, 1H), 3.00 - 2.90 (m, 3H), 2.74 (dd, J = 8.4, 5.7 Hz, 1H), 2.71 (dd, J = 8.5, 5.6 Hz, 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, 491.2540 found 491.2546.

[0084] The resulting material proved to be indeed the derivative 3da.

[0085] Example 5

[0086] Prepared according to the method and procedure of Example 1, with the third step starting material c replaced by compound c-4, and otherwise identical procedure. The target compound 3ea was obtained, with the specific reaction scheme as follows:

[0087]

[0088] The structure is as follows:

[0089]

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

[0091] 1H NMR (600 MHz, CDC13) δ 7.00 (d, J = 8.4 Hz, 2H), 6.91 (d, J = 8.4 Hz, 1H), 6.75 (d, J = 8.5 Hz, 1H), 6.62 (d, J = 8.4 Hz, 2H), 5.48 (d, J = 2.2 Hz, 1H), 3.71 (s, 3H), 3.49 (s, 3H), 3.17 (t, J = 4.5 Hz, 1H), 3.07 (s, 1H), 3.04 (s, 1H), 2.99 - 2.92 (m, 2H), 2.87 (s, 1H), 2.74 (d, J = 6.7 Hz, 1H), 2.71 (d, J = 6.4 Hz, 1H), 2.49 (dtd, J = 12.0, 6.0, 3.6 Hz, 2H), 2.44 (d, J = 6.2 Hz, 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, 491.2540 found 491.2548.

[0092] The resulting material proved to be indeed the derivative 3ea.

[0093] Example 6

[0094] Prepared according to the method and procedure of Example 1, with compound c-5 as the starting material in the third step, and otherwise identical to Example 1. The target compound 3fa was obtained, according to the following scheme:

[0095]

[0096] The structure is as follows:

[0097]

[0098] The compound was characterized by 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 proven that the obtained substance is indeed the derivative 3fa.

[0101] Example 7

[0102] Prepared according to the method and steps of Example 1, except that in the third step, starting material c was replaced with compound c-6, and the remaining methods were the same as in Example 1. The target compound 3ga was obtained, and the specific reaction formula is as follows:

[0103]

[0104] The structure is as follows:

[0105]

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

[0107] 1H NMR (600 MHz, CDC13) δ 7.41 (dd, J = 7.9, 1.5 Hz, 1H), 7.20 (t, J = 7.7 Hz, 1H), 6.91 (d, J = 8.4 Hz, 1H), 6.75 (d, J = 9.8 Hz, 2H), 6.59 - 6.56 (m, 1H), 5.46 (d, J = 2.3 Hz, 1H), 3.69 (s, 3H), 3.47 (s, 3H), 3.17 (t, J = 4.5 Hz, 1H), 3.06 (s, 1H), 3.03 (s, 1H), 3.01 - 2.91 (m, 3H), 2.73 (d, J = 5.5 Hz, 1H), 2.70 (d, J = 5.5 Hz, 1H), 2.50 (dd, J = 4.8, 1.9 Hz, 1H), 2.48 (dd, J = 4.7, 2.0 Hz, 1H), 2.42 (s, 4H), 2.09 (s, 1H), 1.25 (s, 2H). ESI-HRMS: calcd. for C 28 H 31 BrN2O5+H, 556.4765 found 556.4760.

[0108] The resulting material proved to be indeed the derivative 3ga.

[0109] Example 8

[0110] Prepared according to the method and procedure of Example 1, with compound c-7 as the starting material in the third step, and otherwise identical to Example 1. The target compound 3ha was obtained, according to the following scheme:

[0111]

[0112] The structure is as follows:

[0113]

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

[0115] 1H NMR (600 MHz, CDC13) δ 7.06 (d, J = 8.5 Hz, 2H), 6.86 (d, J = 8.5 Hz, 1H), 6.70 (dd, J = 8.4, 6.7 Hz, 1H), 6.56 (d, J = 8.9 Hz, 2H), 5.41 (d, J = 8.7 Hz, 1H), 3.64 (d, J = 12.4 Hz, 3H), 3.53 - 3.47 (m, 1H), 3.42 (d, J = 3.6 Hz, 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 = 11.1 Hz, 1H), 2.67 (s, 1H), 2.65 (s, 1H), 2.45 - 2.41 (m, 1H), 2.40 (d, J = 6.2 Hz, 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] The resulting material proved to be indeed the derivative 3ha.

[0117] Example 9

[0118] Prepared according to the method and procedure of Example 1, with compound c-9 as the starting material in the third step. The target compound 3ia was obtained, with the following specific reaction scheme:

[0119]

[0120] The structure is as follows:

[0121]

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

[0123] 1H NMR (600 MHz, CDC13) δ 7.37 (d, J = 9.4 Hz, 1H), 7.07 (t, J = 7.6 Hz, 1H), 6.84 (d, J = 8.5 Hz, 1H), 6.70 - 6.65 (m, 2H), 6.63 (t, J = 7.5 Hz, 1H), 5.40 (d, J = 2.4 Hz, 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 = 16.3 Hz, 1H), 2.78 - 2.72 (m, 1H), 2.68 (d, J = 5.4 Hz, 1H), 2.65 (d, J = 5.4 Hz, 1H), 2.48 - 2.37 (m, 3H), 2.36 (s, 3H), 2.02 (d, J = 11.6 Hz, 1H), 1.84 - 1.72 (m, 2H). ESI-HRMS: calcd. for C 28 H 32 N2O5S+H, 509.2105 found 509.2109.

[0124] The resulting material proved to be indeed the derivative 3ia.

[0125] Example 10

[0126] Prepared according to the method and procedure of Example 1, with compound c-10 as the starting material in the third step. The target compound 3ja was obtained, with the following specific reaction scheme:

[0127]

[0128] The structure is as follows:

[0129]

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

[0131] 1H NMR (400 MHz, CDC13) δ 6.92 (s, 1H), 6.90 (s, 1H), 6.82 (d, J = 8.0 Hz, 1H), 6.76 (d, J = 8.4 Hz, 2H), 6.64 (d, J = 8.2 Hz, 2H), 5.43 (s, 1H), 3.72 (s, 3H), 3.46 (s, 3H), 3.23 (s, 2H), 3.12 - 2.88 (m, 5H), 2.77 (d, J = 12.4 Hz, 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, 493.5795 found 493.5799.

[0132] The resulting material proved to be indeed the derivative 3ja.

[0133] The antibacterial properties of some of the products were tested and are continued in the next example.

[0134] Example 11

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

[0136] 1. Reagents

[0137] Drug 3ja: laboratory synthesis.

[0138] Fluconazole: Sigma, batch 036M4709V.

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

[0140] The reagents were stored at -20°C. Before the experiment, the drug was taken out and melted in a 35°C incubator, thoroughly mixed, and subjected to pharmacodynamic tests.

[0141] 2. Strains

[0142] The strains used included:

[0143] Drug-resistant strains: drug-resistant C. albicans (103), C. albicans SC5314, C. parapsilosis, C. krusei, C. glabrata, C. neoformans

[0144] All were provided by the Fungus Room of Shanghai Changhai Hospital and identified by morphology and biochemistry.

[0145] All strains were activated by streaking on Sabouraud dextrose agar (SDA) plates, and single colonies were picked and reactivated by streaking on SDA plates. The second single colonies were then inoculated on SDA slants and stored at 4°C after incubation.

[0146] 3. Culture medium / base

[0147] 1) RPMI 1640 liquid medium

[0148] RPMI 1640 (Gibco BRL) 10 g, NaHCO3 2.0 g, morpholine propanesulfonic acid (Sigma) 34.5 g (0.165 mol), dissolved in 900 mL of triple distilled water, adjusted to pH 7.0 (25°C) with 1 mol / L NaOH, and brought to 1000 mL with triple distilled water. Sterilized by 0.22 μM microfiltration membrane, and stored at 4°C after aliquoting.

[0149] 2) Sabouraud dextrose agar solid medium (SDA)

[0150] Proteose peptone 10 g, glucose 40 g, agar 18 g, dissolved in 900 mL of triple distilled water, added with 50 mL of 2 mg / mL chloramphenicol aqueous solution, adjusted to pH 7.0, and brought to 1000 mL with triple distilled water. Sterilized by autoclaving (121°C, 15 min), and stored at 4°C.

[0151] 3) YEPD medium

[0152] Yeast extract 10 g, proteose peptone 20 g, glucose 20 g, dissolved in 900 mL of triple distilled water, added with 50 mL of 2 mg / mL chloramphenicol aqueous solution, and brought to 1000 mL with triple distilled water. Sterilized by autoclaving (121°C, 15 min), and stored at 4°C.

[0153] 4. Instruments and equipment

[0154] Waterproof electric heating constant temperature incubator (Shanghai Yuejin Medical Instrument Factory);

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

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

[0157] 5. 3ja and fluconazole stock solutions

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

[0159] 6. Preparation of bacterial solution

[0160] Before the experiment, a small amount of each fungus was picked from the SDA medium stored at 4°C with a sterile loop, inoculated into 1 mL of YEPD culture solution, and incubated at 35°C with 200 rpm shaking for 16 h to activate the fungi to the late exponential growth phase. Then, each bacterial solution was added to 1 mL of YEPD culture solution, activated again by the above method for 16 h, and counted using a hemocytometer. The concentration of each bacterial solution was adjusted to 1 x 10 3 ~ 5 x 10 3 CFU / mL in RPMI 1640 culture solution.

[0161] 7. Preparation of a drug 3ja and fluconazole combination drug susceptibility plate for multiple fungi:

[0162] Each strain was placed in a sterile 96-well plate, 100 μL of RPMI 1640 liquid medium was added to the first well of each row as a blank control, 100 μL of the above freshly prepared bacterial solution was added to wells 3-12, 198 μL of the bacterial solution was added to well 2, and 100 μL of the bacterial solution was added to well 12 without the drug as a positive growth control. The prepared bacterial solution and the two test drugs were added to wells 2-11, so that the final concentration of fluconazole in each well was 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 in the horizontal direction (the same for each row), and the concentration of 3ja in each well was 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, and 1 μg / mL in the vertical direction (the same for each column). The DMSO content in each well was less than 1%. Each drug susceptibility plate was incubated in a 35°C incubator.

[0163] 8. Determination of MIC values:

[0164] The fungi were incubated in a 35°C incubator for 24 h or 72 h, the experimental results were observed, and the MIC values were determined. When the MIC value of the drug exceeded the determination concentration range, the following method was used for statistics: when the MIC value was higher than the highest concentration of 64 μg / mL, it was counted as >64 μg / mL; when the MIC value was the lowest concentration or below the lowest concentration, there was no difference, and it was counted as ≤0.125 μg / mL. The above experiments were operated in parallel for 3 times, and when the MIC value could be accurately repeated or only differed by one concentration, it was accepted, and the higher concentration was taken as the MIC value; when the MIC value differed by more than two concentrations, the experiment needed to be repeated until the requirements were met.

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

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

[0167] As shown in Table 1, 3ja has an antibacterial synergistic effect when combined with antifungal drug fluconazole. The MIC value of fluconazole alone against drug-resistant C. 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 decreases to 16 μg / mL, and the MIC value of 3ja decreases to 8 μg / mL, which shows the synergistic effect of 3ja on fluconazole.

[0168] Table 1 MIC value of 3ja combined with fluconazole against fungi (μg / mL)

[0169]

[0170] Example 12

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

[0172] Clinical drug-resistant C. albicans 103 was inoculated into 1 mL of YEPD culture solution at a ratio of 1:100, and cultured at 30°C with 200 rpm shaking. After 16 h of activation, the fungi were in the late exponential growth phase. The bacterial solution was added to 1 mL of YEPD culture solution, and activated again for 16 h using the above method. The bacterial solution was then diluted to a concentration of 1×10 5 ~ 5×10 5 CFU / mL, and 1 mL of freshly prepared bacterial solution was placed in a glass tube. 3ja or fluconazole (FLC) was added, and the drug groups were as follows: blank group (without drug) and FLC+3ja at different concentrations:

[0173] The growth of drug-resistant Candida albicans 103 was tested at 24h and 48h using concentrations of (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.

[0174] The experimental results are shown in Figure 1 and Figure 2 ,Depend on Figure 1 It is evident that 3ja (16 μg / mL, 64 μg / mL) and fluconazole (16 μg / mL), when used alone, had no inhibitory effect on the growth of drug-resistant Candida albicans 103, and the turbidity of the bacterial culture was comparable to that of the control group. After 24 h and 48 h of combined administration of different concentrations of 3ja and fluconazole (16 μg / mL), the culture media in the test tubes with 3ja+FLC combination concentrations of (16+16) μg / mL and (64+16) μg / mL remained clear, indicating that the combined use of the two drugs significantly enhanced the anti-drug-resistant Candida albicans effect and had a good synergistic inhibitory effect on drug-resistant Candida albicans.

[0175] Depend on Figure 2 It is evident that when 3ja (8 μg / mL) and different concentrations of fluconazole were used alone, their inhibitory effect on the growth of drug-resistant Candida albicans 103 was minimal, and the bacterial cultures all became turbid. However, after 24 h and 48 h of combined use of 3ja and fluconazole at different concentrations (8+8) μg / mL, (8+16) μg / mL, and (8+64) μg / mL, the culture media in the test tubes remained clear, further demonstrating the synergistic effect of the combined use of 3ja and fluconazole as antifungal agents.

Claims

1. A sinomenine derivative, characterized in that, The structural formula of the sinomenine derivative is as follows: 。 2. A method of synthesizing a sinomenine derivative according to claim 1, wherein, The method comprises the steps of: ​ S1, reacting sinomenine and acryloyl chloride at room temperature in a molar ratio of 1:1.5 under catalysis of triethylamine, then separating and purifying the product in the reaction solution to obtain an intermediate; S2, reacting the intermediate and substituted aniline at room temperature in a molar ratio of 1:4 under catalysis of an organic base for 11-13 hours, then separating and purifying the product in the reaction solution to obtain the sinomenine derivative; The structural formula of the substituted aniline is as follows: wherein R 2-1 is hydroxy.

3. The method for synthesizing the sinomenine derivative according to claim 2, characterized in that, The reactions in S1 and S2 are both carried out in dichloromethane, when separating and purifying the product, the product is first extracted into dichloromethane, then dried by anhydrous sodium sulfate, then dichloromethane is removed by reduced pressure distillation, and finally the product is purified by column chromatography.

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

5. The method for synthesizing the sinomenine derivative according to claim 2, characterized in that, 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 substituted aniline is 10:

1.

6. A sinomenine derivative as claimed in claim 1 for use in the preparation of an antifungal drug.

7. Use according to claim 6, characterized in that, The sinomenine derivative is used for preparing a synergist for use in combination with fluconazole.

8. Use according to claim 6, characterized in that, The fungi include drug-resistant Candida albicans 103, Candida albicans SC5314, Candida parapsilosis, Candida krusei, Candida glabrata and Cryptococcus neoformans.

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