CN102942545B - Nepetalactone-o-bromobenzoic acid ester as well as preparation process and use of nepetalactone-o-bromobenzoic acid ester - Google Patents

Abstract

The invention discloses a nepetalactone o-bromobenzoate and its preparation process and application. The nepetalactone o-bromobenzoate is characterized in that the molecular formula is C ₁₇ H ₁₇ BrO ₄ and the melting point is 184- 186℃, its specific rotation is The invention also discloses its extraction method and its application in the preparation of anti-herpes virus medicine.

Description

A kind of nepetalactone o-bromobenzoate and its preparation process and application

technical field

The invention relates to a nepetalactone o-bromobenzoate, a preparation process and application thereof, and belongs to the technical field of pharmaceutical preparations.

Background technique

Nepetalactone (C ₁₀ H ₁₄ O ₃ ) is a monoterpene lactone extracted from the Chinese herbal medicine Nepeta chinensis. It is a colorless crystal (ethyl acetate), mp: 188-189°C. TLC is red (10% ethanol sulfate), molecular formula is C ₁₀ H ₁₄ O ₃ , and its structural formula is

Chinese patent CN01108186.4 discloses the structural map of nepetalactone and its use, which can be used to prepare anti-cold drugs, anti-inflammatory drugs, antibacterial drugs, analgesics, etc. Chinese patent CN201010217622.1 discloses the structure of nepetalactone An improved preparation process simplifies the preparation operation of nepetalactone, improves the yield and reduces the production cost. Infection with herpes virus can cause a variety of human diseases, such as cold sores, genital herpes, chronic mucosal ulcers, keratoconjunctivitis, meningitis, etc. These clinical symptoms range from mild to severe, and in some cases even life-threatening. Nepetalactone has a weak effect on herpes virus in vitro. In order to obtain a drug with stronger anti-herpes virus effect, it is planned to obtain a nepetalactone derivative with stronger in vitro anti-herpes virus activity through structural modification. So far, there is no report of obtaining nepetalactone o-bromobenzoate from nepetalactone through o-bromobenzoate esterification.

Contents of the invention

The object of the present invention is to provide a kind of nepetalactone o-bromobenzoic acid ester obtained by esterifying nepetalactone with o-bromobenzoic acid ester, its preparation method and its use as medicine.

Technical scheme of the present invention is as follows

Nepetalactone o-bromobenzoate is characterized by a molecular formula of C ₁₇ H ₁₇ BrO ₄ , a melting point of 184-186°C, and a specific rotation of Its structural formula is as follows, the inventor tentatively named nepetalactone o-bromobenzoate:

The preparation method of nepetalactone o-bromobenzoate of the present invention comprises the steps:

A. Take an appropriate amount of nepetalactone, o-bromobenzoic acid and 4-dimethylaminopyridine (DMAP), the ratio of these three substances is 0.5～1.5: 1.5～2.5: 0.5～1.5, and place them in the reaction vessel , add dichloromethane, stir to dissolve completely, add N, N'-dicyclohexylcarbodiimide (DCC) or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide salt salt (EDCI), the amount of these two substances is 1.5 to 2.5 times that of nepetalactone, react at room temperature, TLC checks the reaction end point, and obtains the reaction solution;

B. Add methanol dropwise to the reaction solution obtained in step A to clarify the solution, add silica gel, and distill the solvent to dryness under reduced pressure to obtain a silica gel mixed sample.

C. Pack the silica gel mixed sample obtained in step B into a column for column chromatography, elute with sherwood oil-ethyl acetate with a volume ratio of 4 to 7: 1, and collect the mixture containing nepetalactone o-bromobenzoate The fractions were combined and the eluent was recovered under reduced pressure to obtain a yellow solid of nepetalactone o-bromobenzoate.

The preparation method of nepetalactone o-bromobenzoate in the above step A, the amount ratio of nepetalactone, o-bromobenzoic acid and 4-dimethylaminopyridine three substances is 1:2:1; N, N'-dicyclohexylcarbodiimide or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, the amount of these two substances is that of nepetalactone 2 times; the volume ratio of petroleum ether-ethyl acetate in step C is 5:1.

The in vitro anti-herpes virus pharmacodynamics test of nepetalactone o-bromobenzoate shows that it has good anti-HSV-1 activity, so it can be used to prepare anti-herpes virus drugs.

The nepetalactone o-bromobenzoate of the present invention is a novel menthane-type monoterpenoid compound, which has a simple structure, a simple and cheap preparation method, and strong physiological activity.

Description of drawings

Figure 1 The UV spectrum of the compound nepetalactone o-bromobenzoate, λ _max (CH ₃ OH): 277.6nm, suggesting that the compound has conjugated absorption characteristics

The HPLC purity spectrum of the compound nepetalactone o-bromobenzoate in Fig. 2, the purity of nepetalactone o-bromobenzoate calculated by normalization method is 97.3%.

Detailed ways

Below by way of example, the nepetalactone o-bromobenzoic acid ester that the present invention relates to and preparation method thereof are described in further detail, but this should not be interpreted as the scope of the above-mentioned theme of the present invention being limited to following examples, where The technologies realized based on the above contents of the present invention all belong to the scope of the present invention.

Example 1: Weighing nepetalactone (according to the method disclosed in Chinese patent CN201010217622.1, the commercially available nepeta volatile oil obtained by extracting the flower spikes of the Lamiaceae plant Schizonepeta tenuifolia Briq. by steam distillation was prepared by oxidation And get, purity more than 98%) 18.2mg (0.10mmol), o-bromobenzoic acid 50.2mg (0.25mmol, domestic AR grade), DMAP 12.3mg (0.10mmol, domestic AR grade), it is placed in 50mL round bottom flask Add 4mL of dichloromethane (domestic AR grade), stir to dissolve completely, add DCC 51.6mg (0.25mmol, domestic AR grade), react at room temperature (20-25°C), and check the end point of the reaction by TLC. After the reaction, methanol (domestic AR grade) was added dropwise to clarify the solution, and about 500 mg of silica gel (produced by Qingdao Ocean Chemical Factory, used for column chromatography) was added in three times, and the solvent was distilled under reduced pressure to dryness. Weigh 5g of silica gel (produced by Qingdao Haiyang Chemical Factory, for column chromatography) and pack it into a column, wash with eluent (petroleum ether (60-90°C, domestic AR grade): ethyl acetate (domestic AR grade)=5:1) When the elution is about 40mL, the target compound nepetalactone o-bromobenzoate can be found by TLC tracking. At this time, the eluate is collected and collected every 5mL. When about 30mL, the TLC tracking shows that the target compound has been basically completely eluted. The eluents are combined, and the eluents are recovered under reduced pressure to obtain a yellow powder of nepetalactone o-bromobenzoate with a purity of over 97% and a yield of 81%.

Example 2:

Structural analysis of the nepetalactone o-bromobenzoate prepared in Example 1:

HR-ESI-MS: m/z 366.0415 [M+H] ⁺ (calcd for C ₁₇ H ₁₇ BrO ₄ : 365.2185); [α] ^20.3 _D , +82.5° (c 1.0, CH ₃ OH).

IR (KBr tablet, cm ^-1 ): 3028 (ν _Φ-H ); 2931, 2848 (saturated cyclic hydrocarbon ν _CH ); 1754 (lactone carbonyl ν _C=O ); 1697 (carbonyl ν _C=O ); 1627, 1572, 1503, 1447 (conjugated benzene ring ν _C=C ); 864, 792, 689 (benzene ring meta-substituted γ _Φ-H ).

UV(CH ₃ OH)λ _max : 277.6nm, suggesting that the compound has conjugated absorption characteristics. See Attachment 1

H ¹ -NMR (300MHz, CDCl ₃ , ppm): δ1.01 (d, 3H, J=6.6Hz, 6'-CH ₃ ), 1.11 (dd, 1H, J=4.2, 13.2Hz, 5a-H) , 1.26(m, 1H, 4a-H), 1.90(s, 3H, 3'-H), 1.99-2.10(m, 2H, 5b-H, 6-H), 2.37(m, 1H, 4b-H ), 2.79-2.86(m, 2H, 7-H), 7.32-7.39(m, 2H, Ar-H), 7.65(dd, 1H, J=0.9, 4.5Hz, Ar-H), 7.75(dd, 1H, J = 1.2, 4.5 Hz, Ar-H).

¹³ C NMR (75MHz, CDCl ₃ , ppm): δ171.3 (C-8), 163.4 (C-7'), 157.9 (C-3), 134.3 (C-1'), 132.8, 131.5 (C- 2', C-6'), 127.3, 123.1 (C-3', C-5'), 122.2 (C-4'), 121.3 (C-7), 104.5 (C-4), 45.0 (C- 5), 34.5(C-1), 28.6(C6), 24.4(C-2), 20.9(C-10), 8.3(C-9).

Purity of nepetalactone o-bromobenzoate: detected by HPLC method, the peak area of nepetalactone o-bromobenzoate is 16945496, the total peak area of impurities is 469154, and the normalization method is used to calculate the nepetalactone o-bromobenzoate The purity of lactone o-bromobenzoate was 97.3%. See Attachment 2

Example 3: Weighing nepetalactone (according to the method disclosed in Chinese patent CN201010217622.1, the commercially available nepeta volatile oil obtained by extracting the flower spikes of the Lamiaceae plant Schizonepeta tenuifolia Briq. by steam distillation was prepared by oxidation And get, purity more than 98%) 18.2mg (0.10mmol), o-bromobenzoic acid 50.2mg (0.25mmol, domestic AR grade), DMAP 12.3mg (0.10mmol, domestic AR grade), it is placed in 50mL round bottom flask Add 4mL of dichloromethane (domestic AR grade), stir to dissolve completely, add EDCI 47.9mg (0.25mmol, domestic AR grade), react at room temperature (20-25°C), and check the end point of the reaction by TLC. After the reaction, methanol (domestic AR grade) was added dropwise to clarify the solution, and about 500 mg of silica gel (produced by Qingdao Ocean Chemical Factory, used for column chromatography) was added in three times, and the solvent was distilled under reduced pressure to dryness. Weigh 5g of silica gel (produced by Qingdao Haiyang Chemical Factory, for column chromatography) and pack it into a column, wash with eluent (petroleum ether (60-90°C, domestic AR grade): ethyl acetate (domestic AR grade)=5:1) When the elution is about 40mL, the target compound nepetalactone o-bromobenzoate can be found by TLC tracking. At this time, the eluate is collected and collected every 5mL. When about 30mL, the TLC tracking shows that the target compound has been basically completely eluted. The eluents were combined, and the eluents were recovered under reduced pressure to obtain a yellow solid of nepetalactone o-bromobenzoate with a purity of over 97% and a yield of 83%.

Example 4: Weighing nepetalactone (according to the method disclosed in Chinese patent CN201010217622.1, the commercially available nepeta volatile oil obtained by extracting the flower spikes of the Lamiaceae plant Schizonepeta tenuifolia Briq. by steam distillation was prepared by oxidation And get, purity more than 98%) 24.3mg (0.15mmol), o-bromobenzoic acid 64.3mg (0.32mmol, domestic AR grade), DMAP 18.5mg (0.15mmol, domestic AR grade), it is placed in 50mL round bottom flask Add 4mL of dichloromethane (domestic AR grade), stir to dissolve completely, add DCC 66.1mg (0.32mmol, domestic AR grade), react at room temperature (20-25°C), and check the end point of the reaction by TLC. After the reaction, add methanol (domestic AR grade) dropwise to clarify the solution, add about 550 mg of silica gel (produced by Qingdao Ocean Chemical Factory, for column chromatography) three times the amount, and distill the solvent to dryness under reduced pressure. Weigh 5g of silica gel (produced by Qingdao Ocean Chemical Factory, used for column chromatography) and pack it into a column, wash with eluent (petroleum ether (60-90°C, domestic AR grade): ethyl acetate (domestic AR grade)=6:1) When the elution is about 45mL, the target compound nepetalactone o-bromobenzoate can be found by TLC tracking. At this time, the eluate is collected and collected every 5mL. When it is about 35mL, the TLC tracking shows that the target compound has been basically completely washed out. decompression, combined eluents, and recovered the eluents under reduced pressure to obtain a yellow solid of nepetalactone o-bromobenzoate with a purity of over 97% and a yield of 87%.

Example 5: Weighing nepetalactone (according to the method disclosed in Chinese patent CN201010217622.1, the commercially available nepeta volatile oil obtained by extracting the flower spikes of the Lamiaceae plant Schizonepeta tenuifolia Briq. by steam distillation was prepared by oxidation And get, purity more than 98%) 24.3mg (0.15mmol), o-bromobenzoic acid 64.3mg (0.32mmol, domestic AR grade), DMAP 18.5mg (0.15mmol, domestic AR grade), it is placed in 50mL round bottom flask Add 4mL of dichloromethane (domestic AR grade), stir to dissolve completely, add EDCI 61.3mg (0.32mmol, domestic AR grade), react at room temperature (20-25°C), and check the end point of the reaction by TLC. After the reaction, add methanol (domestic AR grade) dropwise to clarify the solution, add about 550 mg of silica gel (produced by Qingdao Ocean Chemical Factory, for column chromatography) three times the amount, and distill the solvent to dryness under reduced pressure. Weigh 5g of silica gel (produced by Qingdao Ocean Chemical Factory, used for column chromatography) and pack it into a column, wash with eluent (petroleum ether (60-90°C, domestic AR grade): ethyl acetate (domestic AR grade)=6:1) When the elution is about 45mL, the target compound nepetalactone o-bromobenzoate can be found by TLC tracking. At this time, the eluate is collected and collected every 5mL. When it is about 35mL, the TLC tracking shows that the target compound has been basically completely washed out. The eluents were combined, and the eluents were recovered under reduced pressure to obtain a yellow solid of nepetalactone o-bromobenzoate with a purity of over 97% and a yield of 83%.

Example 6: Weighing nepetalactone (according to the method disclosed in Chinese patent CN201010217622.1, the commercially available nepeta volatile oil obtained by extracting the flower spikes of the Lamiaceae plant Schizonepeta tenuifolia Briq. by steam distillation was prepared by oxidation And get, purity more than 98%) 36.5mg (0.20mmol), o-bromobenzoic acid 76.3mg (0.38mmol, domestic AR grade), DMAP 24.5mg (0.20mmol, domestic AR grade), it is placed in 50mL round bottom flask Add 5mL of dichloromethane (domestic AR grade), stir to dissolve completely, add DCC 78.5mg (0.38mmol, domestic AR grade), react at room temperature (20-25°C), and check the end point of the reaction by TLC. After the reaction, methanol (domestic AR grade) was added dropwise to clarify the solution, and about 600 mg of silica gel (produced by Qingdao Ocean Chemical Factory, for column chromatography) was added in three times the amount, and the solvent was distilled to dryness under reduced pressure. Weigh 6g of silica gel (produced by Qingdao Ocean Chemical Factory, used for column chromatography) and pack it into a column, wash with eluent (petroleum ether (60-90°C, domestic AR grade): ethyl acetate (domestic AR grade)=7:1) When about 50mL was eluted, the target compound nepetalactone o-bromobenzoate could be found by TLC tracking. At this time, the eluate was collected and collected once every 5mL. When about 40mL, TLC tracking found that the target compound was basically completely eluted. decompression, combined the eluents, and recovered the eluents under reduced pressure to obtain a yellow solid of nepetalactone o-bromobenzoate with a purity of over 97% and a yield of 85%.

Example 7: Weighing nepetalactone (according to the method disclosed in Chinese patent CN201010217622.1, the commercially available nepeta volatile oil obtained by extracting the flower spikes of the Lamiaceae plant Schizonepeta tenuifolia Briq. by steam distillation was prepared by oxidation And get, purity more than 98%) 36.5mg (0.20mmol), o-bromobenzoic acid 76.3mg (0.38mmol, domestic AR grade), DMAP 24.5mg (0.20mmol, domestic AR grade), it is placed in 50mL round bottom flask Add 5mL of dichloromethane (domestic AR grade), stir to dissolve completely, add EDCI 72.8mg (0.38mmol, domestic AR grade), react at room temperature (20-25°C), and check the end point of the reaction by TLC. After the reaction, methanol (domestic AR grade) was added dropwise to clarify the solution, and about 600 mg of silica gel (produced by Qingdao Ocean Chemical Factory, for column chromatography) was added in three times the amount, and the solvent was distilled to dryness under reduced pressure. Weigh 6g of silica gel (produced by Qingdao Ocean Chemical Factory, used for column chromatography) and pack it into a column, wash with eluent (petroleum ether (60-90°C, domestic AR grade): ethyl acetate (domestic AR grade)=7:1) When about 50mL was eluted, the target compound nepetalactone o-bromobenzoate could be found by TLC tracking. At this time, the eluate was collected and collected once every 5mL. When about 40mL, TLC tracking found that the target compound was basically completely eluted. decompression, combined the eluents, and recovered the eluents under reduced pressure to obtain a yellow solid of nepetalactone o-bromobenzoate with a purity of over 97% and a yield of 86%.

The anti-HSV-1 virus effect of embodiment 8 nepetalactone o-bromobenzoate

1 sample cytotoxicity test

1.1 Preparation of sample concentration

The sample was prepared according to the method of the above-mentioned Example 1, first formulated with dimethyl sulfoxide to 20 mg/mL. Then diluted with Vero cell culture medium to 200μg/mL, 100μg/mL, 50μg/mL, 25μg/mL, 12.5μg/mL, 6.25μg/mL, 3.125μg/mL, 1.562μg/mL.

1.2 Toxicity test

Vero cells were cultured in a 96-well culture plate in a single layer at 37°C and 5% CO ₂ for 24 hours, the supernatant was discarded, and the above-mentioned prepared medicinal solutions of different concentrations were added to 4 wells for each concentration. After culturing in a 37°C, 5% CO ₂ incubator for 72 hours, the cell morphology was observed, and the drug-induced cytotoxicity TC ₅₀ was calculated.

2 cytopathic inhibition test

Vero cells were cultured in a 96-well culture plate in a single layer at 37°C and 5% CO ₂ for 24 hours, discarded the supernatant, washed the cells with the dilution solution, added 100 μL of the washing solution to each well, discarded the washing solution, and added 10TCID ₅₀ virus solution , 37°C, 5% CO ₂ adsorption for 2 hours, the virus was sucked off, and different concentrations of drug solutions were added, with 4 wells for each concentration. After culturing for 72 hours at 37°C and 5% _CO2 , observe CPE (lesions), set up cell control group, virus control group, positive control group (acyclovir) and experimental drug group, observe CPE at the same time, and inhibit lesions by more than 50% , calculate IC ₅₀ (half effective inhibitory concentration) and therapeutic index TI value.

3 results

The compound nepetalactone o-bromobenzoate showed good anti-HSV-1 activity, with IC ₅₀ of 6.25 μg/mL, TC ₅₀ of 29.3 μg/mL, and TI of 4.69. Under the same conditions, nepetalactone did not show better anti-HSV-1 activity, therefore, nepetalactone o-bromobenzoate was significantly better than nepetalactone's anti-HSV-1 activity in vitro. See Table 1.

Table 1 In vitro anti-HSV-1 activity of nepetalactone o-bromobenzoate

Note: "-" indicates that the sample has no inhibition on the virus or is not detected at the concentration of _TC50 .

TI=TC ₅₀ /IC ₅₀ .

Inhibition of the virus or not detected.

TI=TC ₅₀ /IC ₅₀ .

Claims (4)

1. Schizonepetolactone o-bromobenzoic acid ester, has following structure:

2. a preparation method for Schizonepetolactone o-bromobenzoic acid ester claimed in claim 1, is characterized in that comprising the steps:

A. get Schizonepetolactone, o-bromobenzoic acid and DMAP are appropriate, and these three kinds of material amount of substance ratios are 0.5～1.5: 1.5～2.5: 0.5～1.5, be placed in reaction vessel, add methylene dichloride, stirring is dissolved it completely, adds N, N '-dicyclohexylcarbodiimide or 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride, these two kinds of material amount of substances are 1.5～2.5 times of Schizonepetolactone, in room temperature reaction, TLC checks reaction end, obtains reaction solution;

B. by A step gained reaction solution, drip methyl alcohol and make solution clarification, add silica gel, underpressure distillation solvent, to dry, obtains silica gel compound sample;

C. silica obtained B step compound sample dress post is carried out to column chromatography, carry out wash-out with the petroleum ether-ethyl acetate that volume proportion is 4～7: 1, stream part that collection contains Schizonepetolactone o-bromobenzoic acid ester, reclaim under reduced pressure elutriant after merging, can obtain Schizonepetolactone o-bromobenzoic acid ester yellow solid.

3. the preparation method of Schizonepetolactone o-bromobenzoic acid ester as claimed in claim 2, is characterized in that, Schizonepetolactone in steps A, and three kinds of material amount of substance ratios of o-bromobenzoic acid and DMAP are 1: 2: 1; The N adding, N '-dicyclohexylcarbodiimide or 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride, these two kinds of material amount of substances are 2 times of Schizonepetolactone; The volume proportion of step C PetroChina Company Limited. ether-ethyl acetate is 5: 1.

4. a Schizonepetolactone o-bromobenzoic acid ester claimed in claim 1 is in the application of preparing in anti-herpesvirus medicament.