Oxime ester-containing ring-opening steroid derivative as well as preparation method and application thereof
By synthesizing six ring-opening steroidal arylhexime ester derivatives based on vitamin D2, the problems of long synthesis routes and low yields in the prior art are solved, and effective inhibition of specific bacteria is achieved, and potential medicinal value is potential.
Patent Information
- Application Number
- CN202510189494.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-20
- Publication Date
- 2025-05-27
AI Technical Summary
In the prior art, the synthesis route of aromatic heteroxime ester derivatives is long, complicated after-treatment, low yield and high synthesis cost, making it difficult to effectively solve the biocompatibility and biological activity problems of vitamin D2.
By using vitamin D2 as a raw material, six ring-opening steroidal arylhexoester derivatives are synthesized by using steps such as oxidation, oximetry and esterification, and a synthesis method based on such derivatives is provided, including esterification and condensation reaction in anhydrous dichloromethane, and subsequent purification by extraction and column chromatography to obtain the target compound.
The synthesis of compounds with good proliferation inhibition ability against Staphylococcus aureus and E. coli has potential application value for antibacterial drugs, and simplifies the synthesis steps and reduces costs.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of pharmaceutical chemistry, and particularly relates to a class of ring-opening steroid derivatives containing oxime esters, a preparation method thereof, and an application thereof. Background Art
[0002] Vitamin D 2 As an important active substance of vitamin D, its metabolite 1,25-(OH) 2 D in the human body can regulate calcium and phosphorus metabolism, inhibit cancer cell proliferation, regulate blood pressure and immune system, etc. Ergosterol can be converted into vitamin D through a ring-opening reaction under the action of ultraviolet light 2 . Vitamin D 2 Its double bond is easily oxidized and unstable. Long-term use of vitamin D 2 may also cause side effects such as hypercalcemia, resulting in certain limitations in its application. Therefore, structural modification and transformation of it are of great significance for improving biocompatibility and biological activity. In recent years, structural modification of such lead compounds has attracted great attention, and a number of drug molecules with definite curative effects, safety and stability have been discovered, such as: calcitriol, tacalcitol, maxacalcitol, seocalcitol, etc.
[0003] Many naturally occurring biologically active steroids contain oxime-like structures. Squalene-hopene cyclase inhibitors, digitalis, cytochrome P450 inhibitors, etc., as well as synthetic steroid oximes with anticancer activity. Oxime ester compounds are a class of important active compounds with various biological activities such as antibacterial, antiviral, and antitumor. Introducing oxime esters onto lead compounds can not only improve the liposolubility, stability, etc. of lead compounds, but also may reduce toxic side effects and obtain better physiological activities. However, the synthesis of aryl hetero oxime ester derivatives reported in the current literature has deficiencies such as long synthesis routes, cumbersome post-treatment, low yields, and high synthesis costs. Summary of the Invention
[0004] The purpose of the present invention is to provide a class of ring-opening steroid aryl hetero oxime ester derivatives, a preparation method thereof, and an application thereof.
[0005] The technical solution for achieving the purpose of the present invention is: The present invention first provides a class of ring-opening steroid aryl hetero oxime ester derivatives, and their structures are shown as follows: , wherein, R is an unsubstituted phenyl, halogenated phenyl, nitro phenyl, propenyl phenyl or pyridyl.
[0006] Further, the halogenated phenyl is a phenyl mono-substituted by a halogen, and the substitution position is not limited, such as chlorophenyl or iodophenyl; the nitro phenyl is a mono-substituted nitro phenyl, and the substitution position is not limited.
[0007] The present invention also provides a synthesis method of the above-mentioned class of open-ring steroid heteroaryl oxime esters, which includes the following steps: Dissolve compound 3 in anhydrous dichloromethane, add an aromatic carboxylic acid compound, and stir the reaction at room temperature under the catalysis of dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) for esterification condensation. After the reaction is completed, the target compound is obtained through extraction and purification. Among them, the structure of compound 3 is as follows: .
[0008] In the above steps, the aromatic carboxylic acid compound is a compound with a carboxyl group on the side chain of the benzene ring or a compound with a carboxyl group on the side chain of the pyridine ring; further, the aromatic carboxylic acid compound is any one of benzoic acid, 2-chlorobenzoic acid, 3-chlorobenzoic acid, 4-chlorobenzoic acid, 2-iodobenzoic acid, 3-iodobenzoic acid, 4-iodobenzoic acid, 2-nitrobenzoic acid, 3-nitrobenzoic acid, 4-nitrobenzoic acid, cinnamic acid, 2-pyridinecarboxylic acid, nicotinic acid or isonicotinic acid.
[0009] In the above steps, the molar ratio of compound 3 to the aromatic carboxylic acid compound is 1:1.9 - 2.1, and the molar ratio of the aromatic carboxylic acid compound to dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) is 1:1:1.
[0010] In the above steps, the stirring reaction time is not less than 4 hours.
[0011] In the above steps, extraction is carried out using dichloromethane, and purification is carried out using column chromatography purification. Among them, the silica gel used for column chromatography purification is 300 - 400 mesh, and the eluent used is a mixed solution of petroleum ether and ethyl acetate.
[0012] In a specific embodiment of the present invention, six open-ring steroid heteroaryl oxime esters were synthesized, specifically as follows:
[0013] In vitro antibacterial tests confirmed that the open-ring steroid heteroaryl oxime esters provided by the present invention have the effect of inhibiting the growth and proliferation of Gram-negative bacteria - Escherichia coli ( E. coli ) and Gram-positive bacteria - Staphylococcus aureus ( S. aureus ), and have a certain antibacterial effect. It is indicated that the open-ring steroid heteroaryl oxime esters provided by the present invention can be used as antibacterial / antibacterial agents in the fields of daily chemical products, food, etc.; or used for the preparation of antibacterial drugs.
[0014] The present invention provides the application of the above open-ring steroid heteroaryl oxime esters as antibacterial agents or antibacterial agents.
[0015] The present invention provides the use of the above-mentioned open-ring steroid heteroaryloxime esters in the preparation of antibacterial drugs.
[0016] The present invention also provides an antibacterial drug, which comprises the above-mentioned open-ring steroid heteroaryloxime esters as one of the main active ingredients and a pharmaceutically acceptable carrier.
[0017] Compared with the prior art, the present invention has the following remarkable advantages: (1) Six open-ring steroid heteroaryloxime esters with significant physiological activities are synthesized for the first time in the present invention; (2) A synthetic method based on open-ring steroid heteroaryloxime ester derivatives is disclosed for the first time; (3) The synthetic raw materials are easily available, the reaction conditions are mild, the steps are simple and the operation is easy. (4) Preliminary antibacterial kinetic experiments show that this kind of compound has good proliferation inhibitory ability against Staphylococcus aureus and Escherichia coli and may be used for the preparation of novel antibacterial drugs. Description of the Drawings
[0018] Figure 1 is the ( 1 H NMR, 500 MHz, solvent: CDCl 3 ) nuclear magnetic resonance spectrum of compound 4a.
[0019] Figure 2 is the ( 1 C NMR, 126 MHz, solvent: CDCl 3 ) nuclear magnetic resonance spectrum of compound 4a.
[0020] Figure 3 is the ( 1 H NMR, 500 MHz, solvent: CDCl 3 ) nuclear magnetic resonance spectrum of compound 4b.
[0021] Figure 4 is the ( 1 C NMR, 126 MHz, solvent: CDCl 3 ) nuclear magnetic resonance spectrum of compound 4b.
[0022] Figure 5 is the ( 1 HNMR, 500 MHz, solvent: CDCl 3 ) nuclear magnetic resonance spectrum of compound 4c.
[0023] Figure 6 is the ( 1 C NMR, 126 MHz, solvent: CDCl 3 ) nuclear magnetic resonance spectrum of compound 4c.
[0024] Figure 7 is the ( 11H NMR, 500 MHz, Solvent: CDCl 3 ) Nuclear magnetic resonance spectrum.
[0025] Figure 8 For compound 4d's ( 1 13C NMR, 126 MHz, Solvent: CDCl 3 ) Nuclear magnetic resonance spectrum.
[0026] Figure 9 For compound 4e's ( 1 1H NMR, 500 MHz, Solvent: CDCl 3 ) Nuclear magnetic resonance spectrum.
[0027] Figure 10 For compound 4e's ( 1 13C NMR, 126 MHz, Solvent: CDCl 3 ) Nuclear magnetic resonance spectrum.
[0028] Figure 11 For compound 4f's ( 1 1H NMR, 500 MHz, Solvent: CDCl 3 ) Nuclear magnetic resonance spectrum.
[0029] Figure 12 For compound 4f's ( 1 13C NMR, 126 MHz, Solvent: CDCl 3 ) Nuclear magnetic resonance spectrum.
[0030] Figure 13 Effect of compounds 4d and 4f at different concentrations on the viability of NIH 3T3 mouse fibroblasts. Detailed implementation mode
[0031] The following combines examples and drawings to detail the synthesis method of the present invention based on ring-opening steroid heteroaryl oxime ester derivatives.
[0032] The present invention uses the method of splicing bioactive factors to introduce an oxime ester active group into a ring-opening steroid vitamin D 2 active compound for structural modification thereof. Using vitamin D 2 as a raw material, vitamin D 2 ketone was prepared through an oxidation reaction, and then through oximation and esterification, 6 ring-opening steroid heteroaryl oxime ester derivatives were synthesized, with the expectation of obtaining compounds with good proliferation inhibitory ability against Staphylococcus aureus and Escherichia coli.
[0033] The synthesis route of the vitamin D 2 derivatives of the present invention is as follows:
[0034] Among them, (a) aluminum isopropoxide, acetone, toluene; (b) hydroxylamine hydrochloride, triethylamine, ethanol; (c): aromatic carboxylic acid compound; dichloromethane; dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP), at room temperature.
[0035] The synthesis method of the ring-opening steroidal heteroaryloxime ester derivative of the present invention will be described in detail below with reference to examples. The examples do not represent the scope of the invention of this patent.
[0036] Example 1: Synthesis of Compound 2 In a 250 mL round-bottom flask, (1.0 g, 2.52 mmol) vitamin D was added successively 2 , dry toluene (30 mL), acetone (80 mL). After stirring well, (4.0 g, 19.2 mmol) aluminum isopropoxide was slowly added, and the reaction system was heated to reflux for 12 h. The reaction progress was monitored by TLC [V(ethyl acetate):V(petroleum ether)=1:10]. 0.1 mol / L dilute hydrochloric acid was slowly added to the reaction solution to terminate the reaction. After cooling to room temperature, most of the solvent was evaporated, and the mixture was extracted with ethyl acetate (60 mL×3), washed with water (40 mL×2), and washed with saturated sodium chloride solution (30 mL×2). The organic phases were combined and dried over anhydrous sodium sulfate overnight, filtered, and the filtrate was concentrated and purified by column chromatography [V(ethyl acetate):V(petroleum ether)=1:30] to obtain 380 mg of a pale yellow solid compound 2, yield: 38.0%. m.p. 73-75 °C (literature value: 72-73 °C). 1H NMR (500 MHz, CDCl 3)δ 5.92 (s, 1H), 5.41 (s, 1H), 5.33 (s, 1H), 5.28–5.09 (m, 2H), 4.94 (s, 1H),3.15 (t, J = 6.9 Hz, 2H), 2.72 (t, J = 6.9 Hz, 3H), 2.60–2.39 (m, 4H), 1.00(t, J = 10.3 Hz, 6H), 0.92 (d, J = 6.8 Hz, 5H), 0.88–0.74 (m, 10H), 0.56 (s,3H). 13C NMR (126 MHz, CDCl3) δ 199.73, 157.85, 142.32, 141.61, 135.52,131.93, 126.61, 114.91, 77.20, 76.94, 76.69, 56.31, 55.74, 45.12, 42.78,40.29, 40.18, 37.71, 33.02, 31.90, 30.80, 29.62, 28.57, 27.68, 23.16, 22.14,21.05, 19.88, 19.58, 17.55, 12.10, 0.94. MS (ESI, m / z): 395.28 [M+H]+。
[0037] Example 2: Synthesis of Compound 3 Dissolve Compound 2 (0.50 g, 1.27 mmol) in anhydrous ethanol (30 mL) in a 100 mL single-necked flask, add hydroxylamine hydrochloride (0.44 g, 6.35 mmol) and triethylamine (3 mL), heat under reflux with stirring. After the reaction is completed as monitored by TLC, let the reaction mixture cool, then rotary evaporate the solvent under reduced pressure. Then dissolve it in ethyl acetate and extract with ethyl acetate (3×50 mL), wash with water (2×30 mL). Combine the organic phases, dry over anhydrous sodium sulfate, and concentrate to obtain a yellow oil. Purify by column chromatography (silica gel: 300 - 400 mesh, Vethyl acetate: Vpetroleum ether = 1:20) to obtain 354 mg of pale yellow viscous Compound 3, yield: 74.0%. 1 H NMR (500 MHz, CDCl 3) : δ6.77 (s, 1H), 5.24 (s, 1H), 5.19 (t, J = 6.7Hz, 2H), 5.09 (s, 1H), 4.97 (s, 1H), 3.14 (d, J = 7.4 Hz, 2H), 2.65–2.50 (m,3H), 2.45 (dd, J = 8.4, 5.1 Hz, 2H), 2.10–1.90 (m, 5H), 1.85 (d, J = 6.3 Hz,2H), 1.02 (d, J = 6.6 Hz, 4H), 0.58 (s, 4H). 13 CNMR (126 MHz, CDCl 3 ) δ153.86,147.04, 142.45, 141.18, 135.63, 131.84, 116.28, 114.72, 112.85, 77.20, 76.95,76.69, 60.36, 56.34, 55.78, 45.12, 42.77, 40.30, 33.03, 31.74, 30.99, 28.58,28.39, 27.76, 23.78, 23.20, 22.19, 21.03, 19.88, 19.58, 17.55, 14.11, 12.05,7.84. MS (ESI, m / z): 410.26 [M+H]+.
[0038] Example 3: Synthesis of Compound 4a
[0039] 4a In a 50 mL round-bottom flask, compound 3 (60 mg, 0.15 mmol), dicyclohexylcarbodiimide DCC (60 mg, 0.3 mmol), 4-dimethylaminopyridine DMAP (36 mg, 0.3 mmol) and anhydrous dichloromethane (20 mL) were added successively. Benzoic acid (36 mg, 0.3 mmol) was slowly added with stirring, and the reaction was stirred overnight at room temperature. The reaction process was monitored by TLC [V(ethyl acetate):V(petroleum ether)=1:30]. After the reaction was completed, 30 mL of water was added, and the mixture was extracted with dichloromethane (20 mL × 2), washed with water. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a white solid. It was purified by column chromatography (silica gel: 300 - 400 mesh, V(ethyl acetate):V(petroleum ether)=1:50) to obtain 65 mg of white flaky solid, yield: 80.7%. m.p. 103 - 105 °C.1 HNMR (500 MHz, CDCl 3 ): δ 8.13 (m, 2H), 7.58 (t, 1H), 7.46 (t, 2H), 6.34(s, 1H), 5.29 (s, 1H), 5.24 -5.18(t, 2H), 5.16 (s, 1H), 4.99 (t, 1H), 3.14(t, 2H), 2.90 (t, 2H), 2.56(m, 3H), 2.09 – 1.90 (m, 4H), 1.91 – 1.80 (m, 1H),1.78 – 1.67 (m, 2H), 1.68 – 1.55 (m, 4H), 1.55 – 1.40 (m, 4H), 1.39 – 1.30(m, 5H), 1.29 (dd, J = 13.2 Hz, 6H), 1.27 (d, J = 14.2 Hz, 16H), 1.05 – 0.97(m, 3H), 0.96 – 0.88 (m, 5H), 0.88 – 0.79 (m, 11H), 0.56 (s, 3H), 0.08 (s,1H). As Figure 1 shown.
[0040] 13 CNMR (126MHz, CDCl 3 ): δ 162.9, 148.5, 141.0, 140.62 134.7, 132.2,130.9, 128.6, 127.5, 120.0, 114.5, 112.0, 55.4, 54.8, 44.2, 41.9, 39.4, 39.3,32.1, 30.0, 29.2, 28.7, 28.4, 27.6, 26.8, 23.4, 22.2, 21.7, 21.2, 20.1, 19.0,18.7, 16.6, 13.1, 11.2. As Figure 2 shown.
[0041] Example 4: Synthesis of Compound 4b
[0042] 4b Referring to the synthesis method of Compound 4a, the aromatic carboxylic acid compound was 4-chlorobenzoic acid, and 54 mg of a white powdery solid was obtained, yield: 70.3%. m.p. 145 - 147 °C. 1 HNMR (500 MHz, CDCl3 ): δ 8.13 (d, J = 8.5 Hz, 2H), 7.57 (d, J = 8.5 Hz, 2H), 6.45 (s, 1H), 5.42 (s, 1H), 5.31 (dd, J = 13.6, 6.4 Hz, 3H), 5.10 (s, 1H), 3.27 (m, 2H), 3.00 (t, 2H), 2.70 (m, 3H), 2.11 (m, 3H), 1.98 (dd, J = 12.9, 6.6 Hz, 1H), 1.94 (m, 1H), 1.73 (m, 3H), 1.58 (m, 4H), 1.52 – 1.30 (m, 5H), 1.14 (d, J = 6.6 Hz, 3H), 1.04 (d, J = 6.8 Hz, 3H), 0.95 (m, 6H), 0.68 (s, 3H). As Figure 3 shown.
[0043] 13 CNMR (126 MHz, CDCl 3 ): δ 164.3, 163.3, 150.0, 142.2, 141.7, 139.9, 135.8, 132.1, 131.1, 129.1, 121.0, 115.6, 113.3, 58.6, 56.5, 56.0, 45.3, 43.0, 40.6, 40.5, 33.3, 31.1, 30.4, 28.8, 28.0, 24.5, 23.4, 22.4, 21.3, 20.1, 19.8, 18.6, 17.8, 12.4. As Figure 4 shown.
[0044] Example 5: Synthesis of Compound 4c
[0045] 4c
[0046] Referring to the synthesis method of reference compound 4a, the aromatic carboxylic acid compound was 2-iodobenzoic acid, and 64 mg of a pale yellow powdery solid was obtained, yield: 69.3%. m.p. 110 - 112 °C.
[0047] 1 HNMR (500 MHz, CDCl 3 ): δ7.98 (d, J = 7.9 Hz, 1H), 7.71 (d, J = 7.7 Hz,1H), 7.42 (t, J = 7.5 Hz, 1H), 7.17 (t, 1H), 6.32 (s, 1H), 5.28 (s, 1H), 5.20(t, 2H), 5.15 (s, 1H), 4.97 (t, 1H), 3.14 (t, 2H), 2.86 (t, 2H), 2.62 – 2.43(m, 3H), 2.09 – 1.90 (m, 3H), 1.85 (d, J = 6.4 Hz, 1H), 1.72 (d, J = 12.5 Hz,2H), 1.63 (d, J = 11.0 Hz, 3H), 1.46 m, 4H), 1.37 – 1.18 (m, 7H), 1.00 (d, J= 6.7 Hz, 4H), 0.92 (d, J = 6.8 Hz, 4H), 0.83 (t, J = 7.1 Hz, 7H), 0.56 (s,3H). As Figure 5 shown.
[0048] 13 CNMR (126 MHz, CDCl 3 ): δ 163.6, 163.3, 148.9, 141.0, 140.5, 140.0,134.7, 134.5, 131.6, 130.9, 129.7, 126.9, 119.7, 114.5, 112.1, 92.7, 55.4,54.8, 44.2, 41.9, 39.4, 39.3, 32.11 30.0, 29.2, 28.7, 27.6, 26.8, 23.8, 22.3,21.2, 20.1, 19.0, 18.7, 16.6, 11.2. As Figure 6 shown.
[0049] Example 6: Synthesis of Compound 4d
[0050] 4d Referring to the synthesis method of Compound 4a, the aromatic carboxylic acid compound was 4-nitrobenzoic acid, and 63 mg of a white powdery solid was obtained, yield: 76.2%. m.p. 109 - 111 °C. 1 HNMR (500 MHz, CDCl 3 ):δ 8.32 (m, 2H), 8.25 (m, 2H), 6.30 (s, 1H), 5.30 (s, 1H), 5.17 (t, 3H), 4.95 (t, 1H), 3.13 (t, 2H), 2.88 (t, 2H), 2.54 (m, 3H), 2.08 (m, 4H), 1.83 (m, 1H), 1.73 – 1.67 (m, 1H), 1.61 (m, 3H), 1.56 – 1.27 (m, 8H), 1.33 – 1.19 (m, 4H), 0.99 (d, J = 6.6 Hz, 3H), 0.89 (d, J = 6.8 Hz, 4H), 0.80 (t, J = 7.2 Hz, 7H), 0.53 (s, 3H). As Figure 7 shown.
[0051] 13 CNMR (126 MHz, CDCl 3 ): δ 164.9, 162.3, 150.8, 150.7, 142.4, 141.4, 135.8, 132.1, 130.9, 123.9, 120.6, 115.5, 113.7, 56.5, 56.0, 45.4, 43.0, 40.6, 40.5, 33.3, 31.2, 30.3, 28.8, 28.0, 24.6, 23.4, 22.4, 21.3, 20.1, 19.8, 17.8, 12.4. As Figure 8 shown.
[0052] Example 7: Synthesis of Compound 4e
[0053] 4e Referring to the synthesis method of Compound 4a, the aromatic carboxylic acid compound was cinnamic acid, and 53 mg of a white powdery solid was obtained, yield: 63.2%. m.p. 101 - 103 °C. 1 HNMR (500 MHz, CDCl 3): δ 7.84 (d, J = 16.0 Hz, 1H), 7.57 (s, 2H), 7.49 (m, 3H), 6.55 (d, J = 16.0 Hz, 1H), 6.31 (s, 1H), 5.27 (d, J = 17.7 Hz, 1H), 5.21 (t, 2H), 5.16 (s, 1H), 4.99 (t, 1H), 3.14 (m, 2H), 2.84 (m, 2H), 2.55 (m, 3H), 1.97 (m, 3H), 1.87 (m, 1H), 1.74 (m, 1H), 1.63 (m, 2H), 1.58 – 1.41 (m, 5H), 1.39 – 1.23 (m, 4H), 1.03 (d, J = 6.6 Hz, 3H), 0.93 (d, J = 6.8 Hz, 4H), 0.85 (t, 7H), 0.57 (s, 3H). As Figure 9 。
[0054] 13 CNMR (126 MHz, CDCl 3 ): δ 164.9, 163.6, 149.5, 146.1, 142.1, 141.8, 135.9, 134.5, 132.1, 130.7, 129.1, 128.9, 121.3, 116.0, 115.7, 113.1, 56.5, 56.0, 45.3, 43.0, 40.6, 40.5, 33.3, 31.1, 30.4, 28.8, 28.0, 24.4, 23.4, 22.4, 21.3, 20.2, 19.9, 17.8, 12.4. ESI-MS, m / z: 540.25 [M + H]+. As Figure 10 。
[0055] Example 8: Synthesis of Compound 4f
[0056] 4f Referring to the synthesis method of Compound 4a, the aromatic carboxylic acid compound was isonicotinic acid, and 57 mg of a white powdery solid was obtained with a yield of 74.0%. m.p. 96 - 98 °C. 1 HNMR (500 MHz, CDCl 3): δ 8.93 (d, J = 4.8 Hz, 2H), 8.00 (d, J = 5.6 Hz, 2H), 6.44 (s, 1H), 5.44 (s, 1H), 5.31 (m, 3H), 5.09 (t, 1H), 3.27 (t, 2H), 3.01 (m, 2H), 2.69 (t, 3H), 2.09 (d, J = 10.4 Hz, 3H), 1.97 (m, 2H), 1.88 (m, 1H), 1.74 (s, 3H), 1.66 – 1.51 (m, 5H), 1.50 – 1.29 (m, 5H), 1.13 (d, J = 6.6 Hz, 3H), 1.04 (d, J = 6.8 Hz, 4H), 0.95 (t, 7H), 0.68 (s, 3H). As Figure 11 .
[0057] 13 CNMR (126 MHz, CDCl 3 ): δ 164.9, 162.7, 150.8, 150.6, 142.3, 141.5, 136.8, 135.8, 132.1, 123.0, 120.6, 115.5, 113.7, 56.5, 56.0, 45.4, 43.0, 40.6, 40.5, 33.3, 31.2, 30.3, 28.8, 28.0, 24.5, 23.4, 22.4, 21.3, 20.1, 19.8, 17.8, 12.4. ESI-MS, m / z: 515.22 [M + H]+. As Figure 12 .
[0058] Example 9 Antibacterial Test The compounds prepared in Examples 3 - 8 were respectively evaluated for their antibacterial properties against Gram-negative bacteria - Escherichia coli ( E. coli ) and Gram-positive bacteria - Staphylococcus aureus ( S. aureus ). The specific steps are as follows: (1) Preparation of LB liquid medium Weigh 2.5 g of yeast extract, 5 g of tryptone, and 5 g of sodium chloride and add them to a 500 mL beaker. Then add 450 mL of deionized water and stir ultrasonically for 15 min until completely dissolved. Then adjust its pH to 7.2 with 3 mol / L NaOH solution, transfer the solution to a 500 mL volumetric flask, and place it in an autoclave for sterilization at 121 °C with high-pressure steam for 20 min.
[0059] (2)Preparation of LB solid medium Weigh 2.5 g of yeast extract, 5 g of tryptone, 5 g of sodium chloride, and 7.5 g of agar separately and add them to a 500 mL beaker. Then add 450 mL of deionized water and heat until completely dissolved. Then adjust its pH to 7.2 with 3 mol / L NaOH solution, transfer the solution to a 500 mL volumetric flask, and place it in an autoclave for sterilization at 121 °C with high-pressure steam for 20 min.
[0060] (3)Preparation of PBS buffer Weigh 0.27 g of potassium dihydrogen phosphate, 1.42 g of disodium hydrogen phosphate, 8 g of sodium chloride, and 0.2 g of potassium chloride separately and add them to a 1000 mL beaker. Then add 800 mL of ionic water and stir ultrasonically for 15 min until fully dissolved. Then add concentrated hydrochloric acid dropwise to adjust the solution pH to 7.2, transfer the solution to a 1 L volumetric flask, and place it in an autoclave for sterilization at 121 °C with high-pressure steam for 20 min.
[0061] (4)Preparation of plate medium Heat and melt the LB solid medium into a liquid state, quickly add about 10 mL of LB medium to each sterile petri dish, place it horizontally on a sterile workbench, and cool and solidify it at room temperature to make a solid LB plate medium.
[0062] (5)Preparation of bacterial suspension Place the inoculation loop on the flame of an alcohol lamp for sterilization. Use the inoculation loop to pick a single colony from the plate on which bacteria have been cultured and add it to a conical flask containing 100 mL of LB liquid medium. Culture it in a shaker at 37 °C and 140 rpm for 20 h.
[0063] Place 500 μL of the test compound (the compound is dissolved in DMSO and diluted with PBS to a concentration of 1.0 μmol / mL) into 4 mL of the culture solution, and add 500 μL of fresh bacterial solution to the sample dispersion. Start timing from the moment the bacterial solution is added. After contacting for 120 min respectively, take 100 μL of the bacterial solution and 900 μL of PBS solution, and dilute them step by step according to a ratio of 1:10. Take 100 μL of the appropriately diluted solution and spread it on the LB agar plate. Incubate the petri dish at 37 °C for 20 h, count the bacterial colonies formed on the agar plate, and the bacterial growth inhibition rate = (B - A) / B × 100%, where A refers to the number of colonies after the sample contact, and B refers to the number of colonies in the blank group. Calculate the antibacterial efficiency of the sample accordingly. Among them, compound 2 and compound 3 do not have antibacterial activity, and the results of the bacterial growth inhibition rates of compound 4a - 4f and the positive control drug chlortetracycline are shown in Table 1.
[0064] Table 1 Proliferation Inhibition Rates of Compounds 4a - 4f against Staphylococcus aureus and Escherichia coli
[0065] As can be seen from Table 1, after 120 min of contact, all compounds showed certain antibacterial activities. It is speculated that the possible reason is that the oxime ester formed by the combination of vitamin D 2 oxime and aromatic acid compound provides antibacterial ability. Among them, the proliferation inhibition activities of compounds 4d and 4f against two common bacteria, Staphylococcus aureus and Escherichia coli, are better than those of the positive control chlortetracycline. It is speculated that the possible reason is that the combination of nitrobenzoic acid and isonicotinic acid heteroaromatic compounds is more conducive to obtaining antibacterial ability.
[0066] Conclusion: From the data in the table, it can be seen that such ring - opening steroidal heteroaromatic oxime ester derivatives have obvious growth inhibitory effects on the above - mentioned bacteria and have potential medicinal value.
[0067] Example 10 Cytotoxicity Test The preliminary cytotoxicity test was carried out using NIH 3T3 mouse fibroblasts.
[0068] Cell culture: The NIH 3T3 mouse fibroblasts stored in an - 80 °C refrigerator were placed in a 37 °C water bath, and the cryopreservation tube was shaken continuously to melt the cell solution as quickly as possible (to avoid ice crystals entering the cells to form recrystallization due to slow melting and damaging the cells). After the cell solution melted, it was taken out, the outer wall of the cryopreservation tube was wiped with alcohol cotton for sterilization, and the cell solution was transferred into a culture flask that had been pre - added with 3 mL of culture medium (DMEM medium containing 1.5 g / L NaHCO 3 and 10% high - quality fetal bovine serum) and placed in an incubator at 37 °C with 5% CO 2 . The 3T3 cells that had been cultured for 3 - 4 generations and were in a vigorous growth state after resuscitation were aspirated with a pipette gun to remove the old culture medium, rinsed twice with PBS solution, 1 mL of trypsin solution was added, and digested at 37 °C for 1 min. The trypsin solution was aspirated, the side wall of the culture flask was gently patted to make the cells fall off as much as possible, 2 mL of fresh cell culture medium was added, the cells on the bottle wall were blown into the cell solution, and 1 mL of cell culture medium was supplemented. The density of the cells was calculated through a cell counting plate and adjusted to an appropriate concentration.
[0069] MTT assay: (a) Add 200 μL of cell culture medium (about 1×10 4 ) to a 96 - well plate, and place it in an incubator at 37 °C with 5% CO 2Cultivate in a cell incubator for 24 h. (b) Add the test compounds 4d and 4f at appropriate concentrations to the sample group (the compounds are dissolved in DMSO and diluted with PBS to prepare different concentrations, and 3 replicate wells are set for each concentration) and cultivate for 48 h. In the control group, only add an equal amount of PBS solution. (c) Incubate the 96-well plate in a cell incubator at 37 °C with 5% CO 2 air and 100% humidity for an appropriate time. (d) Dilute 5×MTT with Dilution Buffer to 1×MTT. (e) Add 20 μL of 1×MTT to each well and incubate at 37 °C for 4 hours to reduce MTT to formazan. (f) Aspirate the supernatant, add 200 μL of DMSO to each well to dissolve the formazan, and shake well with a plate shaker. (g) Detect the absorbance of each well at a wavelength of 570 nm with an enzyme-linked immunosorbent assay (ELISA) reader. The test results are shown in Figure 13 .
[0070] Relative cell survival rate % = (OD 样品 / OD 参照 ) × 100% OD 样品 : Absorbance of the cell solution with different concentrations of the sample added OD 参照 : Absorbance of the cell solution in the blank reference group.
[0071] Compared with the blank control group, with the increase in the concentration of the test compound, the cell survival rate decreased to a certain extent. Even when its concentration reached 100 μg / mL, the cell survival rate could still remain above 80%. According to the cell toxicity evaluation classification standard: in tests such as MTT, when the cell viability decreased by less than 10% compared with the control group, it could be judged as grade 0; in the MTT test, when the cell viability decreased by 10% - 20%, it could be judged as grade 1. It shows that the in vitro toxicity of the test compounds 4d and 4f to NIH3T3 mouse fibroblasts is below grade 1, and the cell compatibility is good.
[0072] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.
Claims
1. A class of open-ring steroidal derivatives containing oxime esters, characterized in that: Its structure is as follows: ， Wherein, R is unsubstituted phenyl, halogenated phenyl, nitrophenyl, propenylphenyl or pyridyl.
2. The ring-opening steroidal derivative containing oxime ester according to claim 1, characterized in that: The halogenated phenyl group is a monosubstituted chlorophenyl group or a monosubstituted iodophenyl group; and the nitrophenyl group is a monosubstituted nitrophenyl group.
3. A method for synthesizing a ring-opening steroidal derivative containing an oxime ester as claimed in claim 1, characterized in that: The method comprises the following steps: dissolving compound 3 in anhydrous dichloromethane, adding an aromatic carboxylic acid compound, reacting under stirring at room temperature under the catalysis of dicyclohexylcarbodiimide and 4-dimethylaminopyridine, and extracting and purifying to obtain a target compound after the reaction is completed, wherein the aromatic carboxylic acid compound is a compound containing a carboxyl group on the side chain of a benzene ring or a compound containing a carboxyl group on the side chain of a pyridine ring; the structure of compound 3 is as follows: 。 4. The method for synthesizing the ring-opening steroidal derivative containing oxime ester according to claim 3, characterized in that: The aromatic carboxylic acid compound is any one of benzoic acid, 2-chlorobenzoic acid, 3-chlorobenzoic acid, 4-chlorobenzoic acid, 2-iodobenzoic acid, 3-iodobenzoic acid, 4-iodobenzoic acid, 2-nitrobenzoic acid, 3-nitrobenzoic acid, 4-nitrobenzoic acid, cinnamic acid, 2-pyridinecarboxylic acid, nicotinic acid or isonicotinic acid.
5. The method for synthesizing the ring-opening steroidal derivative containing oxime ester according to claim 3, characterized in that: The molar ratio of compound 3 to the aromatic carboxylic acid compound is 1:1.9-2.
1.
6. The method for synthesizing the open-ring steroidal derivative containing oxime ester according to claim 3, characterized in that: The molar ratio of the aromatic carboxylic acid compound to dicyclohexylcarbodiimide and 4-dimethylaminopyridine is 1:1:
1.
7. The method for synthesizing the ring-opening steroidal derivative containing oxime ester according to claim 3, characterized in that: The stirring reaction time is not less than 4 hours; and / or, the extraction adopts dichloromethane; and / or, the purification adopts silica gel column chromatography, the silica gel is 300-400 mesh, and the eluent is a mixed solution of petroleum ether and ethyl acetate.
8. Use of the open-ring steroidal derivative containing oxime ester according to claim 1 or 2 as an antibacterial agent or bacteriostatic agent.
9. Use of the open-ring steroidal derivative containing oxime ester according to claim 1 or 2 in the preparation of antibacterial drugs.
10. An antibacterial drug, characterized in that: The invention comprises the ring-opening steroidal derivative containing oxime ester as claimed in claim 1 or 2 as one of the main active ingredients, and a pharmaceutically acceptable carrier.