Phenalide compound and preparation method and application thereof

Abstract

The invention discloses phthalide compounds as well as a preparation method and application thereof. The phthalide compounds comprise a compound 1 and a compound 2 as well as a preparation method and application thereof. The two compounds are separated and purified from the ethyl acetate extract of the traditional Chinese medicine ligusticum wallichii for the first time. An in-vitro biological activity test shows that the two compounds have obvious inhibitory activity on xanthine oxidase, which indicates that the two compounds can be used as novel potential candidate drugs for treating hyperuricemia and gout. The embodiment of the invention provides a preparation method, structural identification and pharmacological action of the two phthalide compounds, and provides a new strategy for treatment of hyperuricemia and gout diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of natural medicinal chemistry, in particular to phthalide compounds and preparation methods and applications thereof. Background Art

[0002] Hyperuricemia (HUA) is a purine metabolism disorder, collectively known as the "four highs" syndrome along with hypertension, hyperglycemia, and hyperlipidemia. Its pathogenesis is influenced by both genetic and environmental factors. Studies have shown that HUA increases the risk of various diseases, including gouty arthritis, renal impairment, and cardiovascular disease, and significantly impacts patients' employment, economic status, and mental health. Data indicate that approximately 41 million people worldwide suffer from gout, and their onset is directly related to elevated serum uric acid levels. Notably, elevated serum uric acid levels are becoming increasingly prominent in people aged 20-30 years, becoming a pressing public health issue. Xanthine oxidase, a key enzyme that catalyzes the conversion of xanthine and hypoxanthine to uric acid, has become a target for drug development in the treatment of HUA and gout. Currently, several anti-hyperuricemic drugs, such as allopurinol and febuxostat, reduce uric acid production by inhibiting xanthine oxidase (XO) activity. However, these drugs can cause adverse reactions, including allergic reactions and liver and kidney damage, to varying degrees. Therefore, the development of safe and effective xanthine oxidase inhibitors is urgently needed.

[0003] Traditional Chinese medicine (TCM) is favored for its ability to alleviate symptoms, regulate metabolic imbalances, and minimize adverse reactions. TCM classifies gout as a type of "bi syndrome." Treatment utilizes herbs that dispel wind, relieve pain, and promote blood circulation, taking into account the progression of the disease. Chuanxiong, the rhizome of the Apiaceae plant Ligusticum chuanxiong Hort., is listed in the 2020 edition of the Chinese Pharmacopoeia as possessing the effects of promoting qi and blood circulation, dispelling wind, and relieving pain. Therefore, TCM often incorporates Chuanxiong into gout treatment formulas, such as Sangdang Decoction and Tongbi Decoction.

[0004] Our previous studies have shown that oral administration of an ethyl acetate extract of Chuanxiong rhizome can reduce serum uric acid levels and inhibit renal pathological changes in hyperuricemic mice. This suggests that the ethyl acetate extract of Chuanxiong rhizome has the potential to lower uric acid levels. However, the exact components of the ethyl acetate extract of Chuanxiong rhizome that exert this uric acid-lowering effect remain unknown. The chemical components isolated and purified from Chuanxiong rhizome primarily include volatile oils, organic acids, and nitrogen-containing compounds. Phthalate compounds have been shown to be promising bioactive natural products. Therefore, identifying and developing safe and effective phthalide compounds from the traditional Chinese medicine Chuanxiong rhizome for the treatment of hyperuricemia and gout holds great promise.

[0005] During the systematic and in-depth study of the chemical constituents and pharmacological effects of the ethyl acetate extract of Ligusticum chuanxiong Hort., two new phthalide compounds were isolated and identified for the first time, and these two compounds showed significant inhibitory activity against xanthine oxidase. So far, there are no reports on Compound 1 and Compound 2, no reports on pharmaceutical compositions with Compound 1 and Compound 2 as active ingredients, and no reports on the use of Compound 1 and Compound 2 and their compositions for the treatment of hyperuricemia and gout.

[0006] However, the present invention details the preparation methods, structural identification and pharmacological effects of Compound 1 and Compound 2, providing a new strategy for the treatment of hyperuricemia and gout diseases. Summary of the Invention

[0007] In view of this, the present invention provides phthalide compounds, their preparation methods and applications.

[0008] To solve the above technical problems, the present invention adopts the following technical solutions:

[0009] Phthalide compounds, the compounds are new Compound 1 and Compound 2, and their structural formulas are:

[0010]

[0011] Preferably, the molecular formulas of Compound 1 and Compound 2 are C 16 H 22 O5.

[0012] Preferably, Compound 1 and Compound 2 are obtained by separation and purification from the ethyl acetate extract of Ligusticum chuanxiong Hort.

[0013] Preparation method of phthalide compounds, comprising the following steps:

[0014] S1. Make the Ligusticum chuanxiong Hort. medicinal materials into powder, soak in ethyl acetate, filter, and concentrate the filtrate under reduced pressure to obtain the ethyl acetate extract of Ligusticum chuanxiong Hort.;

[0015] S2. The ethyl acetate extract of Ligusticum chuanxiong Hort. is subjected to silica gel column chromatography to obtain five fractions, Fr.1 - 5; fraction Fr.2 is subjected to silica gel column chromatography to obtain four fractions, Fr.2-1 - Fr.2-4;

[0016] S3. Subject fraction Fr.2-4 to gel column chromatography, and use methanol as the eluent to obtain eight fractions, Fr.2-4-1 - Fr.2-4-8;

[0017] S4. Subject fraction Fr.2-4-6 to semi-preparative HPLC purification to obtain six fractions, Fr.2-4-6-1 - Fr.2-4-6-6; subject fraction Fr.2-4-6-3 to semi-preparative HPLC purification to obtain Compound 1 and Compound 2.

[0018] Preferably, in step S1, the dosage of Ligusticum chuanxiong Hort. is 18 kg, the soaking time in ethyl acetate is 2 days, and the ratio of the mass of the medicinal material to the volume of the solvent is 1:4.

[0019] Preferably, in step S2, the addition amount of the ethyl acetate extract is 100 g; the addition amount of fraction Fr.2 is 16.35 g; silica gel column chromatography is eluted with a petroleum ether - ethyl acetate gradient.

[0020] Preferably, in step S3, the addition amount of fraction Fr.2 - 4 is 2.97 g; the stationary phase of the gel column chromatography is Sephadex LH - 20.

[0021] Preferably, in step S4, the conditions for purifying fraction Fr.2 - 4 - 6 by semi - preparative HPLC to obtain six components Fr.2 - 4 - 6 - 1 to Fr.2 - 4 - 6 - 6 are: the chromatographic column is a C18 chromatographic column, the mobile phase is acetonitrile - water, the volume ratio of acetonitrile to water is 70:30, and the flow rate is 3 mL / min.

[0022] Preferably, in step S4, the conditions for purifying fraction Fr.2 - 4 - 6 - 3 by semi - preparative HPLC to obtain compound 1 and compound 2 are: the chromatographic column is a phenyl column, the mobile phase is methanol - water, the volume ratio of methanol to water is 70:30, and the flow rate is 3 mL / min.

[0023] Application of phthalide compounds, application of the said compound 1 and compound 2 in treating hyperuricemia and gout activity. Specifically, the said compound 1 and compound 2 have obvious inhibitory activity on xanthine oxidase in vitro and can be used as new potential candidate drugs for treating hyperuricemia and gout.

[0024] The present invention has achieved the following technical effects compared with the prior art:

[0025] (1) The test results of the inhibitory activity of the compounds of the present invention on xanthine oxidase in vitro show that the two compounds show obvious inhibitory effects on XO. The IC 50 values of compound 1 and compound 2 for inhibiting XO are 18.00 ± 1.11 μM and 29.89 ± 2.04 μM respectively; the results show that the two compounds can be used as new potential candidate drugs for treating hyperuricemia and gout;

[0026] (2) Compound 1 and compound 2 of the present invention can be made into any pharmaceutically acceptable dosage form, such as tablets, dripping pills, ointments, sprays, capsules, etc., and excipients commonly used in the pharmaceutical industry, such as flavoring agents, disintegrants, solubilizing agents, buffering agents, etc., can be added. Description of the Drawings

[0027] Figure 1 is the 1 1 H-NMR spectrum of Compound 1 proposed by the present invention;

[0028] Figure 2 is the 13 13 C-NMR spectrum of Compound 1 proposed by the present invention;

[0029] Figure 3 is the HSQC spectrum of Compound 1 proposed by the present invention;

[0030] Figure 4 is the HMBC spectrum of Compound 1 proposed by the present invention;

[0031] Figure 5 is the 1 1 H– 1 H COSY spectrum of Compound 1 proposed by the present invention;

[0032] Figure 6 is the ROESY spectrum of Compound 1 proposed by the present invention;

[0033] Figure 7 is the HR-ESI-MS spectrum of Compound 1 proposed by the present invention.

[0034] Figure 8 is the 1 1 H-NMR spectrum of Compound 2 proposed by the present invention;

[0035] Figure 9 is the 13 13 C-NMR spectrum of Compound 2 proposed by the present invention;

[0036] Figure 10 is the HSQC spectrum of Compound 2 proposed by the present invention;

[0037] Figure 11 is the HMBC spectrum of Compound 2 proposed by the present invention;

[0038] Figure 12 is the 1 1 H– 1 H COSY spectrum of Compound 2 proposed by the present invention;

[0039] Figure 13 is the ROESY spectrum of Compound 2 proposed by the present invention;

[0040] Figure 14 is the HR-ESI-MS spectrum of Compound 2 proposed by the present invention. Detailed implementation manners

[0041] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0042] The present invention discloses phthalide compounds, which are new compound 1 and compound 2, and their structural formulas are as follows:

[0043]

[0044] The molecular formulas of compound 1 and compound 2 are C 16 H 22 O5.

[0045] Compound 1 and compound 2 are obtained by separation and purification from the ethyl acetate extract of Ligusticum chuanxiong Hort.

[0046] The present invention also discloses a preparation method of phthalide compounds, including the following steps:

[0047] S1. Make 18 kg of Ligusticum chuanxiong Hort. medicinal materials into powder, soak them in ethyl acetate for 2 days, the ratio of the mass of the medicinal materials to the volume of the solvent is 1:4, filter, and concentrate the filtrate under reduced pressure to obtain the ethyl acetate extract of Ligusticum chuanxiong Hort.

[0048] S2. Subject 100 g of the ethyl acetate extract of Ligusticum chuanxiong Hort. to silica gel column chromatography to obtain five fractions Fr.1 - 5; subject 16.35 g of fraction Fr.2 to silica gel column chromatography to obtain four fractions Fr.2-1 - Fr.2-4; among them, silica gel column chromatography is eluted with a petroleum ether - ethyl acetate gradient.

[0049] S3. Subject 2.97 g of fraction Fr.2-4 to gel column chromatography, and use methanol as the eluent to obtain eight fractions Fr.2-4-1 - Fr.2-4-8; among them, the stationary phase of gel column chromatography is Sephadex LH-20.

[0050] S4. The fraction Fr.2-4-6 was purified by semi-preparative HPLC to obtain six fractions: Fr.2-4-6-1 to Fr.2-4-6-6; the fraction Fr.2-4-6-3 was purified by semi-preparative HPLC to obtain Compound 1 and Compound 2. Among them, the conditions for purifying the fraction Fr.2-4-6 by semi-preparative HPLC to obtain six fractions Fr.2-4-6-1 to Fr.2-4-6-6 were as follows: the chromatographic column was a C18 chromatographic column, the mobile phase was acetonitrile-water, the volume ratio of acetonitrile to water was 70:30, and the flow rate was 3 mL / min; the conditions for purifying the fraction Fr.2-4-6-3 by semi-preparative HPLC to obtain Compound 1 and Compound 2 were as follows: the chromatographic column was a phenyl column, the mobile phase was methanol-water, the volume ratio of methanol to water was 70:30, and the flow rate was 3 mL / min.

[0051] The present invention also discloses the application of phthalide compounds, the application of Compound 1 and Compound 2 in the treatment of hyperuricemia and gout activities. Specifically, Compound 1 and Compound 2 have obvious inhibitory activities on xanthine oxidase in vitro and can be used as new potential candidate drugs for the treatment of hyperuricemia and gout.

[0052] Example 1:

[0053] Preparation of Compound 1 and Compound 2:

[0054] S1. 18 kg of Ligusticum chuanxiong herbs were made into powder, soaked in ethyl acetate at a material-liquid ratio of 1:4 (W / V) for 2 days, and the filtrate was concentrated under reduced pressure to obtain the ethyl acetate extract of Ligusticum chuanxiong.

[0055] S2. 100 g of the ethyl acetate extract was subjected to silica gel column chromatography (gradient elution with petroleum ether-ethyl acetate) to obtain five fractions: Fr.1 to Fr.5; 16.35 g of the fraction Fr.2 was subjected to silica gel column chromatography (gradient elution with petroleum ether-ethyl acetate) to obtain four fractions: Fr.2-1 to Fr.2-4.

[0056] S3. 2.97 g of the fraction Fr.2-4 was subjected to gel column chromatography (Sephadex LH-20), and methanol was used as the eluent to obtain eight fractions: Fr.2-4-1 to Fr.2-4-8.

[0057] S4. The fraction Fr.2-4-6 was purified by semi-preparative HPLC [C18 chromatographic column, acetonitrile-water (V / V, 70:30), 3 mL / min] to obtain six fractions: Fr.2-4-6-1 to Fr.2-4-6-6; further, the fraction Fr.2-4-6-3 was purified by semi-preparative HPLC [phenyl column, methanol-water (V / V, 70:30), 3 mL / min] to obtain Compound 1 and Compound 2.

[0058] Example 2:

[0059] Structural Identification of Compound 1 and Compound 2

[0060] High-resolution mass spectrometry was determined using a UPLC-Q / TOF (WATERS I-Class VION IMS Q-Tof, USA) mass spectrometer. Nuclear magnetic resonance spectra were determined using a Bruker Avance III-600 nuclear magnetic resonance spectrometer (Bruker, Germany), with TMS (tetramethylsilane) as the internal standard. The specific rotation was determined using a Jasco model 1020 polarimeter (Horiba, Tokyo, Japan). Electronic circular dichroism spectra (ECD spectra) were determined using a J-815 circular dichroism spectrometer (Japan). Ultraviolet spectra were determined using a UV-2600 spectrophotometer (Shimadzu, Kyoto, Japan). Infrared spectra were determined using a Bruker VERTEX 70 infrared spectrometer (Germany).

[0061] Compound 1, a colorless oil, has the molecular formula C 16 H 22 O5, and HR-ESIMS (+) m / z is 317.1355 [M+Na] + (calculated value is 317.1359); -205.8 (c 0.06, MeOH); UV (MeOH) λ max (logε) 275 nm (4.33); IR v max 3453, 2958, 2876, 1766, 1682, 1642, 1260, 1176, 1049 cm -1 ; The NMR data are shown in Table 1.

[0062] Compound 2, a yellow oil, has the molecular formula C 16 H 22 O5, and HR-ESIMS (+) m / z is 317.1356 [M+Na] + (calculated value is 317.1359); -144.0 (c 0.05, MeOH); UV (MeOH) λ max (logε) 274 nm (4.33); IR v max 3500, 2970, 2880, 1765, 1680, 1640, 1250, 1180, 1040, 802 cm -1 ; The NMR data are shown in Table 1.

[0063]

[0064] Table 1. For Compound 1 and Compound 2 1 H-NMR (600 MHz) and 13 C-NMR (150 MHz) data

[0065]

[0066]

[0067] a Overlapped

[0068] Example 3:

[0069] In vitro inhibitory activity test of Compound 1 and Compound 2 against XO:

[0070] First, phosphate buffer (90 μL, pH 7.5), Compound 1 and Compound 2 (10 μL, dissolved in DMSO), and XO enzyme solution (50 μL, 0.25 U / mL, dissolved in phosphate buffer) were added in sequence. After pre-incubating at 37 °C for 3 minutes, the reaction was initiated by adding the substrate solution (50 μL, 480 μM xanthine, dissolved in phosphate buffer), and incubated at 37 °C for 5 minutes; the absorbance at 295 nm was measured every 30 seconds using a microplate reader; allopurinol was used as a positive control; 3 replicates were set for each group, and the inhibition rate was calculated using the following formula:

[0071]

[0072] ΔA E 、ΔA S and ΔA B represent the differences in absorbance of the enzyme group, sample group, and blank group from t = 0 minute to t = 5 minutes, respectively.

[0073] The results of the activity test showed that the IC 50 values of Compound 1 and Compound 2 for inhibiting XO were 18.00 ± 1.11 μM and 29.89 ± 2.04 μM, respectively (the IC 50 value of allopurinol was 10.71 ± 0.16 μM), and the results indicated that Compound 1 and Compound 2 could be used as new potential candidate drugs for the treatment of hyperuricemia and gout.

[0074] As described above, it is only a preferred embodiment of the present invention, and does not impose any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A phthalide compound, characterized in that, The compounds are new compound 1 and compound 2, and their structural formulas are as follows:

2. The phthalide compound according to claim 1, wherein The molecular formulas of the said Compound 1 and Compound 2 are both C 16 H 22 O5.

3. The phthalide compound according to claim 1, wherein Compound 1 and compound 2 are obtained by separation and purification from the ethyl acetate extract of Ligusticum chuanxiong Hort.

4. Preparation method of phthalide compounds, characterized in that, It includes the following steps: S1. Make the Ligusticum chuanxiong Hort. medicinal materials into powder, soak them in ethyl acetate, filter, and concentrate the filtrate under reduced pressure to obtain the ethyl acetate extract of Ligusticum chuanxiong Hort. S2. The ethyl acetate extract of Ligusticum chuanxiong Hort. is subjected to silica gel column chromatography to obtain five fractions, Fr.1 - 5; fraction Fr.2 is subjected to silica gel column chromatography to obtain four fractions, Fr.2 - 1 to Fr.2 - 4. S3. Fraction Fr.2 - 4 is subjected to gel column chromatography, and eight fractions, Fr.2 - 4 - 1 to Fr.2 - 4 - 8, are obtained using methanol as the eluent. S4. Fraction Fr.2 - 4 - 6 is purified by semi - preparative HPLC to obtain six fractions, Fr.2 - 4 - 6 - 1 to Fr.2 - 4 - 6 - 6; fraction Fr.2 - 4 - 6 - 3 is purified by semi - preparative HPLC to obtain compound 1 and compound 2.

5. The preparation method of the phthalide compound according to claim 4, characterized in that, In the above step S1, the dosage of Ligusticum chuanxiong Hort. medicinal materials is 18 kg, the soaking time in ethyl acetate is 2 days, and the ratio of the mass of the medicinal materials to the volume of the solvent is 1:

4.

6. The preparation method of the phthalide compound according to claim 4, wherein, In the above step S2, the addition amount of the ethyl acetate extract is 100 g; the addition amount of fraction Fr.2 is 16.35 g; the silica gel column chromatography is eluted with a petroleum ether - ethyl acetate gradient.

7. The method for preparing the phthalide compound according to claim 4, wherein, In the above step S3, the addition amount of fraction Fr.2 - 4 is 2.97 g; the stationary phase of the gel column chromatography is Sephadex LH - 20.

8. The preparation method of the phthalide compound according to claim 4, characterized in that, In the above step S4, the conditions for purifying fraction Fr.2 - 4 - 6 by semi - preparative HPLC to obtain six fractions, Fr.2 - 4 - 6 - 1 to Fr.2 - 4 - 6 - 6, are as follows: the chromatographic column is a C18 chromatographic column, the mobile phase is acetonitrile - water, the volume ratio of acetonitrile to water is 70:30, and the flow rate is 3 mL / min.

9. The preparation method of the phthalide compound according to claim 4, wherein, In the above step S4, the conditions for purifying fraction Fr.2 - 4 - 6 - 3 by semi - preparative HPLC to obtain compound 1 and compound 2 are as follows: the chromatographic column is a phenyl column, the mobile phase is methanol - water, the volume ratio of methanol to water is 70:30, and the flow rate is 3 mL / min.

10. Use of phthalide compounds, characterized in that, The application of compound 1 and compound 2 in the treatment of hyperuricemia and gout activities. Specifically, compound 1 and compound 2 have obvious inhibitory activities on xanthine oxidase in vitro and can be used as new potential candidate drugs for the treatment of hyperuricemia and gout.

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