Cyclohexenol compound as well as preparation method and application thereof

Abstract

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

Description

Technical Field

[0001] The present invention relates to the technical field of natural medicine chemistry, and particularly relates to cyclohexenol compounds, a preparation method thereof, and an application thereof. Background Art

[0002] Chuanxiong Rhizoma is the dried rhizome of the umbelliferous plant Ligusticum chuanxiong Hort., and is a famous traditional Chinese medicine. It is recorded in the Chinese Pharmacopoeia that Chuanxiong Rhizoma has the effects of promoting blood circulation and qi movement, dispelling wind and relieving pain. Modern pharmacological studies have shown that Chuanxiong Rhizoma can treat various diseases such as gout, osteoarthritis, rheumatoid arthritis, arteriosclerosis, etc. In addition, extracts and components of Chuanxiong Rhizoma also exhibit various activities, including anti-asthmatic, anti-inflammatory, antibacterial, antioxidant, neuroprotective, and pro-apoptotic effects.

[0003] Gout is a joint inflammation caused by an increase in the level of uric acid in the serum, which then leads to the deposition of sodium urate crystals in the joints, causing severe pain. Gout has imposed a significant burden on personal health and national healthcare. Recent studies have shown that gout can cause chronic kidney disease, hypertension, cardiovascular and cerebrovascular diseases, diabetes, and even death. Among them, reducing uric acid is an important strategy for treating gout. The uric acid precursors hypoxanthine and xanthine can be converted into uric acid under the action of xanthine oxidase (XO). Therefore, inhibiting the activity of XO can reduce the uric acid level and treat gout. Currently, allopurinol and febuxostat are the most commonly used first-line uric acid-lowering drugs in clinical practice, which reduce uric acid production by inhibiting XO. However, side effects such as allergic reactions, headache, fever, liver and kidney toxicity, and gastrointestinal discomfort limit their widespread application. Therefore, there is an urgent need to develop new and effective XO inhibitors with fewer side effects. Developing safe and effective XO inhibitors from natural products is an important strategy.

[0004] We previously clarified through animal experiments that the main active part of Chuanxiong Rhizoma for treating gout is the ethyl acetate part. However, the pharmacodynamic substances of the ethyl acetate part of Chuanxiong Rhizoma that play a role in treating gout are still unclear. The present invention for the first time isolates and identifies two new cyclohexenol compounds from the ethyl acetate extract of Chuanxiong Rhizoma, and these two compounds exhibit good inhibitory activity against xanthine oxidase in vitro. 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 using Compound 1 and Compound 2 and their compositions for treating gout.

[0005] However, the present invention details the preparation methods, structural identification, and pharmacological activities of Compound 1 and Compound 2, providing a new strategy for the treatment of gout diseases. Summary of the Invention

[0006] In view of this, the present invention provides cyclohexenol compounds, a preparation method thereof and an application thereof.

[0007] To solve the above technical problems, the present invention adopts the following technical solutions:

[0008] Cyclohexenol compounds, the compounds are new compound 1 and compound 2, and their structural formulas are:

[0009]

[0010] Preferably, the molecular formulas of the compound 1 and the compound 2 are C 11 H 18 O2 and C 11 H 20 O2.

[0011] Preferably, the compound 1 and the compound 2 are obtained by separation and purification from the ethyl acetate extract of Ligusticum chuanxiong.

[0012] A preparation method of cyclohexenol compounds, comprising the following steps:

[0013] S1. Making the Ligusticum chuanxiong medicinal materials into powder, soaking in ethyl acetate, filtering, and concentrating the filtrate under reduced pressure to obtain the ethyl acetate extract of Ligusticum chuanxiong;

[0014] S2. Subjecting the ethyl acetate extract of Ligusticum chuanxiong to silica gel column chromatography to obtain five fractions of Fr.1 to 5; subjecting the fraction Fr.2 to silica gel column chromatography to obtain four fractions of Fr.2-1 to Fr.2-4;

[0015] S3. Subjecting the fraction Fr.2-4 to gel column chromatography and using methanol as an eluent to obtain eight fractions of Fr.2-4-1 to Fr.2-4-8;

[0016] S4. Purifying the fraction Fr.2-4-6 by semi-preparative HPLC to obtain six fractions of Fr.2-4-6-1 to Fr.2-4-6-6;

[0017] Purifying the fraction Fr.2-4-6-1 by semi-preparative HPLC to obtain four fractions of Fr.2-4-6-1-1 to Fr.2-4-6-1-4;

[0018] S5. Purifying the fraction Fr.2-4-6-1-3 by semi-preparative HPLC to obtain compound 1;

[0019] Purifying the fraction Fr.2-4-6-1-2 by semi-preparative HPLC to obtain compound 2.

[0020] Preferably, in the step S1, the dosage of the Ligusticum chuanxiong 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.

[0021] Preferably, in the step S2, the addition amount of the ethyl acetate extract is 100 g; the addition amount of the component Fr.2 is 16.35 g; the silica gel column chromatography is eluted with a petroleum ether-ethyl acetate gradient.

[0022] Preferably, in the step S3, the addition amount of the component Fr.2-4 is 2.97 g; the stationary phase of the gel column chromatography is Sephadex LH-20.

[0023] Preferably, in the step S4, the conditions for purifying the component Fr.2-4-6 by semi-preparative HPLC to obtain six components 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;

[0024] The conditions for purifying the component Fr.2-4-6-1 by semi-preparative HPLC to obtain four components Fr.2-4-6-1-1 to Fr.2-4-6-1-4 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 50:50, and the flow rate is 3 mL / min.

[0025] Preferably, in the step S5, the conditions for purifying the component Fr.2-4-6-1-3 by semi-preparative HPLC to obtain compound 1 are as follows: the chromatographic column is a phenyl column, the mobile phase is acetonitrile-water, the volume ratio of acetonitrile to water is 45:55, and the flow rate is 3 mL / min;

[0026] The conditions for purifying the component Fr.2-4-6-1-2 by semi-preparative HPLC to obtain compound 2 are as follows: successively passing through semi-preparative HPLC [phenyl column, acetonitrile-water (V / V, 45:55)] and semi-preparative HPLC [phenyl column, methanol-water (V / V, 60:40)], and the flow rate is 3 mL / min for both.

[0027] Applications of cyclohexenol compounds, applications of the compound 1 and the compound 2 in the treatment of gout activity. Specifically, the compound 1 and the compound 2 have good inhibitory activity against xanthine oxidase in vitro and can be used as new potential candidate drugs for the treatment of gout.

[0028] The present invention has achieved the following technical effects compared with the prior art:

[0029] (1) The test results of the inhibitory activity of the compounds of the present invention against xanthine oxidase in vitro show that the two compounds show good inhibitory effects on XO. The IC of the compound 1 and the compound 2 in inhibiting XO 50They were 34.92±1.56 μM and 41.20±3.63 μM respectively; the results indicated that the two compounds could be used as novel potential candidate drugs for the treatment of gout;

[0030] (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, solubilizers, buffers, etc., can be added. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is the 1 1H-NMR spectrum of Compound 1 proposed by the present invention;

[0032] Figure 2 is the 13 13C-NMR spectrum of Compound 1 proposed by the present invention;

[0033] Figure 3 is the HSQC spectrum of Compound 1 proposed by the present invention;

[0034] Figure 4 is the HMBC spectrum of Compound 1 proposed by the present invention;

[0035] Figure 5 is the 1 1H– 1 1H COSY spectrum of Compound 1 proposed by the present invention;

[0036] Figure 6 is the HR-ESI-MS spectrum of Compound 1 proposed by the present invention.

[0037] Figure 7 is the 1 1H-NMR spectrum of Compound 2 proposed by the present invention;

[0038] Figure 8 is the 13 13C-NMR spectrum of Compound 2 proposed by the present invention;

[0039] Figure 9 is the HSQC spectrum of Compound 2 proposed by the present invention;

[0040] Figure 10 is the HMBC spectrum of Compound 2 proposed by the present invention;

[0041] Figure 11 is the 1 1H– 1 1H COSY spectrum of Compound 2 proposed by the present invention;

[0042] Figure 12 is the ROESY spectrum of Compound 2 proposed by the present invention;

[0043] Figure 13 It is the HR-ESI-MS spectrum of Compound 2 proposed by the present invention. Detailed implementation manners

[0044] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to 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.

[0045] The present invention discloses cyclohexenol compounds, which are new Compound 1 and Compound 2, and their structural formulas are:

[0046]

[0047] The molecular formulas of Compound 1 and Compound 2 are C 11 H 18 O2 and C 11 H 20 O2.

[0048] Compound 1 and Compound 2 are obtained by separation and purification from the ethyl acetate extract of Ligusticum chuanxiong Hort.

[0049] The present invention also discloses a preparation method of cyclohexenol compounds, including the following steps:

[0050] S1. Make 18 kg of Ligusticum chuanxiong Hort. medicinal materials into powder, soak 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.

[0051] 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;

[0052] Subject 16.35 g of fraction Fr.2 to silica gel column chromatography to obtain four fractions Fr.2-1 - Fr.2-4;

[0053] Among them, the silica gel column chromatography is eluted with a petroleum ether - ethyl acetate gradient;

[0054] 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;

[0055] Among them, the stationary phase of the gel column chromatography is Sephadex LH-20;

[0056] 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;

[0057] The fraction Fr.2-4-6-1 was purified by semi-preparative HPLC to obtain four fractions: Fr.2-4-6-1-1 to Fr.2-4-6-1-4;

[0058] 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;

[0059] The conditions for purifying the fraction Fr.2-4-6-1 by semi-preparative HPLC to obtain four fractions: Fr.2-4-6-1-1 to Fr.2-4-6-1-4 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 50:50, and the flow rate was 3 mL / min.

[0060] S5. The fraction Fr.2-4-6-1-3 was purified by semi-preparative HPLC to obtain compound 1; the fraction Fr.2-4-6-1-2 was purified by semi-preparative HPLC to obtain compound 2;

[0061] Among them, the conditions for purifying the fraction Fr.2-4-6-1-3 by semi-preparative HPLC to obtain compound 1 were as follows: the chromatographic column was a phenyl column, the mobile phase was acetonitrile-water, the volume ratio of acetonitrile to water was 45:55, and the flow rate was 3 mL / min;

[0062] The conditions for purifying the fraction Fr.2-4-6-1-2 by semi-preparative HPLC to obtain compound 2 were as follows: successively through semi-preparative HPLC [phenyl column, acetonitrile-water (V / V, 45:55)] and semi-preparative HPLC [phenyl column, methanol-water (V / V, 60:40)], and the flow rate was 3 mL / min for both.

[0063] The present invention also discloses the application of cyclohexenol compounds, the application of compound 1 and compound 2 in the treatment of gout activity. Specifically, compound 1 and compound 2 have good inhibitory activity against xanthine oxidase in vitro and can be used as new potential candidate drugs for the treatment of gout.

[0064] Example 1:

[0065] Preparation of compound 1 and compound 2:

[0066] S1. 18kg of Chuanxiong herbal powder was 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 an ethyl acetate extract of Chuanxiong;

[0067] S2. 100 g of the ethyl acetate extract was purified by silica gel column chromatography (petroleum ether-ethyl acetate gradient elution) to obtain five fractions Fr.1 to 5;

[0068] 16.35 g of component Fr.2 was purified by silica gel column chromatography (petroleum ether-ethyl acetate gradient elution) to obtain four components Fr.2-1 to Fr.2-4;

[0069] S3. 2.97 g of fraction Fr.2-4 was subjected to gel column chromatography (Sephadex LH-20); eight fractions Fr.2-4-1 to Fr.2-4-8 were obtained using methanol as the eluent;

[0070] S4. Component Fr.2-4-6 was purified by semi-preparative HPLC [C18 column, acetonitrile-water (V / V, 70:30), 3 mL / min] to obtain six components, Fr.2-4-6-1 to Fr.2-4-6-6;

[0071] Furthermore, the component Fr.2-4-6-1 was purified by semi-preparative HPLC [C18 column, acetonitrile-water (V / V, 50:50), 3 mL / min] to obtain four components Fr.2-4-6-1-1 to Fr.2-4-6-1-4;

[0072] S5. The component Fr.2-4-6-1-3 was purified by semi-preparative HPLC [phenyl column, acetonitrile-water (V / V, 45:55)] to obtain compound 1;

[0073] The component Fr.2-4-6-1-2 was purified by semi-preparative HPLC [phenyl column, acetonitrile-water (V / V, 45:55)] and semi-preparative HPLC [phenyl column, methanol-water (V / V, 60:40)] to obtain compound 2.

[0074] Example 2:

[0075] Structural identification of compound 1 and compound 2:

[0076] The high-resolution mass spectrometry was determined by an UPLC-Q / TOF (WATERS I-Class VION IMS Q-Tof, USA) mass spectrometer. The nuclear magnetic resonance spectrum was determined by a Bruker Avance III-600 nuclear magnetic resonance spectrometer (Bruker, Germany), with TMS (tetramethylsilane) as the internal standard. The specific rotation was determined by a Jascomodel 1020 polarimeter (Horiba, Tokyo, Japan). The electronic circular dichroism spectrum (ECD spectrum) was determined by a J-815 circular dichroism spectrometer (Japan). The ultraviolet spectrum was determined by a UV-2600 spectrophotometer (Shimadzu, Kyoto, Japan). The infrared spectrum was determined by a Bruker VERTEX 70 infrared spectrometer (Germany).

[0077] Compound 1, colorless oil, with the molecular formula C 11 H 18 O2, HR-ESIMS m / z 181.1204 [M-H] - (calculated value is 181.1234); -450.4 (c 0.05, MeOH); UV (MeOH) λ max (logε) 217 nm (3.93); IR v max 3400, 2940, 2860, 1673 cm -1 ; The nuclear magnetic resonance data are shown in Table 1.

[0078] Compound 2, colorless oil, with the molecular formula C 11 H 20 O2, HR-ESIMS m / z 183.1379 [M-H] - (calculated value is 183.1391); -381.2 (c 0.06, MeOH); UV (MeOH) λ max (logε) 202 nm (3.35); IR v max 3372, 2960, 2870, 1618 cm -1 ; The nuclear magnetic resonance data are shown in Table 1.

[0079]

[0080] Table 1. 1 1H-NMR (600 MHz) and 13 13C-NMR (150 MHz) data of Compound 1 and Compound 2

[0081]

[0082] a Overlapped

[0083] Example 3:

[0084] In vitro inhibitory activity test of Compound 1 and Compound 2 against XO:

[0085] First, add 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) in sequence. After pre-incubating at 37 °C for 3 minutes, add the substrate solution (50 μL, 480 μM xanthine, dissolved in phosphate buffer) to initiate the reaction, and incubate at 37 °C for 5 minutes; use a microplate reader to measure the absorbance at 295 nm every 30 seconds; allopurinol is used as a positive control; set 3 replicates for each group, and calculate the inhibition rate using the following formula:

[0086]

[0087] ΔA E 、ΔA S and ΔA B respectively represent the differences in absorbance between the enzyme group, the sample group, and the blank group from t = 0 minute to t = 5 minutes.

[0088] The results of the activity test show that the IC 50 values of Compound 1 and Compound 2 for inhibiting XO are 34.92 ± 1.56 μM and 41.20 ± 3.63 μM respectively (the IC 50 of allopurinol is 10.71 ± 0.16 μM). The results indicate that Compound 1 and Compound 2 can be used as new potential candidate drugs with anti-gout effects.

[0089] 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 cyclohexenol compound, characterized in that, The compounds are novel compound 1 and compound 2, and their structural formulas are as follows:

2. The cyclohexenol compound according to claim 1, wherein The molecular formulas of Compound 1 and Compound 2 are C 11 H 18 O2 and C 11 H 20 O2, respectively.

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

4. A method for preparing cyclohexenol compounds, characterized in that, It includes the following steps: S1. The Ligusticum chuanxiong Hort. medicinal materials are made into powder, soaked in ethyl acetate, filtered, and the filtrate is concentrated 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 - Fr.2-4. S3. Fraction Fr.2-4 is subjected to gel column chromatography, and methanol is used as the eluent to obtain eight fractions Fr.2-4-1 - Fr.2-4-8. S4. Fraction Fr.2-4-6 is purified by semi-preparative HPLC to obtain six fractions Fr.2-4-6-1 - Fr.2-4-6-6. Fraction Fr.2-4-6-1 is purified by semi-preparative HPLC to obtain four fractions Fr.2-4-6-1-1 - Fr.2-4-6-1-4. S5. Fraction Fr.2-4-6-1-3 is purified by semi-preparative HPLC to obtain compound 1. Fraction Fr.2-4-6-1-2 is purified by semi-preparative HPLC to obtain compound 2.

5. The preparation method of the cyclohexenol 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 cyclohexenol 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; petroleum ether - ethyl acetate gradient elution is used for silica gel column chromatography.

7. The preparation method of the cyclohexenol 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 for gel column chromatography is Sephadex LH-20.

8. The method for preparing a cyclohexenol compound according to claim 4, wherein 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 - 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. The conditions for purifying fraction Fr.2-4-6-1 by semi-preparative HPLC to obtain four fractions Fr.2-4-6-1-1 - Fr.2-4-6-1-4 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 50:50, and the flow rate is 3 mL / min.

9. The preparation method of the two cyclohexenol compounds according to claim 4, characterized in that, In the above step S5, the conditions for purifying fraction Fr.2-4-6-1-3 by semi-preparative HPLC to obtain compound 1 are as follows: the chromatographic column is a phenyl column, the mobile phase is acetonitrile - water, the volume ratio of acetonitrile to water is 45:55, and the flow rate is 3 mL / min. The conditions for purifying fraction Fr.2-4-6-1-2 by semi-preparative HPLC to obtain compound 2 are as follows: successively through semi-preparative HPLC [phenyl column, acetonitrile - water (V / V, 45:55)] and semi-preparative HPLC [phenyl column, methanol - water (V / V, 60:40)], and the flow rate is 3 mL / min for both.

10. Use of cyclohexenol compounds, characterized in that, Applications of the said Compound 1 and Compound 2 in the treatment of gout activity. Specifically, the said Compound 1 and Compound 2 have good inhibitory activity against xanthine oxidase in vitro and can be used as new potential candidate drugs for the treatment of gout.

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