CN103012099A - Fluorenone compounds and preparation method and application thereof - Google Patents

Abstract

The invention discloses a fluorenone compound and its preparation method and application. The structural formula of the fluorenone compound is: ; said R ₁ is -H, R ₂ is -OH, its molecular formula is C ₁₉ H ₁₈ O ₆ , and the compound is named gramniphenolD; said R ₁ is -OH, R ₂ is -H, and its molecular formula is C ₁₉ H ₁₈ O ₆ , the compound is named gramniphenol E. Said method is obtained by taking dried bamboo leaf orchid branches, leaves or fruits as raw materials, extracting from extract, extracting with organic solvent, silica gel column chromatography and high pressure liquid chromatography. Application of the fluorenone compounds in the preparation of anti-AIDS drugs. GramniphenolsD and gramniphenolE are the first discovered natural fluorenones substituted with 2-hydroxy-3-methylbut-3-enyloxy. According to the cytotoxicity test on C8166 host cells and the inhibition test of HIV-1IIIB-induced CPE in C8166 cells, the two fluorenone compounds have good anti-HIV-1 activity, and the EC ₅₀ values are 1.46±0.15, respectively. μg/mL and 1.58±0.20μg/mL, the therapeutic index (TI) values were 137 and 126.6. The compound of the invention has a novel structure and good activity, and can be used as a leading compound of an anti-AIDS drug.

Description

A kind of fluorenone compound and its preparation method and application

technical field

The invention belongs to the technical field of extraction of effective plant components, and in particular relates to a fluorenone compound and a preparation method and application thereof.

Background technique

Orchidaceae (Orchidaceae) are monocotyledonous plants, and Arundina (Arundina) is a genus of Orchidaceae, which is a terrestrial orchid. There are about 5 species in this genus, which are distributed in tropical Asia to some islands in Oceania. Among them, A. graminifolia and A. stenopetala are also produced in southern my country. Bamboo leaf orchid, plant height 40-80cm, sometimes up to 1m or more; underground rhizomes are often oval-shaped and enlarged at the base of the connecting stems, resembling pseudobulbs, stems are upright, often clustered or grown in sheets, produced in Zhejiang, Jiangxi, Fujian, Taiwan, southern Hunan, Guangdong, Hainan, Guangxi, southern Sichuan (Miyi), Guizhou (Rongjiang, Xingyi), Yunnan (Dengchuan, Fengqing, Jinghong, Xichou, Pingbian, etc.) and Southeast Tibet (Medog). Growing on grass slopes, beside valleys, under bushes or in forests, at an altitude of 400-2800 meters. It is also distributed in Nepal, Sikkim, Bhutan, India, Sri Lanka, Myanmar, Vietnam, Laos, Cambodia, Thailand, Malaysia, Indonesia, Ryukyu Islands and Tahiti. The medicinal parts of Bamboo Orchid are rhizomes and stems and leaves, which are bitter in nature and flat. Heat-clearing and toxic substances removing, expelling wind and dampness, relieving pain, diuresis. For the treatment of jaundice, hot stranguria, beriberi edema, hernia abdominal pain, rheumatic arthralgia, stomach pain, urinary tract infection, snake bites, sores, carbuncles, swollen toxins, traumatic injuries, etc. Bamboo leaf orchid is a herbal medicine commonly used by the Dai people in Xishuangbanna area. The local Dai compatriots call the bamboo leaf orchid with beautiful flowers "Nong Shang Hei", which is a well-known detoxification medicine.

Fluorenone benzofuran has many types of plant fluorenone components, complex stereochemistry, and various biological activities. Research in this field is very active at home and abroad. Whether it is naturally occurring or artificially synthesized fluorenone compounds, have aroused widespread concern among chemists.

Contents of the invention

The first object of the present invention is to provide a fluorenone compound; the second object is to provide a preparation method of the fluorenone compound; the third object is to provide the application of the fluorenone compound in the preparation of anti-AIDS drugs.

The first object of the present invention is achieved in that the described fluorenone compound takes dry bamboo leaf orchid branches, leaves or fruit as raw material, extracts through extract, organic solvent extraction, silica gel column chromatography, high pressure liquid chromatography Separated and obtained, its structural formula is:

。

.

Said R ₁ is -H, R ₂ is -OH, its molecular formula is C ₁₉ H ₁₈ O ₆ , and the compound is named gramniphenol D.

Said R ₁ is -OH, R ₂ is -H, its molecular formula is C ₁₉ H ₁₈ O ₆ , and the compound is named gramniphenol E.

The second purpose of the present invention is achieved in this way. It is obtained by extracting the dried bamboo leaf orchid branches, leaves or fruits through extracting, organic solvent extraction, silica gel column chromatography, and high-pressure liquid chromatography. Specifically, :

A. Extraction of extract: Coarsely crush the branches, leaves or fruits of Bamboo orchid to 20-40 mesh, ultrasonically extract 2-4 times with organic solvent, 30-60 minutes each time, combine the extracts; filter the extracts, concentrate under reduced pressure When the extract reaches 1/4 ~ 1/2 volume, let it stand still, filter out the sediment, and concentrate into extract a;

B. Organic solvent extraction: Add 1 to 2 times the amount of water by weight to the extract a, extract 3 to 5 times with an organic solvent equal to the volume of water, combine the organic solvent extraction phase, and concentrate under reduced pressure to form extract b;

C. Silica gel column chromatography: dissolve the extract b with acetone with a weight ratio of 1.5 to 3 times, then mix the sample with 80 to 100 mesh silica gel with a weight of 0.8 to 1.2 times the extract, and then perform silica gel column chromatography. The column silica gel is 160-200 mesh, and the amount used is 6-8 times the weight of the extract b; the organic solvent solution with a volume ratio of 1:0-0:1 is used for gradient elution, and the gradient eluate is collected, concentrated, and monitored by TLC , merge the same parts;

D. Reversed-phase column chromatography: put the 9:1 part of the eluent in step C to reverse-phase column chromatography, and the reverse-phase column is packed with reverse-phase material C-18; methanol with a volume content of 20-100% The aqueous solution was subjected to gradient elution, and each part of the eluate was collected and concentrated, monitored by TLC, and the same parts were combined;

E. High-performance liquid chromatography separation: the 45%~60% methanol aqueous solution eluting part of the eluent in step D is separated and purified by high-performance liquid chromatography to obtain the fluorenone compounds;

The high-performance liquid chromatography separation and purification described in F and E steps is to use 40~60% methanol as mobile phase, flow velocity 10~14ml/min, 21.2' 250 mm, 5mm Zorbax PrepHT GF reversed-phase preparation column as stationary phase, The detection wavelength of the ultraviolet detector is 254 nm, 50-60 mL is injected each time, the chromatographic peaks are collected for 10-25 min, and evaporated to dryness after repeated accumulation to obtain the fluorenone compound gramniphenol D.

The high-performance liquid chromatography separation and purification described in G, E step is to be mobile phase with the methyl alcohol of 40～60%, flow velocity 10～14ml/min, 21.2 ' 250 mm, the Zorbax PrepHT GF reversed-phase preparation column of 5mm is stationary phase, The detection wavelength of the ultraviolet detector is 254 nm, 50-60mL is injected each time, the chromatographic peaks are collected for 25-40min, and evaporated to dryness after repeated accumulation to obtain the fluorenone compound gramniphenol E.

The third object of the present invention is achieved in this way, the application of the fluorenone compound in the preparation of anti-AIDS drugs.

The fluorenone compound of the present invention is isolated for the first time, and is determined to be a fluorenone compound by nuclear magnetic resonance and mass spectrometry methods, and its specific structure is characterized as:

。

.

Its isomer compounds gramniphenol D and gramniphenol E can be separated by the method of the present invention. Gramniphenols D and gramniphenol E are the first discovered natural fluorenones substituted with 2-hydroxy-3-methylbut-3-enyloxy. Using gramniphenol D and gramniphenol E as raw materials, the two fluorenone compounds have good anti-HIV-1 activity through the cytotoxicity test on C8166 host cells and the inhibition test on HIV-1IIIB-induced C8166 cytopathy (CPE) , the EC ₅₀ values were 1.46±0.15μg/mL and 1.58±0.20μg/mL, and the therapeutic index (TI) values were 137 and 126.6. The above results reveal that the compound of the present invention has a novel structure and good activity, can be used as a leading compound of anti-AIDS drugs, and has a good application prospect in the preparation of anti-AIDS drugs.

Description of drawings

Figure 1 is the carbon nuclear magnetic resonance spectrum ( ¹³ C NMR) of the compound gramniphenol D;

Figure 2 is the hydrogen nuclear magnetic resonance spectrum ( ¹ H NMR) of the compound gramniphenol D;

Figure 3 is the carbon nuclear magnetic resonance spectrum ( ¹³ C NMR) of the compound gramniphenol E;

Figure 4 is the hydrogen nuclear magnetic resonance spectrum ( ¹ H NMR) of the compound gramniphenol E;

Figure 5 shows the main HMBC ( → ) and ¹ H- ¹ H COZY ( – ) correlation of the compound gramniphenol D.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings, but the present invention is not limited in any way, and any changes or improvements based on the teaching of the present invention fall within the protection scope of the present invention.

The fluorenone compound described in the present invention is obtained by extracting from extract, organic solvent extraction, silica gel column chromatography, and high-pressure liquid chromatography using dried bamboo leaf orchid branches, leaves or fruits as raw materials, and its structural formula is:

.

Said R ₁ is -H, R ₂ is -OH, its molecular formula is C ₁₉ H ₁₈ O ₆ , and the compound is named gramniphenol D.

Said R ₁ is -OH, R ₂ is -H, its molecular formula is C ₁₉ H ₁₈ O ₆ , and the compound is named gramniphenol E.

The preparation method of fluorenone compounds described in the present invention is obtained by extracting from extract, organic solvent extraction, silica gel column chromatography, and high-pressure liquid chromatography by using dried bamboo leaf orchid branches, leaves or fruits as raw materials, specifically: :

A. Extraction of extract: Coarsely crush the branches, leaves or fruits of Bamboo orchid to 20-40 mesh, ultrasonically extract 2-4 times with organic solvent, 30-60 minutes each time, combine the extracts; filter the extracts, concentrate under reduced pressure When the extract reaches 1/4 ~ 1/2 volume, let it stand still, filter out the sediment, and concentrate into extract a;

B. Organic solvent extraction: Add 1 to 2 times the amount of water by weight to the extract a, extract 3 to 5 times with an organic solvent equal to the volume of water, combine the organic solvent extraction phase, and concentrate under reduced pressure to form extract b;

C. Silica gel column chromatography: dissolve the extract b with acetone with a weight ratio of 1.5 to 3 times, then mix the sample with 80 to 100 mesh silica gel with a weight of 0.8 to 1.2 times the extract, and then perform silica gel column chromatography. The column silica gel is 160-200 mesh, and the amount used is 6-8 times the weight of the extract b; the organic solvent solution with a volume ratio of 1:0-0:1 is used for gradient elution, and the gradient eluate is collected, concentrated, and monitored by TLC , merge the same parts;

D. Reversed-phase column chromatography: put the 9:1 part of the eluent in step C to reverse-phase column chromatography, and the reverse-phase column is packed with reverse-phase material C-18; methanol with a volume content of 20-100% The aqueous solution was subjected to gradient elution, and each part of the eluate was collected and concentrated, monitored by TLC, and the same parts were combined;

E. High-performance liquid chromatography separation: the 45%~60% methanol aqueous solution eluting part of the eluent in step D is separated and purified by high-performance liquid chromatography to obtain the fluorenone compounds;

The high-performance liquid chromatography separation and purification described in F and E steps is to use 40~60% methanol as mobile phase, flow velocity 10~14ml/min, 21.2' 250 mm, 5mm Zorbax PrepHT GF reversed-phase preparation column as stationary phase, The detection wavelength of the ultraviolet detector is 254 nm, 50-60 mL is injected each time, the chromatographic peaks are collected for 10-25 min, and evaporated to dryness after repeated accumulation to obtain the fluorenone compound gramniphenol D.

The high-performance liquid chromatography separation and purification described in G, E step is to be mobile phase with the methyl alcohol of 40～60%, flow velocity 10～14ml/min, 21.2 ' 250 mm, the Zorbax PrepHT GF reversed-phase preparation column of 5mm is stationary phase, The detection wavelength of the ultraviolet detector is 254 nm, 50-60mL is injected each time, the chromatographic peaks are collected for 25-40min, and evaporated to dryness after repeated accumulation to obtain the fluorenone compound gramniphenol E.

The organic solvent described in step A is one of 70-100% acetone, ethanol or methanol.

The organic solvent described in step B is one of ethyl acetate, chloroform, ether, sherwood oil or benzene.

The organic solvent solution described in step C is one of n-hexane-acetone, chloroform-acetone, chloroform-methanol, petroleum ether-acetone or petroleum ether-ethyl acetate.

The volume ratio of the organic solvent solution described in step C is 1:0, 20:1, 9:1, 8:2, 3:2, 1:1, 1:2, 0:1.

The high-performance liquid chromatography separation and purification described in step E uses 40-60% methanol as the mobile phase, a flow rate of 10-14ml/min, a 21.2' 250 mm, 5mm Zorbax PrepHT GF reverse-phase preparation column as the stationary phase, and ultraviolet detection The detection wavelength of the detector is 254 nm, each injection is 50-60 mL, the chromatographic peaks are collected for 10-40 min, and evaporated to dryness after repeated accumulation.

Application of the fluorenone compound described in the present invention in the preparation of anti-AIDS drugs.

Bamboo leaf orchid of the present invention is not restricted by region and species, and can realize the present invention.

Example 1

Take 5.8 kg of dried bamboo leaf orchid branches, leaves and/or fruits, coarsely crush them to 40 meshes, use 70% acetone to ultrasonically extract 4 times, each time for 60 minutes, and combine the extracts; filter the extracts, concentrate under reduced pressure to volume 1/4; stand, filter the precipitate, concentrate into 674g extract a; add 630g water to extract a, extract 5 times with ethyl acetate equal to the volume of water, combine the extract phases, concentrate under reduced pressure to 496g Extract b; use 1900g of 200 mesh silica gel to pack the column, add 478.5g of acetone to the extract b to dissolve, then add 496g of 100 mesh silica gel to mix the sample, and put the sample on the column after mixing; the volume ratio is 1:0, 20: 1. Gradient elution with chloroform-methanol organic solvent solution of 9:1, 8:2, 3:2, 1:1, 1:2, 0:1, collecting the gradient eluate, concentrating, monitoring by TLC, combining the same The part, obtains 6 parts, and the eluent c of the chloroform-methanol organic solvent solution of volume ratio 9:1 is 81.2g; Use reversed-phase material C-18 packing column, reversed-phase column on eluent c, with volume Carry out gradient elution with methanol aqueous solution with a content of 20-100%, collect and concentrate each part of the eluate, monitor by TLC, and combine the same part; take the eluate obtained by eluting with a volume content of 45-60% methanol aqueous solution, Then use 55% methanol as mobile phase, flow rate 10ml/min, 21.2'250mm, 5mm Zorbax PrepHT GF reverse phase preparative column as stationary phase, UV detector detection wavelength is 254 nm, inject 50mL each time, collect 19min The chromatographic peaks were accumulated several times and then evaporated to dryness to obtain the fluorenone compound gramniphenol D; the chromatographic peaks collected for 28 minutes were accumulated and evaporated to dryness to obtain the fluorenone compound gramniphenol E.

Example 2

Take 5 kg of dried bamboo leaf orchid branches, leaves and/or fruits, coarsely crush them to 20 mesh, use 100% ethanol to ultrasonically extract twice, each time for 50 minutes, and combine the extracts; filter the extracts, and concentrate under reduced pressure to volume 1/3; stand, filter the precipitate, and concentrate into 590g of extract a; add 590g of water to extract a, extract 3 times with chloroform equal to the volume of water, combine the extract phases, concentrate under reduced pressure into 438g of extract Cream b; use 3504g of 160 mesh silica gel to pack the column, add 1314g of acetone to the extract b to dissolve, then add 350.4g of 80 mesh silica gel to mix the sample, and put the sample on the column after mixing; the volume ratio is 1:0 and 20:1 respectively , 9:1, 8:2, 3:2, 1:1, 1:2, 0:1 gradient elution of n-hexane-acetone organic solvent solution, the gradient eluate was collected, concentrated, monitored by TLC, and combined with the same Part of the reversed-phase material C-18 was used to pack the column, and the eluent c was applied to the reversed-phase column, and the gradient elution was carried out with a methanol aqueous solution with a volume content of 20-100%, and each part of the eluate was collected and concentrated, and monitored by TLC , combined the same parts; take the eluate obtained by eluting with 45~60% methanol aqueous solution, and then use 45% methanol as the mobile phase, flow rate 14ml/min, 21.2′250mm, 5mm Zorbax PrepHT GF reverse phase The preparation column is a stationary phase, and the detection wavelength of the ultraviolet detector is 254 nm. Each injection is 55 mL, and the chromatographic peaks are collected for 22 minutes. peak, evaporated to dryness after adding up several times to obtain the fluorenone compound gramniphenol E.

Example 3

Take 6kg of dried bamboo leaf orchid branches, leaves and/or fruits, roughly crush them to 30 mesh, use 80% methanol to ultrasonically extract 4 times, each time for 30min, and combine the extracts; filter the extracts, and concentrate under reduced pressure to 1% of the volume. /2; stand, filter out the sediment, concentrate into 700g extract a; add 1400g of water to extract a, extract 4 times with ether equal to the volume of water, combine the extraction phases, concentrate under reduced pressure into 520g extract b; use 3638g of 180 mesh silica gel to pack the column, add 1040g of acetone to the extract b to dissolve, then add 624g of 90 mesh silica gel to mix the sample, and put the sample on the column after mixing; the volume ratio is 1:0, 20:1, 9 respectively :1, 8:2, 3:2, 1:1, 1:2, 0:1 chloroform-acetone organic solvent solution gradient elution, collect gradient eluate, concentrate, monitor by TLC, merge the same part; Pack the column with reversed-phase material C-18, put the eluent c on the reversed-phase column, and carry out gradient elution with methanol aqueous solution with a volume content of 20-100%, collect and concentrate each part of the eluate, monitor by TLC, and combine the same Take the eluate obtained by eluting with 45~60% methanol aqueous solution by volume content, then use 60% methanol as mobile phase, flow rate 12ml/min, 21.2′250mm, 5mm Zorbax PrepHT GF reverse-phase preparation column is The stationary phase, the detection wavelength of the ultraviolet detector is 254nm, each injection is 60mL, the chromatographic peak is collected for 10min, evaporated to dryness after repeated accumulation, and the fluorenone compound gramniphenol D is obtained; the chromatographic peak of 26min is collected, and the chromatographic peak is repeatedly Add up and evaporate to dryness to obtain the fluorenone compound gramniphenol E.

Example 4

Take 5.5 kg of dried bamboo leaf orchid branches, leaves and/or fruits, coarsely crush them to 40 mesh, and ultrasonically extract 3 times with 90% ethanol, each time for 45 minutes, and combine the extracts; filter the extracts, concentrate under reduced pressure to volume 1/4; stand, filter the precipitate, concentrate into 640g of extract a; add 960g of water to extract a, extract 4 times with petroleum ether equal to the volume of water, combine the extract phases, and concentrate under reduced pressure to 475g Extract b; use 2850g of 160 mesh silica gel to pack the column, add 712.5g of acetone to the extract b to dissolve, then add 475g of 80 mesh silica gel to mix the sample, and put the sample on the column after mixing; the volume ratio is 1:0, 20: 1. 9:1, 8:2, 3:2, 1:1, 1:2, 0:1 petroleum ether-acetone organic solvent solution gradient elution, gradient eluent collected, concentrated, monitored by TLC, combined The same part; use the reverse phase material C-18 to pack the column, put the eluent c on the reverse phase column, and carry out gradient elution with methanol aqueous solution with a volume content of 20~100%, collect and concentrate each part of the eluate, and pass through TLC Monitor and combine the same parts; take the eluate obtained by eluting with 45~60% methanol aqueous solution, then use 50% methanol as the mobile phase, flow rate 10ml/min, 21.2′250mm, 5mm Zorbax PrepHT GF reaction The phase preparation column is a stationary phase, and the detection wavelength of the ultraviolet detector is 254nm. Collect the chromatographic peaks for 21 minutes, evaporate to dryness after repeated accumulation, and obtain the fluorenone compound gramniphenol D; collect the chromatographic peaks for 35 minutes, and after repeated accumulations Evaporate to dryness to obtain the fluorenone compound gramniphenol E.

Example 5

Take 5 kg of dried bamboo leaf orchid branches, leaves and/or fruits, coarsely crush them to 20 mesh, use 70% methanol to ultrasonically extract 4 times, each time for 35 minutes, and combine the extracts; filter the extracts, and concentrate under reduced pressure to 1% of the volume. /2; leave standstill, filter out the sediment, concentrate into 580g extract a; add 1160g of water to extract a, extract 5 times with benzene equal to the volume of water, combine the extraction phases, concentrate under reduced pressure to become 430g extract b; use 200 mesh silica gel 3010 g to pack the column, add 1290 g of acetone to the extract b to dissolve, then add 100 mesh silica gel 344 g to mix the sample, and put the sample on the column after mixing; the volume ratio is 1:0, 20:1, 9:1, 8:2, 3:2, 1:1, 1:2, 0:1 petroleum ether-ethyl acetate organic solvent solution gradient elution, the gradient eluate was collected, concentrated, monitored by TLC, combined The same part; use the reverse phase material C-18 to pack the column, put the eluent c on the reverse phase column, and carry out gradient elution with methanol aqueous solution with a volume content of 20~100%, collect and concentrate each part of the eluate, and pass through TLC Monitor and combine the same parts; take the eluate obtained by eluting with 45~60% methanol aqueous solution, then use 40% methanol as the mobile phase, flow rate 12ml/min, 21.2′250mm, 5mm Zorbax PrepHT GF reaction The phase preparation column is a stationary phase, and the detection wavelength of the ultraviolet detector is 254nm. Collect the chromatographic peaks for 25 minutes, evaporate to dryness after repeated accumulation, and obtain the fluorenone compound gramniphenol D; collect the chromatographic peaks for 40 minutes, and accumulate them several times. Evaporate to dryness to obtain the fluorenone compound gramniphenol E.

Example 6

The compound gramniphenol D prepared in Example 1 is a red jelly; the optical rotation value [a] is 24.8 D - 16.8 (the solvent is methanol c 0.25); the determination method is: use nuclear magnetic resonance, combined with other spectral techniques to identify the structure.

(1) UV spectrum (the solvent is methanol), λ _max (log e ): 210 (3.86), 268 (3.82), 320 (2.58), 346 (2.89) nm;

(2) Infrared spectrum (potassium bromide tablet), n _max 3328, 2972, 2895, 1698, 1610, 1548, 1456, 1187, 1114 cm ^-1 ;

(3) HRESIMS shows that the quasi-molecular ion peak of the compound of the present invention is m/z 365.1002 [M + H] ⁺ (calculated value is 365.1001), combined with ¹³ C and ¹ H NMR spectra (Figure 1 and Figure 2, carbon spectrum hydrogen spectrum data attribution See Table 1) giving its molecular formula C ₁₉ H ₁₈ O ₆ . See Table 1 for ¹ H NMR (C ₅ D ₅ N, 500 MHz) and ¹³ C NMR (C ₅ D ₅ N, 125 MHz) data.

The infrared absorption of Gramniphenol D shows that there are hydroxyl groups (3328 cm ^-1 ), carbonyl groups (1698 cm ^-1 ), and aromatic rings (1610, 1548, 1456 cm ^-1 ) in its structure. The ultraviolet absorption spectrum at 268, 320, 346 nm also shows the presence of aromatic rings. ¹ H and ¹³ C NMR spectra showed 19 carbon signals and 18 hydrogen signals, indicating 1,4,5,7-oxidized fluorenone structures (H-2, H-3, H-6, H-8, and C-1 - C-9a), 1 methoxy group ( d _C 57.0, d _H 3.91), 2 phenolic hydroxyl protons ( δ _H 10.50, 11.02), and 1 2-hydroxy-3-methylbut-3 - enyloxy unit[-OCH ₂ CH(OH)C(CH ₂ )(CH ₃ )] (H-1￠, H-2￠, H-4￠, H-5￠, and C-1￠ - C- 5￠). The correlation between the methoxy proton ( δ _H 3.91) and C-5 ( δ _C 159.5) in the HMBC spectrum indicates that the methoxy group is at the C-5 position. In addition, the HMBC correlation of H-1￠ ( d _H 3.95 and 4.12) with C-7 ( d _C 152.0) indicated that the 2-hydroxy-3-methylbut-3-enyloxy group was substituted at the C-7 position. Finally, the hydroxyl signal ( dH _11.02 ) correlates with the HMBC _of C-1 ( dC 150.5), C-2 ( _{dC 118.3} ), and C-9a ( dC _116.7 ), as well as another hydroxyl signal ( dH 10.50) to C-3 ( d _C 129.1), C-4 ( d _C 145.0), and C-4a ( d _C 125.1), positioning the 2 phenolic hydroxyl protons at C-1 and C-4, respectively. By comparing with the NMR spectrum and optical rotation value of the known compound stachylines A, the configuration of C-2' is S . So far the structure of the compound has been confirmed.

Example 7

The compound gramniphenol E prepared in Example 1 is a red jelly; the optical rotation value [a] is 25.0 D - 14.6 (the solvent is methanol c 0.25); the determination method is: use nuclear magnetic resonance, combined with other spectral techniques to identify the structure.

(1) UV spectrum (methanol as solvent), λ _max (log e ): 210 (3.79), 266 (3.86), 320 (2.52), 346 (2.73) nm;

(2) Infrared spectrum (potassium bromide pellet), n _max 3325, 2967, 2897, 1701, 1611, 1546, 1452, 1182, 1120 cm ^-1 ;

(3) HRESIMS shows that the quasi-molecular ion peak of the compound of the present invention is m/z 365.1009 [M + H] ⁺ (calculated value is 365.1001), combined with ¹³ C and ¹ H NMR spectra (Figure 3 and Figure 4, see the table for carbon spectrum data attribution 1) Give its molecular formula C ₁₉ H ₁₈ O ₆ . See Table 1 for ¹ H NMR (C ₅ D ₅ N, 500 MHz) and ¹³ C NMR (C ₅ D ₅ N, 125 MHz) data.

The molecular formula C ₁₉ H ₁₈ O ₆ of Gramniphenol E is the same as that of Gramniphenol D, and the ¹ H and ¹³ C NMR spectra are also very similar to those of Gramniphenol D. Through comparison, it is found that the difference between these two compounds lies in the substitution on the fluorenone ring. ^{The 1} H NMR signals of d _H 6.57 (d, J = 1.9 Hz), 6.28 (d, J = 1.9 Hz), 6.67 (d, J = 1.7 Hz), and 6.83 (d, J = 1.7 Hz) illustrate gramniphenol E For 2,4,5,7-oxidized substituted fluorenones. The methoxy signal ( δ _H 3.85) and C-5 ( δ _C 153.3) in the HMBC spectrum indicated that the methoxy group was at the C-5 position. In addition, _the HMBC correlation of H-1¢ ( dH _3.93 and 4.22) with C-7 ( δC 157.3) indicated that ( S )-2-hydroxy-3-methylbut-3-enyloxy was at the C-7 position. The hydroxyl signal ( d _H 11.11) in the HMBC spectrum is related to C-1 ( d _C 105.9), C-2 ( d _C 159.5), and C-3 ( d _C 110.6), and another hydroxyl ( d _H 10.83) is related to C-3 ( dC 110.6), C-4 ( dC 154.3), and C-4a ( _dC 124.1) are related, indicating two _hydroxyl substitutions at C-2 and C-4 _, respectively. So far the structure of the compound has been determined.

Table 1 ¹ H and ¹³ C NMR data of compounds (solvent is C ₅ D ₅ N)

Example 8

The compounds gramniphenol D and gramniphenol E prepared in Example 2 were subjected to structural determination according to the methods in Examples 6 and 7 respectively, and the results were: the structures were the same as in Examples 6 and 7, and the molecular formulas were C ₁₉ H ₁₈ O ₆ and C ₁₉ H ₁₈ O ₆ .

Example 9

The compounds gramniphenol D and gramniphenol E prepared in Example 3 were subjected to structural determination according to the methods in Examples 6 and 7 respectively, and the results were: their structures were the same as in Examples 6 and 7, and the molecular formulas were C ₁₉ H ₁₈ O ₆ and C ₁₉ H ₁₈ O ₆ .

Example 10

The compounds gramniphenol D and gramniphenol E prepared in Example 4 were subjected to structural determination according to the methods in Examples 6 and 7 respectively, and the results were: the structures were the same as in Examples 6 and 7, and the molecular formulas were C ₁₉ H ₁₈ O ₆ and C ₁₉ H ₁₈ O ₆ .

Example 11

The compounds gramniphenol D and gramniphenol E prepared in Example 5 were subjected to structural determination according to the methods in Examples 6 and 7 respectively, and the results were: their structures were the same as in Examples 6 and 7, and the molecular formulas were C ₁₉ H ₁₈ O ₆ and C ₁₉ H ₁₈ O ₆ .

Example 12

Take the fluorenone compound (gramniphenol D) prepared in Example 1 and carry out the anti-HIV-1 activity test, as follows:

Determination of drugs and compounds: the samples to be tested were dissolved in DMSO, and the positive control compound azidethymidine (AZT, 3′-Azido-3′-deoxythymidine) was purchased from Sigma Company, dissolved in RPMI-1640 complete medium, filtered through 0.22 μm Sterilize by membrane filtration and store at -20°C after aliquoting.

Reagents: HEPES (N-2(2-Hydroxyethyl) piperazine-N'-(2-ethanesufonic acid), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), DMF (N, N′-Dimethyl formamine), penicillin (Penicillin), streptomycin sulfate (Streptomycin sulfate), glutamine (Glutamine) were purchased from Sigma; DMSO, 2-mercaptoethanol (2-Me, 2-Mercaptoethanol ) are products of Bio-Rad. RPMI-1640 and newborn calf serum are products of Gibco.

Medium: RPMI-1640 complete medium, containing 10% newborn calf serum, 2 mM L-glutamine, 10 mM HEPES, 50 μM 2-mercaptoethanol, 100,000 IU penicillin, 100 μg/mL streptomycin.

Cells and viruses: human T lymphocyte cell line C8166, MT4 and HIV-1 experimental strain HIV-1IIIB are all from the British Medical Research Council, AIDS Reagent Project. All cells and viruses were cultured in complete RPMI-1640 medium containing 10% calf serum. Prepare HIV-1IIIB according to conventional methods, titrate and calculate the TCID ₅₀ of the virus. After aliquoting the virus stock solution, store it at -70°C. Cells and viruses were frozen and recovered according to conventional methods.

HIV-1 infectivity titration: perform titration according to the modified method described by Johnson & Byington, briefly described as follows: make 4-fold dilutions of HIV-1IIIB stock solution on a 96-well plate, 10 gradients, 6 replicate wells for each gradient, and set a control at the same time Holes 6 holes. Add 50 μL of C8166 cells (4×10 ⁵ /mL) to each well, and the final volume of each well is 200 μL. Incubate at 37°C, 5% _CO2 . On the third day, 100 μL of fresh RPMI-1640 complete medium was added, and on the seventh day, the cytopathic effect (Cytopathic Effect, CPE) induced by HIV-1 in each well was observed under an inverted microscope to check whether there was syncytium (Syncytium) in each well. The formation of the virus was determined; the TCID ₅₀ (50% Tissue Culture Infection Dose) of the virus was calculated according to the Reed & Muench method.

Cytotoxicity detection of samples to C8166 host cells: 100 μL of 4×10 ⁵ /mL C8166 cell suspension was mixed with different solutions of the compounds to be tested, and three replicate holes were set. At the same time, control wells without compounds were set up, cultured at 37°C, 5% CO ₂ for 3 days, and the cytotoxicity was detected by MTT colorimetry. The OD value was measured by ELx800 ELISA instrument, the measurement wavelength was 595 nm, and the reference wavelength was 630 nm. Calculate the CC ₅₀ value (50% Cytotoxic Concentration), which is the concentration of the compound that produces toxicity to 50% of the normal T lymphocyte line C8166.

Inhibition test of samples on HIV-1IIIB-induced C8166 cell pathology (CPE): Inoculate 50 μL/well of 8×10 ⁵ /mL C8166 cells on a 96-well cell culture plate containing 100 μL/well of doubly diluted compound, and then add 50 μL of HIV-1IIIB diluted supernatant (MOI0.0016). Set up 3 replicate holes. At the same time, set up normal cell control wells without compounds, culture at 37°C, 5% CO ₂ for 3 days, and count the formation of syncytia under an inverted microscope (100×). EC ₅₀ (50% Effective Concentration) is the concentration of the compound that inhibits syncytia formation by 50%.

Calculation formula: draw a dose-response curve based on the experimental results, and calculate the 50% effective concentration (EC ₅₀ ) of the compound for inhibiting virus, 50% concentration for inhibiting cell growth (CC ₅₀ ) and the therapeutic index TI value of anti-HIV-1 activity according to the Reed & Muench method (Therapeutic index) is: TI = CC ₅₀ /EC ₅₀ .

Test results: After the cytotoxicity detection of C8166 host cells and the inhibition test of HIV-1IIIB-induced C8166 cytopathy (CPE), fluorenone compounds (gramniphenol D) have good anti-HIV-1 activity, EC ₅₀ The value was 1.46±0.15 μg/mL, and the therapeutic index (TI) values were 137 (see Table 2). The above results reveal that the compound of the present invention has a novel structure and good activity, can be used as a leading compound of anti-AIDS drugs, and has a good application prospect in the preparation of anti-AIDS drugs.

Example 13

The fluorenone compound (gramniphenol E) prepared in Example 1 was tested for anti-HIV-1 activity by the same method as in Example 12. The test results: the cytotoxicity of C8166 host cells was detected, and the induction of C8166 by HIV-1IIIB Cytopathic pathology (CPE) inhibition test, fluorenone compound (gramniphenol E) has good anti-HIV-1 activity, EC ₅₀ value is 1.58±0.2μg/mL, therapeutic index (TI) values are 137 (see table 2). The above results reveal that the compound of the present invention has a novel structure and good activity, can be used as a leading compound of anti-AIDS drugs, and has a good application prospect in the preparation of anti-AIDS drugs.

Table 2 Anti-HIV activity of compounds

 

 

Claims (9)

1. A kind of fluorenone compound, it is characterized in that: described fluorenone compound is raw material with dry bamboo leaf orchid branch, leaf or fruit, extracts through extract, organic solvent extraction, silica gel column chromatography, high-pressure liquid Obtained by phase chromatographic separation, its structural formula is:

. 2. The fluorenone compound according to claim 1, characterized in that: said R ₁ is -H, R ₂ is -OH, its molecular formula is C ₁₉ H ₁₈ O ₆ , and the compound is named gramniphenol D. 3. The fluorenone compound according to claim 1, characterized in that: said R ₁ is -OH, R ₂ is -H, its molecular formula is C ₁₉ H ₁₈ O ₆ , and the compound is named gramniphenol E. 4. A preparation method of fluorenone compounds as claimed in claim 1, characterized in that the dry bamboo leaf orchid branches, leaves or fruits are used as raw materials, extracted through extracts, organic solvent extraction, silica gel column chromatography, high-pressure liquid Obtained by phase chromatographic separation, specifically: A. Extraction of extract: Coarsely crush the branches, leaves or fruits of Bamboo orchid to 20-40 mesh, ultrasonically extract 2-4 times with organic solvent, 30-60 minutes each time, combine the extracts; filter the extracts, concentrate under reduced pressure When the extract reaches 1/4 ~ 1/2 volume, let it stand still, filter out the sediment, and concentrate into extract a; B. Organic solvent extraction: Add 1 to 2 times the amount of water by weight to the extract a, extract 3 to 5 times with an organic solvent equal to the volume of water, combine the organic solvent extraction phase, and concentrate under reduced pressure to form extract b; C. Silica gel column chromatography: dissolve the extract b with acetone with a weight ratio of 1.5 to 3 times, then mix the sample with 80 to 100 mesh silica gel with a weight of 0.8 to 1.2 times the extract, and then perform silica gel column chromatography. The column silica gel is 160-200 mesh, and the amount used is 6-8 times the weight of the extract b; the organic solvent solution with a volume ratio of 1:0-0:1 is used for gradient elution, and the gradient eluate is collected, concentrated, and monitored by TLC , merge the same parts; D. Reversed-phase column chromatography: put the 9:1 part of the eluent in step C to reverse-phase column chromatography, and the reverse-phase column is packed with reverse-phase material C-18; methanol with a volume content of 20-100% The aqueous solution was subjected to gradient elution, and each part of the eluate was collected and concentrated, monitored by TLC, and the same parts were combined; E. High-performance liquid chromatography separation: the eluent obtained by eluting with an aqueous methanol solution with a volume content of 45-60% is separated and purified by high-performance liquid chromatography to obtain the fluorenone compounds; The high-performance liquid chromatography separation and purification described in F and E steps is to use 40-60% methanol as mobile phase, flow rate 10-14ml/min, 21.2 × 250 mm, 5 μm Zorbax PrepHT GF reverse-phase preparative column as stationary phase, The detection wavelength of the ultraviolet detector is 254 nm, each injection is 50-60 μL, and the chromatographic peaks are collected for 10-25 minutes, and evaporated to dryness after repeated accumulation to obtain the fluorenone compound gramniphenol D; The HPLC separation and purification described in the G and E steps is to use 40-60% methanol as mobile phase, flow rate 10-14ml/min, 21.2 × 250 mm, 5 μm Zorbax PrepHT GF reverse-phase preparative column as stationary phase, The detection wavelength of the ultraviolet detector is 254 nm, 50-60 μL is injected each time, the chromatographic peaks are collected for 25-40 minutes, and evaporated to dryness after repeated accumulation to obtain the fluorenone compound gramniphenol E. 5. the preparation method of fluorenone compound according to claim 2 is characterized in that: the organic solvent described in step A is a kind of in acetone, ethanol or methyl alcohol of 70～100%. 6. the preparation method of fluorenone compound according to claim 2 is characterized in that: the organic solvent described in step B is the one in ethyl acetate, chloroform, ether, sherwood oil or benzene. 7. the preparation method of fluorenone compound according to claim 2 is characterized in that: the organic solvent solution described in C step is n-hexane-acetone, chloroform-acetone, chloroform-methanol, sherwood oil-acetone or sherwood oil - One of ethyl acetate. 8. the preparation method of fluorenone compound according to claim 2 is characterized in that: the volume ratio of the organic solvent solution described in step C is 1:0, 20:1, 9:1, 8:2, 3:2, 1:1, 1:2, 0:1. 9. A use of the fluorenone compound according to claim 1, 2 or 3 in the preparation of anti-AIDS drugs.

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