CN114805382B - A sesquiterpene ketone compound and its separation and application in the preparation of anti-pancreatic cancer drugs - Google Patents

Abstract

The invention discloses a structure and a separation method of sesquiterpene chromone compounds, and discovers that the sesquiterpene chromone compounds separated from Xinjiang asafetida have good pancreatic cancer resisting activity through an MTT (methyl thiazolyl tetrazolium) experiment, and can be used as an active ingredient for preparing medicines for treating pancreatic cancer. The structural formula of the sesquiterpene chromone compound is as follows:

Description

A sesquiterpene ketone compound and its separation and preparation of anti-pancreatic cancer drugs Applications

technical field

The present invention relates to a natural sesquiterpene ketone compound and its separation method, and also relates to the anti-pancreatic cancer biological activity of the natural sesquiterpene ketone compound and its application as an active substance for preparing anti-pancreatic cancer drugs, belonging to natural field of medicinal chemistry.

Background technique

Pancreatic cancer (PC) is highly invasive and fatal, and is one of the malignant tumors with the worst prognosis currently known. In recent years, the incidence of pancreatic cancer in my country has been on the rise, and its mortality rate ranks among the top 10 malignant tumors, with a 5-year survival rate of only 10%. About 80%-85% of pancreatic cancer patients are unresectable or have metastasized. Even for patients who can be locally surgically resected, the prognosis is still poor, and only 10%-15% of patients can be controlled by surgery. Drug therapy is one of the main means of pancreatic cancer treatment, but due to drug resistance and lack of drug specificity, the overall effective rate of drugs is less than 20%. In order to prolong the survival period of patients with pancreatic cancer, it is urgent to develop safer and more effective chemotherapy drugs for pancreatic cancer.

As an important source of drugs, natural products play an important role in the discovery and development of new drugs. More than 60% of antineoplastic drugs are closely related to natural products. Although small-molecule targeted drugs dominate cancer therapy. Therefore, searching for natural active compounds and discovering new anti-pancreatic cancer drugs will help promote the search for highly effective anti-pancreatic cancer drugs. Xinjiang Ferula ( Ferula sinkiangensis KM Shen) is a plant of the genus Ferula in the family Umbelliferae. It is a perennial herb that bears fruit once. It is 0.5-1.5 meters high. The whole plant has a strong onion and garlic-like odor. Root spindle-shaped or conical, stout, with withered leaf sheath fibers remaining on the root neck. The oil gum resin secreted by Ferulicum ferulae in Xinjiang has a special onion and garlic-like odor, which has been recorded in successive dynasties of herbal medicines and "Pharmacopia of the People's Republic of China". , Insect accumulation abdominal pain and other diseases.

Contents of the invention

The object of the present invention is to provide a kind of sesquiterpene ketone compound and separation method thereof;

Another object of the present invention is to study the anti-pancreatic cancer activity of the above-mentioned isolated sesquiterpene ketones, in order to prepare anti-pancreatic cancer drugs.

1. Separation of sesquiterpene ketones

The separation of sesquiterpene ketones of the present invention comprises the following steps:

(1) Extract the dried Xinjiang ferulae root with methanol at room temperature for 3 times, each time for 7 days, combine the extracts, evaporate and concentrate until there is no alcohol smell, and obtain the total extract;

(2) After dispersing the total extract with water, extract with petroleum ether (60~90℃), dichloromethane, ethyl acetate and n-butanol respectively to obtain petroleum ether phase, dichloromethane phase, ethyl acetate phase, n-butanol phase and water phase;

(3) The ethyl acetate phase was separated by silica gel chromatography and eluted with petroleum ether-ethyl acetate (40:1~1:1) to obtain 12 components FA-FL in sequence.

Silica gel column chromatography was performed on component FH, and eluted with dichloromethane-ethyl acetate (50:1), to obtain five components FH-1 to FH-5;

Fraction FH-1 was subjected to silica gel column chromatography and eluted with petroleum ether-ethyl acetate (10:1) to obtain 12 fractions FH-1-1 to FH-1-12.

Fraction FH-1-7 was eluted with dichloromethane-ethyl acetate (60:1) to obtain eight fractions FH-1-7-1 to FH-1-7-8;

Use dichloromethane-ethyl acetate (40:1) for component FH-1-7-5 to obtain four components FH-1-7-5-1 to FH-1-7-5-4;

(4) Purify fraction FH-1-7-5-1 by HPLC (C ₁₈ , MeOH/H ₂ O=76/24, flow rate=4 ml/min) to obtain compound 1;

(5) Compound 1 was purified by chiral HPLC (Chiral CD-Ph, petroleum ether: ethyl acetate = 84/16, flow rate = 3ml/min) to obtain compound (+)-1 and compound (- )-1 in sequence .

Compound ( ⁺ )-1 and ^compound (-)-1 is analyzed, and its 1H (400 MHz) and 13C (100MHz) NMR data are shown in Table 1, and its structural formula is determined as follows:

Compound (+ ) -1 and compound (- ) ^-1 are colorless oils, and the molecular formula is C ₂₄ H ₃₀ O ₅ saturation. Strong infrared absorption at 3424 ^-1 and 1723.9 cm ^-1 indicated the presence of hydroxyl and carbonyl groups. 1D NMR data indicated 5,6,8-trisubstituted phenyl ring [δH7.77(d, J=8.5Hz, H-5), 6.50(dd, J=8.5,2.16Hz, H-6), 6.37(d , J=2.04Hz, H-8) and δC129.5(C-5), 110.3(C-6), 162.4(C-7), 103.6(C-8), 114.39C-9), 160.69C- 10)], and two trisubstituted olefin bonds [5.11 (d, J=9.76Hz, H-2, H-6') and δ _C 126.1(C-2'), 129.3(C-6')]. Two H protons [δH3.14(d, J=10.5Hz, H-3), 3.14(d, J=10.52Hz, H-10') and δC55.0(C-3), 83.7(C-10 ')]. 1D NMR data also showed 24 carbon signals, including one carbonyl δC 191.34 (C-4), four methyl groups [δH 1.19 (s, H3-13'), 1.29 (s, H3-12'), 1.51 ( s, H3-14') and 1.53 (s, H3-15')].

The planar structures of compound (+)-1 and compound (- )-1 were further elucidated by comprehensive interpretation of the 2D NMR spectra. The analysis of ¹ H- ¹ H COZY spectrum shows that there are four related systems, namely CH(10')-CH(8'), CH(5)-CH(6), CH(9')-CH(1' ), CH(6')-CH(5'). The correlation of HMBC data at H-5/C-4/C-7/C-9 and H-6/C-8/C-10, and H-8/C-6/C-10 indicates that the A ring Exist (as shown in Figure 1), in H-3/C-/C-2'/C-2/C-1', H-2'/C-4'/C-15', H ₃ -15 '/C-3'/C-2'/C-4', H ₃ -14'/C-7'/C-8', H-6'/C-14'/C-8', H- 4'/C-6', H-10'/C-11'/C-8', H ₃ -13'/C-10'/C-11'/C-12' and H-12'/C The -13'/C-11' correlation indicated the presence of B, C, and D loops (as shown in Figure 1). The absolute configuration was determined by ECD calculations (Fig. 2-Fig. 7).

2. Anti-pancreatic cancer activity of sesquiterpene ketones

In this experiment, human pancreatic cancer cells SW1990 cells, CFPAC-1 cells, Capan-2 cells and PANC-1 cells were used, and SW1990 cells, CFPAC-1 cells, Capan-2 cells and PANC-1 cells in logarithmic growth phase were used to inoculate In a 96-well plate (1×10 ⁴ cells per well), after the cells adhere to the wall, add the compound to be tested in a gradient concentration range of 0.1-100 µM, incubate for 72 h, and then add 10 µL CCK-8 to each well The solution was incubated for 4 hours, shaken for 10 min, and the absorbance value was measured at 450 nm by a microplate reader. The MTT method was used to detect the effects of Xinjiang ferula sesquiterpene ketones (+)-1 and (-)-1 on SW1990 cells, CFPAC- 1 cell, Capan-2 cell and PANC-1 cell proliferation inhibitory effect (Figure 6), the cell survival rate was calculated according to the absorbance value, and the IC ₅₀ value of the test compound was calculated. Among them, paclitaxel (Taxol) was used as a positive control drug.

Table 2 shows the inhibitory effects of compounds (+)-1 and (-)-1 on pancreatic cancer SW1990 cells, CFPAC-1 cells, Capan-2 cells and PANC-1 cells. Among them, compound (+)-1 inhibited SW1990 cells, CFPAC-1 cells, Capan-2 cells and PANC-1 cells with IC ₅₀ values of 11.77±1.83 µM , 6.12±0.52 µM , 8.57±0.59 µM , 2.24 ±0.83 µM , compound (-)-1 inhibited SW1990 cells, CFPAC-1 cells, Capan-2 cells and PANC-1 cells with IC ₅₀ values of 15.67±1.53 µM , 19.13±2.99 µM , 14.57±0.65 µM M, 0.70±0.47 µM ; The results showed that compounds (+)-1 and (-)-1 had significant inhibitory effects on pancreatic cancer SW1990 cells, CFPAC-1 cells, Capan-2 cells and PANC-1 cells , indicating that compounds (+)-1 and (-)-1 can significantly inhibit the proliferation of pancreatic cancer cells, have potential anti-pancreatic cancer effects, and have the potential to develop therapeutic drugs for pancreatic cancer, and can be used as active ingredients for the preparation of anti-pancreatic cancer drugs . That is to use Xinjiang ferulicum sesquiterpene ketones as active components, and make internal preparations or injections with pharmaceutically or physiologically acceptable excipients and conventional pharmaceutical preparations. The internal preparations are powders, granules, capsules, soft capsules, powders, pills, tablets, and oral liquids.

Description of drawings

Figure 1 shows the ¹ H ^-1 H COZY (black bold) and HMBC (black arrow) of compound (+)-1 and compound (-)-1.

Figure 2 is the absolute configuration of compound (+)-1.

Figure 3 is the ECD spectrum of compound (-)-1.

Figure 4 is the CD spectrum of compound (-)-1.

Figure 5 shows the absolute configuration of compound (+)-1.

Figure 6 is the ECD spectrum of compound (+)-1.

Figure 7 is the CD spectrum of compound (+)-1.

Figure 8 shows the inhibition of the survival rate of pancreatic cancer cells by compounds (+)-1 and (-)-1.

Detailed ways

(1) Extract the dried Xinjiang ferulae root with methanol at room temperature for 3 times, each time for 7 days, combine the extracts, evaporate and concentrate until there is no alcohol smell, and obtain the total extract;

(2) Disperse and dissolve the total extracts in water and then extract with petroleum ether, dimethyl chloride, ethyl acetate and n-butanol to obtain petroleum ether phase, dichloromethane phase, ethyl acetate phase, n-butanol phase and water box;

(3) The ethyl acetate phase (260 g) was chromatographically separated on a silica gel column, using petroleum ether: ethyl acetate (the volume ratio of petroleum ether: ethyl acetate was 40:1, 30:1, 20:1, 10 :1, 8:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1) for elution to obtain 12 components FA-FL in sequence;

Component FH (61.3g) was subjected to silica gel column chromatography, eluted with dichloromethane:ethyl acetate (50:1), to obtain five components FH-1 to FH-5 in sequence;

Component FH-1 (14.05 g) was subjected to silica gel column chromatography, eluted with petroleum ether: ethyl acetate (10:1), and 12 components FH-1-1 to FH-1-12 were obtained in sequence;

Fraction FH-1-7 (393.6 mg) was eluted with dichloromethane:ethyl acetate (60:1) to obtain eight fractions FH-1-7-1 to FH-1-7-8;

Fraction FH-1-7-5 (67mg) was used dichloromethane:ethyl acetate (40:1) to obtain four fractions FH-1-7-5-1 to FH-1-7-5-4 ;

(4) Fraction FH-1-7-5-1 (24 mg) was purified by HPLC (C ₁₈ , MeOH/H ₂ O = 76/24, flow rate = 4 ml/min) to obtain compound 1 (4 mg , t _R = 65.052 min);

(5) Compound 1 was purified by chiral HPLC (Chiral CD-Ph, petroleum ether: ethyl acetate = 84/16, flow rate = 3ml/min), and compound (+)-1 (1 mg, t _R =26.180 min) and compound (-)-1 (0.9 mg, t _R =31.917 min).

Compound [(±)-1]: white oil; mp 218-221 °C; UV (CH ₃ CN) λmax (log ε) 204(3.131)nm, 274(2.356) nm; IR (KBr) ν _max = 3424 , 2926.9, 1723.9, 1612.2,1463.6, 1272.3, 1272.3, 1111.5, 1022.5, 1000.8, 972.0, 973.7 cm ⁻¹ ; ¹ H NMR and ¹³ CNMR data are shown in Table 1; spectrum m/z 399.2158 [M + H] ⁺ (C ₂₄ H ₃₀ O ₅ , 399.2158).

+52.381° (c 0.1, CH₃OH); ECD (CH₃OH) λmax (Δε):207(Δε -38.385), 257 (Δε -2.98), 268 (Δε -4.96), 301 (Δε 6.35), 328 (Δε-1.44) nm。(+)-1: white oil;

+52.381° (c 0.1, CH ₃ OH); ECD (CH ₃ OH) λ max (Δε):207(Δε -38.385), 257 (Δε -2.98), 268 (Δε -4.96), 301 (Δε 6.35) , 328 (Δε-1.44) nm.

-48.531° (c 0.1, CH₃OH); ECD (CH₃OH)λmax (Δε):208 (Δε 33.67), 249 (Δε 0.77), 271 (Δε 3.67), 300 (Δε -5.54), 327(Δε0.96) nm。(-)-1: white oil;

-48.531° (c 0.1, CH ₃ OH); ECD (CH ₃ OH) λ max (Δε):208 (Δε 33.67), 249 (Δε 0.77), 271 (Δε 3.67), 300 (Δε -5.54), 327 (Δε0.96) nm.

Claims (10)

1. a sesquiterpene chromone compound has the structural formula as follows:

。

2. the method for separating sesquiterpene chromone compounds according to claim 1, comprising the steps of:

(1) Extracting dried Ferula sinica root with methanol at room temperature for 3 times each for 7 days, mixing extractive solutions, and evaporating and concentrating until no alcohol smell exists to obtain total extractive solution;

(2) Dispersing the total extract with water, and extracting with petroleum ether, dichloromethane, ethyl acetate and n-butanol at 60-90 ℃ to obtain petroleum ether phase, dichloromethane phase, ethyl acetate phase, n-butanol phase and water phase;

(3) Separating ethyl acetate phase by silica gel chromatography, eluting with petroleum ether-ethyl acetate, and sequentially obtaining 12 components FA-FL;

subjecting component FH to silica gel column chromatography, eluting with dichloromethane-ethyl acetate to obtain five components FH-1 to FH-5;

subjecting component FH-1 to silica gel column chromatography, eluting with petroleum ether-ethyl acetate to obtain 12 components FH-1-1 to FH-1-12;

eluting the components FH-1-7 with dichloromethane-ethyl acetate to obtain eight components FH-1-7-1 to FH-1-7-8;

the components FH-1-7-5 were treated with methylene chloride-ethyl acetate to give four components FH-1-7-5-1 to FH-1-7-5-4;

(4) Purification of component FH-1-7-5-1 by HPLC gave Compound 1; HPLC purification conditions: c (C) ₁₈ ，MeOH/H ₂ O=76/24, flow rate=4 ml/min;

(5) Purifying the compound 1 by chiral HPLC to obtain a compound (+) -1 and a compound (-) -1 in sequence; chiral HPLC purification conditions: chiral CD-Ph, petroleum ether ethyl acetate=84/16, flow rate=3 ml/min.

3. The method for separating sesquiterpene chromone compounds according to claim 2, wherein: in the eluent petroleum ether-ethyl acetate of the ethyl acetate phase in the step (3), the volume ratio of petroleum ether to ethyl acetate is 40:1-1:1.

4. The method for separating sesquiterpene chromone compounds according to claim 2, wherein: in the eluent dichloromethane-ethyl acetate of the component FH in the step (3), the volume ratio of dichloromethane to ethyl acetate is 50:1.

5. The method for separating a hemiterpene chromone compound according to claim 2, wherein: in the eluent petroleum ether-ethyl acetate of the FH-1 component in the step (3), the volume ratio of petroleum ether to ethyl acetate is 10:1.

6. The method for separating sesquiterpene chromone compounds according to claim 2, wherein: in the eluent dichloromethane-ethyl acetate of the component FH-1-7 in the step (3), the volume ratio of dichloromethane to ethyl acetate is 60:1.

7. The method for separating sesquiterpene chromone compounds according to claim 2, wherein: in the eluent dichloromethane-ethyl acetate of the component FH-1-7-5 in the step (3), the volume ratio of dichloromethane to ethyl acetate is 40:1.

8. The use of a sesquiterpene chromone compound according to claim 1 for preparing an anti-pancreatic cancer medicament.

9. The use of the sesquiterpene chromones according to claim 8 for preparing an anti-pancreatic cancer drug, wherein: the sesquiterpene chromone compound is used as an active component, and is prepared into an oral preparation or an injection by using pharmaceutically or physiologically acceptable auxiliary materials and the preparation process of a conventional pharmaceutical preparation.

10. The use of the sesquiterpene chromones according to claim 9 for preparing an anti-pancreatic cancer drug, characterized in that: the oral preparation is powder, granule, capsule, soft capsule, powder, pill, tablet, and oral liquid.

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