阅读说明：本技术 一类c-4位取代香豆素类化合物及其制备方法和应用 (C-4 substituted coumarin compounds and preparation method and application thereof ) 是由 李鲜 周峰旭 李飞 陈晨 曹婷婷 李旭 李勇 谢惠定 于 2020-11-23 设计创作，主要内容包括：本发明涉及从薄叶红厚壳植物的干燥茎叶中分离得到一类新的C-4位取代香豆素类化合物及其制备方法和应用。以藤黄科(Guttiferae)红厚壳属(Calophyllum)植物薄叶红厚壳(Calophyllum membranaceum Garda.et Champ.)的干燥茎叶为原料,经提取、硅胶柱色谱、反相ODS柱色谱、Sephadex LH-20、HPLC半制备等多种分离方法获得到五种新的C-4位取代,C-3、C-4位间双键被还原的香豆素类化合物Ⅰ：化合物Ⅱ：化合物Ⅲ：化合物Ⅳ和化合物Ⅴ本发明所述化合物具有靶向抑制CYP1B1酶活性,可用于改善或者预防代谢性疾病如肿瘤、肿瘤耐药、肥胖、高血压和动脉粥样硬化,具有潜在的临床应用价值。(The invention relates to a novel C-4 substituted coumarin compound separated from dried stems and leaves of Calophyllum gracile and a preparation method and application thereof. Taking dried stem and leaf of Calophyllum Membranaceae (Guttiferae) Calophyllum plant Calophyllum Membranaceae (Calophyllum Garda. et Champ.) as raw material, extracting, performing silica gel column chromatography, and performing reversed phase ODS column chromatographyFive new coumarin compounds I which are substituted at C-4 position and have reduced C-3 and C-4 position double bonds are obtained by various separation methods such as chromatography, Sephadex LH-20, HPLC semi-preparation and the like: compound ii: compound iii: compound IV And compound V)

1. The C-4 substituted coumarin compound has the following structure:

2. the method for preparing C4-substituted coumarin compounds according to claim 1, which comprises the following steps:

(1) taking dried stem and leaf of Calophyllum inophyllum, pulverizing, extracting with water and/or alcohol, and concentrating;

(2) dissolving the concentrate obtained in the step (1) with water, filtering, mixing with polyamide, eluting by an MCI column, sequentially carrying out gradient elution by using 0%, 10%, 30%, 50%, 70%, 95% methanol-water mixed solvent and acetone, wherein each gradient elution is 2-5 column volumes, and concentrating to obtain Fr A-F components;

(3) mixing the Fr E obtained in the step (2) with silica gel, mixing the mixture with petroleum ether: performing gradient elution on ethyl acetate, V, 100:1,50:1,30:1,15:1,8:1,4:1,2:1,1:1 and 0:1, wherein each gradient elution is 2-5 column volumes, and performing TLC (thin layer chromatography) detection to combine the same components to obtain 27 component segments of Fr 1-27;

(4) combining the Fr6 and Fr 7 obtained in the step (3), separating by gel Sephadex LH-20 column chromatography with methanol as an eluent, eluting for 1-3 column volumes, monitoring by TLC after the sample is completely eluted, and combining the same components to obtain Fr 6-1-6-15 groups of segments;

(5) and (3) carrying out HPLC chromatography on the Fr 6-4 component obtained in the step (4) by using a methanol-water mixed solvent with the concentration of 85-100% as an eluent to obtain a compound I and a compound II, wherein the flow rate v is 2mL/min, the detection wavelength lambda is 210nm, and the peak-off time t is_Ⅰ＝17min，t_Ⅱ＝22min；

(6) Separating the Fr 8 component obtained in the step (3) by gel Sephadex LH-20 column chromatography with acetone as an eluent, eluting for 1-3 column volumes, monitoring by TLC after the sample is completely eluted, and combining the same components to obtain Fr 8-1-8;

(7) the Fr 8-4 component obtained in the step (6) adopts 80-90% of methanol-water mixed solventPerforming HPLC chromatographic isocratic elution to obtain compound III and compound IV at flow rate v of 2mL/min and detection wavelength λ of 210nm and peak-off time t_Ⅲ＝14min，t_Ⅳ＝25min；

(8) And (3) combining the Fr10 obtained in the step (3) with Fr 11 and Fr 12, and then performing forward silica gel column chromatography, wherein the weight ratio of petroleum ether: a dichloromethane mixed solvent, V, 5:1,4:1,3:1,2:1,1:1, eluting 5-10 column volumes per gradient, performing synchronous TLC detection, and combining the same components to obtain Fr 10-1-10-17 components;

(9) subjecting the Fr 10-9 fraction obtained in step (8) to gel Sephadex LH-20 column chromatography with dichloromethane: separating by using an eluent with v: v ═ 3:1 and using the mixed solvent of methanol as an eluent, eluting by 1-3 column volumes at equal intervals, monitoring by adopting TLC after a sample is completely eluted, and combining the same components to obtain Fr 10-9-1-Fr 10-9-20;

(10) performing HPLC on the Fr 10-9-8 component obtained in the step (9) by using 80-90% of methanol-water mixed solvent to obtain a compound V, wherein the flow rate v is 2mL/min, the detection wavelength lambda is 210nm, and the peak-off time t is_Ⅴ＝16min。

3. The method for preparing C4-substituted coumarins according to claim 2, wherein:

leaching by using 95% ethanol water solution, and concentrating;

step (2) eluting 3 column volumes per gradient;

step (3) eluting 3 column volumes per gradient;

eluting 1 column volume;

eluting by using a 90% methanol-water mixed solvent;

eluting 1 column volume;

eluting by using a methanol-water mixed solvent with the concentration of 85 percent;

step (8) elute 5 column volumes per gradient;

step (9), isocratically eluting for 1 column volume;

step (10) was eluted with a mixed solvent of 87% methanol and water.

4. The use of the C-4 substituted coumarins of claim 1 in the preparation of CYP1B1 enzyme inhibitors.

5. The use according to claim 4, wherein the CYP1B1 enzyme inhibitor is a medicament for the prophylaxis or treatment of a metabolic disorder.

6. Use according to claim 5, characterized in that said metabolic disorders are selected from the group consisting of tumors, obesity, hypertension and atherosclerosis.

7. The use of claim 6, wherein said tumor comprises a drug resistant tumor.

Technical Field

The invention relates to the technical field of medicines, in particular to a coumarin compound separated from dry stems and leaves of Calophyllum gracile Linn, a separation preparation method and application thereof.

Background

Cytochrome P4501B 1(Cytochrome P4501B 1, CYP1B1) is a heme-thiolate monooxygenase catalyzing substrate epoxidation and hydroxylation, and belongs to one of three members of Cytochrome P450 superfamily 1. In recent years, screening studies surrounding cytochrome P4501B 1 function and inhibitors have become a leading hot spot for biomedical and pharmacological research. Research shows that CYP1B1 regulates the metabolism of a plurality of endogenous substances including steroid hormones, fatty acids, melatonin and vitamins as well as exogenous substances such as drugs, pro-carcinogens and teratogens, and the enzyme maintains the dynamic balance of important physiological substances in vivo through interaction with nuclear receptors such as Peroxisome Proliferator Activated Receptors (PPARs), Retinoic Acid Receptors (RAR) and Estrogen Receptors (ER), and is closely related to the occurrence and development of various metabolic diseases such as tumors, tumor resistance, obesity, hypertension and atherosclerosis in human beings. Research shows that CYP1B1 enzyme is becoming a new target in research of tumor prevention, treatment and drug resistance overcoming, and screening of CYP1B1 enzyme inhibitor becomes a new way for discovery of novel tumor drugs, and has potential clinical application value. The coumarin compound obtained from the traditional medicinal plants is probably one of the sources for obtaining safe and effective CYP1B1 enzyme inhibitors.

Calophyllum Membranaceae Garda. et Champ.) belongs to Calophyllum of Guttiferae (Guttiferae), and is named as Calophyllum hybridum, Dieda general, and Piper hancei. In distribution, the thin leaf and the thick red shell are mainly distributed in China and the north part of Somali, and the stems and the barks of the thin leaf and the thick red shell are commonly used for treating rheumatism, arthritis and lumbago in Chinese folk medicine.

Disclosure of Invention

The invention carries out systematic separation on the ethanol extract of the stem and leaf of the Calophyllum gracile so as to obtain a natural lead compound with better biological activity, thereby providing scientific basis for the development and application of Calophyllum gracile resources.

The specific technical scheme of the invention is as follows:

the C-4 substituted coumarin compound has the following structure:

the invention also aims to provide a preparation method of the C4-substituted coumarin compound, which is obtained by separating dried stems and leaves of Calophyllum Membranaceae (Guttiferae) Calophyllum plant Calophyllum Membranaceae Garda. et Champ. by silica gel column chromatography, reversed phase ODS column chromatography, gel column chromatography and HPLC chromatography in sequence.

Preferably, the method comprises the following steps:

(1) pulverizing dried stem and leaf of Calophyllum inophyllum Linn, extracting with water and/or alcohol, preferably one or more of water, methanol and ethanol, more preferably 95% ethanol water solution, and concentrating;

(2) dissolving the concentrate obtained in the step (1) with water, filtering, mixing with polyamide, eluting by an MCI column, sequentially carrying out gradient elution by using 0%, 10%, 30%, 50%, 70%, 95% methanol-water mixed solvent and acetone, wherein each gradient elution is 2-5 column volumes (preferably 3 column volumes), and concentrating to obtain Fr A-F components;

(3) mixing the Fr E obtained in the step (2) with silica gel, mixing the mixture with petroleum ether: gradient elution is carried out on ethyl acetate (V: V, 100:1,50:1,30:1,15:1,8:1,4:1,2:1,1:1,0:1), each gradient elution is carried out for 2-5 column volumes (preferably 3 column volumes), and 27 group segments in total of Fr 1-27 are obtained after TLC detection and combination of the same components.

(4) Combining Fr6 and Fr 7 obtained in the step (3), separating by gel Sephadex LH-20 column chromatography with methanol as an eluent, eluting for 1-3 column volumes (preferably 1 column volume), monitoring by TLC after the sample is completely eluted, and combining the same components to obtain Fr 6-1-6-15 groups of segments;

(5) subjecting the Fr 6-4 fraction obtained in step (4) to HPLC chromatography using 85-100% methanol-water mixed solvent, preferably 90% methanol-water mixed solvent to obtain compound I and compound II, preferably using Zorbax SB-C18(Agilent,9.4 mm. times.250 mL) as chromatographic column, with flow rate v 2mL/min, detection wavelength λ 210nm, and peak-off time t_Ⅰ＝17min，t_Ⅱ＝22min；

(6) Separating the Fr 8 component obtained in the step (3) by gel Sephadex LH-20 column chromatography with acetone as an eluent, eluting for 1-3 column volumes (preferably 1 column volume), monitoring by TLC after the sample is completely eluted, and combining the same components to obtain Fr 8-1-8;

(7) and (3) isocratically eluting the Fr 8-4 fraction obtained in the step (6) by using a methanol-water mixed solvent with the concentration of 80-90%, preferably 85%, by using HPLC chromatography to obtain a compound III and a compound IV, wherein the preferable chromatographic column is Zorbax SB-C18(Agilent,9.4 mm. times.250 mL), the flow rate v is 2mL/min, the detection wavelength lambda is 210nm, and the peak-off time t is_Ⅲ＝14min，t_Ⅳ＝25min；

(8) And (3) combining the Fr10 obtained in the step (3) with Fr 11 and Fr 12, and then performing forward silica gel column chromatography, wherein the weight ratio of petroleum ether: dichloromethane mixed solvent (V: V, 5:1,4:1,3:1,2:1,1:1), each gradient elution is performed for 5-10 column volumes (preferably 5 column volumes), synchronous TLC detection is performed, and the same components are combined to obtain Fr10-1 to Fr 10-17 components;

(9) subjecting the Fr 10-9 fraction obtained in step (8) to gel Sephadex LH-20 column chromatography with dichloromethane: separating by using a methanol mixed solvent (V: V ═ 3:1) as an eluent, eluting by using 1-3 column volumes (preferably 1 column volume) at equal intervals, monitoring by using TLC (thin layer chromatography) after a sample is completely eluted, and combining the same components to obtain Fr 10-9-1-Fr 10-9-20;

(10) subjecting the Fr 10-9-8 fraction obtained in step (9) to HPLC using a methanol-water mixed solvent of 80-90%, preferably 87%, to obtain Compound V, preferably using Zorbax SB-C18(Agilent,9.4 mm. times.250 mL) as a column, at a flow rate of 2mL/min, at a detection wavelength of 210nm, and at a peak-off time t_Ⅴ＝16min。

The invention also aims to provide application of the C-4 substituted coumarin compound in preparation of CYP1B1 enzyme inhibitors. The inhibition activity of the compound on cytochrome P4501 series CYP1B1 enzyme, CYP1A1 enzyme and CYP1A2 enzyme is determined by adopting enzyme incubation reaction, and the result shows that the compound can selectively inhibit the CYP1B1 enzyme activity, but hardly has the inhibition activity on the CYP1A1 enzyme and CYP1A2 enzyme, so that the compound has strong targeting property, small side effect and higher clinical application value, and can be used for improving or preventing metabolic diseases such as tumor, tumor drug resistance, obesity, hypertension, atherosclerosis and the like.

The invention has the advantages that:

(1) the invention takes dried stems and leaves of Calophyllum Membranaceae (Guttiferae) Calophyllum plant Calophyllum Membranaceae (Calophyllum) Calophyllum Garda. et Champ. as raw materials, and the C-4 substituted coumarin compounds are obtained by separation of silica gel column chromatography, reversed phase ODS column chromatography, gel column chromatography and HPLC chromatography in sequence, and the compounds are one of sources for obtaining safe and effective CYP1B1 enzyme inhibitors.

(2) The compound can selectively inhibit the activity of CYP1B1 enzyme, but has almost no inhibitory activity to CYP1A1 enzyme and CYP1A2 enzyme, and shows that the compound has strong targeting property, small side effect and higher clinical application value.

(3) The compounds II, III and V have strong activity of inhibiting CYP1B1 enzyme, can be used for improving or preventing metabolic diseases such as tumors, tumor drug resistance, hypertension and atherosclerosis, and have potential clinical application value.

Drawings

FIG. 1 is a graph of HR-ESI-MS of Compound I.

FIG. 2 shows NMR of Compound I¹H NMR spectrum.

FIG. 3 shows NMR of Compound I¹³C NMR spectrum.

FIG. 4 shows NMR of Compound I¹H-¹H COSY spectrogram

FIG. 5 shows the NMR HSQC spectrum of compound I.

FIG. 6 shows the NMR HMBC spectrum of compound I.

FIG. 7 is a HR-ESI-MS diagram of Compound II.

FIG. 8 shows NMR of Compound II¹H NMR spectrum.

FIG. 9 shows NMR of Compound II¹³C NMR spectrum.

FIG. 10 shows NMR of Compound II¹H-¹H COSY spectrogram

FIG. 11 shows the NMR HSQC spectrum of compound II.

FIG. 12 shows the NMR HMBC spectrum of compound II.

FIG. 13 is a HR-ESI-MS diagram of Compound III.

FIG. 14 NMR of Compound III¹H NMR spectrum.

FIG. 15 shows NMR of Compound III¹³C NMR spectrum.

FIG. 16 shows NMR of Compound III¹H-¹H COSY spectrogram

FIG. 17 shows the NMR HSQC spectrum of compound III.

FIG. 18 shows the NMR spectrum of compound III with HMBC.

FIG. 19 is a HR-ESI-MS graph of Compound IV.

FIG. 20 shows NMR of Compound IV¹H NMR spectrum.

FIG. 21 shows NMR of Compound IV¹³C NMR spectrum.

FIG. 22 shows NMR of Compound IV¹H-¹H COSY spectrum.

FIG. 23 shows the NMR HSQC spectrum of Compound IV.

FIG. 24 shows the NMR HMBC spectrum of compound IV.

FIG. 25 is a HR-ESI-MS plot of Compound V.

FIG. 26 NMR of Compound V¹H NMR spectrum.

FIG. 27 NMR of Compound V¹³C NMR spectrum.

FIG. 28 NMR of Compound V¹H-¹H COSY spectrogram

FIG. 29 is the NMR HSQC spectrum of Compound V.

FIG. 30 shows the NMR HMBC spectrum of compound V.

Detailed Description

The present invention is further described with reference to the following examples, but the present invention is not limited to the following examples, and it is anticipated that one skilled in the art may make various modifications in combination with the prior art.

Specific rotation is measured by JASCO P-1020 full-automatic digital polarimeter; measuring the UV spectrum by using a Shimadzu UV-2401PC type ultraviolet spectrometer; measuring the IR spectrum by a BRUKER sensor-27 Fourier transform mid-infrared spectrometer type infrared spectrometer, and tabletting by KBr; ESI-MS and HRESI-MS were measured with an Agilent G6230 time-of-flight mass spectrometer; FAB-MS was measured using a Thermo Fisher Scientific DFS fast atom bombardment ion source mass spectrometer; NMR was measured using a Brucker AM-4Avance model III 600 NMR spectrometer with TMS as an internal standard, δ representing the chemical shift (in ppm) and J representing the coupling constant (in Hz).

The HPLC analytical instruments are LC-5510 type analysis and semi-preparative high performance liquid chromatograph (Beijing east west analysis) and Waters1525 high performance liquid chromatograph, and the chromatographic columns are Zorbax SB-C18(Agilent,9.4mm × 250mL,5 μm) and Waters SunAire C18 reversed phase chromatographic column; the normal phase silica gel plate for thin layer chromatography, the silica gel (80-100 meshes) for sample mixing and the silica gel (200-300 meshes) for column chromatography are produced in Qingdao ocean factories; the reverse phase filling material RP-18 is 40-60 μm, produced by Merk corporation; the macroporous adsorption resin is D101 polystyrene type macroporous adsorption resin produced by Mitsubishi corporation of Japan; the gel is Sephadex LH-20(GE Healthcare); the MCI filling material is MCI-gel CHP-20P; developer of 10% H₂SO₄-an ethanol solution.

Example 1

(1) Taking 15.0Kg of dried stems and leaves of the Calophyllum gracile L.var.gracile L.gracile.

(2) Dissolving the concentrated extract in water, filtering, mixing with polyamide, performing MCI column chromatography, performing gradient elution with 0%, 10%, 30%, 50%, 70%, 95% methanol-water mixed solvent and acetone sequentially, wherein each gradient elution has about 3 column volumes, and concentrating to obtain Fr A-F components.

(3) Fr E was sampled, and the samples were stirred with silica gel, and mixed with petroleum ether: gradient elution is carried out on ethyl acetate (V: V, 100:1,50:1,30:1,15:1,8:1,4:1,2:1,1:1,0:1), each gradient elution is about 3 column volumes, and 27 group segments in total are obtained after TLC detection and combination of the same components.

(4) And (3) combining Fr6 and Fr 7, separating by gel Sephadex LH-20 column chromatography, taking methanol as an eluent at the flow rate of 1d/4s for 9min, taking a bottle as a group of segments, monitoring by TLC after the sample is completely eluted, and combining the same components to obtain Fr 6-1-6-15 components.

(5) Segmenting the elution component Fr 6-4 group in the step (4), adopting a 90% methanol-water mixed solvent, and performing HPLC chromatography to obtain a compound I and a compound II, wherein a chromatographic column is Zorbax SB-C18(Agilent,9.4mm multiplied by 250mL), the flow rate v is 2mL/min, the detection wavelength lambda is 210nm, and the peak-off time t is_Ⅰ＝17min，t_Ⅱ＝22min。

A compound I:compound ii:

(6) and (3) separating the Fr 8 component in the step (3) by gel Sephadex LH-20 column chromatography with acetone as an eluent, eluting at 1 column volume by isocratic elution and at the flow rate of 1d/4s for 9min, taking a bottle as a group of segments, monitoring by TLC after the sample is completely eluted, and combining the same components to obtain Fr 8-1-8.

(7) And (4) performing HPLC (high performance liquid chromatography) half-preparation on the elution component Fr 8-4 in the step (6) by adopting a methanol-water mixed solvent with the concentration of 85% to obtain a compound III and a compound IV. The column was Zorbax SB-C18(Agilent,9.4 mm. times.250 mL), the flow rate v was 2mL/min, the detection wavelength λ was 210nm, and the peak-off time t was_Ⅲ＝14min，t_Ⅳ＝25min。

Compound iii:a compound IV:

(8) and (3) combining the Fr10, Fr 11 and Fr 12 in the step (3), and then carrying out forward silica gel column chromatography, wherein the weight ratio of petroleum ether: dichloromethane (V: V, 5:1,4:1,3:1,2:1,1:1), eluting 5 column volumes per gradient, detecting by synchronous TLC, and combining the same components to obtain each component Fr 10-1-10-17.

(9) Subjecting the Fr 10-9 fraction obtained in step (8) to gel Sephadex LH-20 column chromatography with dichloromethane: separating by using methanol (v: v ═ 3:1) as an eluent, eluting by using an equal elution speed of 1 column volume and a flow rate of 1d/4s for 9min, connecting bottles into a group of segments, monitoring by using TLC after a sample is completely eluted, and combining the same components to obtain Fr 10-9-1-Fr 10-9-20.

(10) And (3) performing HPLC half-preparation on the Fr 10-9-8 component in the step (9) by using a mixed solvent of 87% methanol and water to obtain a compound V. The column was Zorbax SB-C18(Agilent,9.4 mm. times.250 mL), the flow rate v was 2mL/min, the detection wavelength λ was 210nm, and the peak-off time t was_Ⅴ＝16min。

Compound v:and (3) structural identification:

using means including nuclear magnetic resonance spectroscopy (¹H-NMR、¹³C-NMR, HSQC, HMBC) and mass spectrometry (HR-ESI-MS) to identify the structure of the compound.

(1) The compound I is an orange oily substance and is easily dissolved in chloroform; [ alpha ] to]2_D5 ═ -103.08(c ═ 0.160, MeOH); HR-ESI-MS gives the peak M/z of the excimer ion 417.2275[ M + H ]]⁺(calcd.for C₂₄H₃₁O₆417.2272); bonding of¹An H-NMR spectrum of the sample solution,¹³C-NMR spectrum, determination of the formula C₂₄H₃₂O₆The unsaturation degree was 9. Meanwhile, the signal attribution of all carbon atoms and hydrogen atoms and the chemical structure of the compound are determined by measuring a two-dimensional H-C correlation spectrum (HSQC) and an H-C remote correlation spectrum (HMBC).¹H NMR and¹³the C NMR data are shown in tables 1 and 2.

FIG. 1 is a graph of HR-ESI-MS of Compound I, illustrating the molecular weight of Compound I. FIG. 2 shows NMR of Compound I¹H NMR spectrum shows the attribution of each hydrogen in the structure of the compound I. FIG. 3 shows NMR of Compound I¹³And C NMR spectrum shows the attribution of each carbon in the structure of the compound I. FIG. 4 shows NMR of Compound I¹H-¹H COSY spectrogram, which shows the related hydrogen attribution in the structure of the compound I. FIG. 5 is a nuclear magnetic resonance HSQC spectrum of compound I, illustrating the relegation of carbon and hydrogen in the structure of compound IBelongs to the field of medicine. FIG. 6 shows the NMR spectrum of HMBC, which shows the connection position of each substituent in the structure of compound I.

(2) The compound II is yellow oily substance and is easily dissolved in chloroform; [ alpha ] to]² _D ⁵-102.9(c ═ 0.140, MeOH); HR-ESI-MS gives the excimer peak m/z: 425.1931[ M + Na ]]⁺(calcd.for C₂₃H₃₀NaO₆425.1935); bonding of¹An H-NMR spectrum of the sample solution,¹³C-NMR spectrum, determination of the formula C₂₃H₃₀O₆The unsaturation degree was 9. Meanwhile, the signal attribution of all carbon atoms and hydrogen atoms and the chemical structure of the compound are determined by measuring a two-dimensional H-C correlation spectrum (HSQC) and an H-C remote correlation spectrum (HMBC).¹HNMR and¹³the C NMR data are shown in tables 1 and 2.

FIG. 7 is a graph of HR-ESI-MS of Compound II, illustrating the molecular weight of Compound II. FIG. 8 shows NMR of Compound II¹And H NMR spectrum shows the attribution of each hydrogen in the structure of the compound II. FIG. 9 shows NMR of Compound II¹³And C NMR spectrum shows the attribution of each carbon in the structure of the compound II. FIG. 10 shows NMR of Compound II¹H-¹H COSY spectrogram, which shows the related hydrogen attribution in the structure of the compound II. FIG. 11 is a NMR HSQC spectrum of compound II, illustrating the relative assignment of carbon and hydrogen in the structure of compound II. FIG. 12 is a nuclear magnetic resonance HMBC spectrum of compound II illustrating the attachment position of each substituent in the structure of compound II.

(3) The compound III is yellow oily matter and is easily dissolved in chloroform; [ alpha ] to]² _D ³-18.95(c ═ 0.170, MeOH); HR-ESI-MS gives the excimer peak m/z: 403.2132[ M-H]^-(calcd.for C₂₃H₃₁O₆403.2126); bonding of¹An H-NMR spectrum of the sample solution,¹³C-NMR spectrum, from which the formula C can be deduced₂₃H₃₂O₆The unsaturation degree is 8. Meanwhile, the signal attribution of all carbon atoms and hydrogen atoms and the chemical structure of the compound are determined by measuring a two-dimensional H-C correlation spectrum (HSQC) and an H-C remote correlation spectrum (HMBC).¹H NMR and¹³the C NMR data are shown in tables 1 and 2.

FIG. 13 is a graph of HR-ESI-MS of Compound III, illustrating the molecular weight of Compound III. FIG. 14 NMR of Compound III¹H NMR spectrum shows the attribution of each hydrogen in the structure of the compound III. FIG. 15 shows NMR of Compound III¹³C NMR spectrum shows the attribution of each carbon in the structure of the compound III. FIG. 16 shows NMR of Compound III¹H-¹H COSY spectrogram, which shows the related hydrogen attribution in the structure of the compound III. FIG. 17 is a NMR HSQC spectrum of compound III, illustrating the relative assignment of carbon and hydrogen in the structure of compound III. FIG. 18 shows the NMR spectrum of HMBC of compound III, illustrating the attachment position of each substituent in the structure of compound III.

(4) The compound IV is a yellow oily substance and is easily dissolved in chloroform; [ alpha ] to]2_D3 ═ -178.55(c ═ 0.120, MeOH); HR-ESI-MS gives the excimer peak m/z: 403.2132[ M-H]^-(calcd.for C₂₃H₃₁O₆403.2126); bonding of¹An H-NMR spectrum of the sample solution,¹³C-NMR spectrum, from which the formula C can be deduced₂₃H₃₂O₆The unsaturation degree is 8. Meanwhile, the signal attribution of all carbon atoms and hydrogen atoms and the chemical structure of the compound are determined by measuring a two-dimensional H-C correlation spectrum (HSQC) and an H-C remote correlation spectrum (HMBC).¹H NMR and¹³the C NMR data are shown in tables 1 and 2.

FIG. 19 is a graph of HR-ESI-MS of Compound IV, illustrating the molecular weight of Compound IV. FIG. 20 shows NMR of Compound IV¹And H NMR spectrum shows the attribution of each hydrogen in the structure of the compound IV. FIG. 21 shows NMR of Compound IV¹³And C NMR spectrum shows the attribution of each carbon in the structure of the compound IV. FIG. 22 shows NMR of Compound IV¹H-¹H COSY spectrogram, which illustrates the related hydrogen attribution in the structure of the compound IV. FIG. 23 is a nuclear magnetic resonance HSQC spectrum of compound IV, illustrating the relative assignment of carbon and hydrogen in the structure of compound IV. FIG. 24 is a nuclear magnetic resonance HMBC spectrum of compound IV, illustrating the attachment position of each substituent in the structure of compound IV.

(5) Book (I)The compound V is colorless crystal and is easily dissolved in chloroform; [ alpha ] to]² _D ⁵-64.17(c ═ 0.180, MeOH); HR-ESI-MS gives the excimer peak m/z: 437.1979[ M-H]^-(calcd.for C₂₆H₂₉O₆437.1970); bonding of¹An H-NMR spectrum of the sample solution,¹³C-NMR spectrum, from which the formula C can be deduced₂₆H₃₀O₆The unsaturation degree was 12. Meanwhile, the signal attribution of all carbon atoms and hydrogen atoms and the chemical structure of the compound are determined by measuring a two-dimensional H-C correlation spectrum (HSQC) and an H-C remote correlation spectrum (HMBC).¹H NMR and¹³the C NMR data are shown in tables 1 and 2.

FIG. 25 is a graph of HR-ESI-MS of Compound V, illustrating the molecular weight of Compound V. FIG. 26 NMR of Compound V¹And H NMR spectrum shows the attribution of each hydrogen in the structure of the compound V. FIG. 27 NMR of Compound V¹³And C NMR spectrum shows the attribution of each carbon in the structure of the compound V. FIG. 28 NMR of Compound V¹H-¹H COSY spectrogram, which illustrates the related hydrogen attribution in the structure of the compound V. FIG. 29 is a NMR HSQC spectrum of Compound V, illustrating the assignment of the relevant carbon and hydrogen in the structure of Compound V. FIG. 30 is a NMR HMBC spectrum of compound V, illustrating the attachment positions of the substituents in the structure of compound V.

TABLE 1 of Compounds I, II, III¹H NMR data (Acetone-d)₆)

Remarking: delta in ppm, J in Hz.¹H-NMR:600MHz。

TABLE 2 of the compounds I, II, III¹³C NMR data (CDCl)₃)

Remarking: delta in ppm of the amount of the acid derivative,¹³C-NMR:150MHz。

EXAMPLE 2 screening of the Compounds I, II, III, IV of the invention for inhibiting CYP1B1 enzymatic Activity

1. Experimental Material

Nicotinamide Adenine Dinucleotide Phosphate (NADPH), Mouse Liver Microsomes (MLM), beta-estradiol, resveratrol, acetonitrile.

2. Experimental methods

2.1 Experimental reaction systems

The reaction system contained β -estradiol (20.0uM), resveratrol (10.0uM) or test compound (compounds i, ii, iii, iv, v, 10.0 μ M), MLM (0.5mg/mL), buffer (PBS, pH 7.4). And after the reaction system is incubated, adding NADPH for reaction, stopping the reaction, centrifuging, and taking supernate to be tested. Incubation systems were performed in triplicate. (ii) positive control group: resveratrol and estradiol are also present. Negative control group: NADPH was absent and replaced by an equal volume of PBS. Experiment group: with estradiol and test compound. Fourthly, blank group: estradiol alone.

2.2 UPLC-ESI-QTOFMS analysis

Analysis of all microsomal samples was performed on an Agilent 1290 series UPLC system equipped with a 1290 quaternary pump (Agilent, Santa Clara, CA) and drug metabolites were detected by XDB-C18 column (2.1 × 100mm, 1.8mm, Agilent, Santa Clara, CA). The liquid flow rate was 0.3 mL/min. Phase A was 0.01% formic acid in water and phase B was acetonitrile containing 0.01% formic acid. The elution gradient was as follows: 0-12min, 2-98% B; 12-14min, 98% B; 14-16min, 98% A. The column temperature was 45 ℃. The data were in positive ion mode. The flow rates of the collision gas and the drying gas were 9L/min. The capillary voltage was 3.5kV, the temperature was 350 ℃ and the atomizer pressure was 35 psi. The target ions scanned are 273.1849 and 289.1798.

2.3 multivariate data analysis and statistical analysis

Chromatographic and spectroscopic data analysis was performed using the Mass Hunter Workstsion data software Collection software (Agilent, Santa Clara, Calif., USA). All values are expressed as mean values and statistical analysis was performed using Prism v.6. The enzyme activity inhibition ratio of the objective compound was calculated as follows.

CYP1B1 enzyme activity inhibition rate (positive control group or experimental group-blank group)/(negative control group-blank group) × 100%

3. Results of the experiment

The results of the experiment are shown in table 3. The results show that the compounds I, II, III, IV and V all have CYP1B1 enzyme inhibition activity, the inhibition rate of the compound I is 38.04%, the inhibition rate of the compound IV is 19.37%, the inhibition rate of the compound II is 43.20% which is equivalent to that of positive control resveratrol (the inhibition rate is 43.84%), the inhibition rate of the compound III is 49.72%, the inhibition rate of the compound V is 47.00% which is superior to that of resveratrol.

EXAMPLE 3 screening of CYP1A1 enzyme Activity inhibition by Compounds I, II, III and V of the invention

1. Experimental Material

Nicotinamide Adenine Dinucleotide Phosphate (NADPH), Mouse Liver Microsomes (MLM), granisetron, alpha-naphthoflavone, acetonitrile.

2. Experimental methods

2.1 Experimental reaction systems

The reaction system contained granisetron (20.0uM), alpha-naphthoflavone (1.0. mu.M) or compounds (compounds I, II, III, V, 10. mu.M described in the present invention), MLM (0.5mg/mL), and buffer (PBS, pH 7.4). And after the reaction system is incubated, adding NADPH for reaction, stopping the reaction, centrifuging, and taking supernate to be tested. Incubation systems were performed in triplicate. (ii) positive control group: and granisetron and alpha-naphthyl flavone. Negative control group: NADPH was absent and replaced by an equal volume of PBS. Experiment group: granisetron and the test compound (compound 1 or compound 3) are present together. Fourthly, blank group: only granisetron.

2.2 UPLC-ESI-QTOFMS analysis

Analysis of all microsomal samples was performed on an Agilent 1290 series UPLC system equipped with a 1290 quaternary pump (Agilent, Santa Clara, CA) and drug metabolites were detected by XDB-C18 column (2.1 × 100mm, 1.8mm, Agilent, Santa Clara, CA). The liquid flow rate was 0.3 mL/min. Phase A was 0.01% formic acid in water and phase B was acetonitrile containing 0.01% formic acid. The elution gradient was as follows: 0-12min, 2-98% B; 12-14min, 98% B; 14-16min, 98% A. The column temperature was 45 ℃. The data were in positive ion mode. The flow rates of the collision gas and the drying gas were 9L/min. The capillary voltage was 3.5kV, the temperature was 350 ℃ and the atomizer pressure was 35 psi. The target ions scanned are 273.1849 and 289.1798.

2.3 multivariate data analysis and statistical analysis

Chromatographic and spectroscopic data analysis was performed using the Mass Hunter Workstsion data software Collection software (Agilent, Santa Clara, Calif., USA). All values are expressed as mean values and statistical analysis was performed using Prism v.6. The enzyme activity inhibition ratio of the objective compound was calculated as follows.

CYP1a1 enzyme activity inhibition rate (positive control group or experimental group-blank group)/(negative control group-blank group) × 100%

3. Results of the experiment

The experimental results are shown in table 3, and the results show that the inhibition rate of the positive control alpha-naphthoflavone is 14.85%, the inhibition rate of the compound I (inhibition rate of 10.97%), the inhibition rate of the compound II (inhibition rate of-4.20%), the inhibition rate of the compound III (inhibition rate of 1.23%), and the inhibition rate of the compound V (inhibition rate of-23.00%) on CYP1A1 enzyme is obviously lower than the inhibition rate on CYP1B1 enzyme, so that the compounds I, II, III and V have higher targeting property on CYP1B1, and can selectively inhibit CYP1B1 enzyme.

EXAMPLE 4 screening of CYP1A2 enzyme Activity inhibition by Compounds I, III and V of the invention

1. Experimental Material

Nicotinamide Adenine Dinucleotide Phosphate (NADPH), Mouse Liver Microsomes (MLM), phenacetin, alpha-naphthoflavone, acetonitrile.

2. Experimental methods

2.1 Experimental reaction systems

The reaction system contained phenacetin (20.0. mu.M), α -naphthalenaflavone (1.0. mu.M) or compound (compounds I, II, III, V, 10.0. mu.M described in the present invention), MLM (0.5mg/mL), buffer (PBS, pH 7.4). And after the reaction system is incubated, adding NADPH for reaction, stopping the reaction, centrifuging, and taking supernate to be tested. Incubation systems were performed in triplicate. (ii) positive control group: both alpha-naphthalenones and phenacetin. Negative control group: HLM alone, without NADPH, was replaced with an equal volume of PBS. Experiment group: there is phenacetin and the test compound at the same time. Fourthly, blank group: only phenacetin.

2.2 UPLC-ESI-QTOFMS analysis

Analysis of all microsomal samples was performed on an Agilent 1290 series UPLC system equipped with a 1290 quaternary pump (Agilent, Santa Clara, CA) and drug metabolites were detected by XDB-C18 column (2.1 × 100mm, 1.8mm, Agilent, Santa Clara, CA). The liquid flow rate was 0.3 mL/min. Phase A was 0.01% formic acid in water and phase B was acetonitrile containing 0.01% formic acid. The elution gradient was as follows: 0-12min, 2-98% B; 12-14min, 98% B; 14-16min, 98% B. The column temperature was 45 ℃. The data were in positive ion mode. The flow rates of the collision gas and the drying gas were 9L/min. The capillary voltage was 3.5kV, the temperature was 350 ℃ and the atomizer pressure was 35 psi. The target ions scanned are 273.1849 and 289.1798.

2.3 multivariate data analysis and statistical analysis

Chromatographic and spectroscopic data analysis was performed using the Mass Hunter Workstsion data software Collection software (Agilent, Santa Clara, Calif., USA). All values are expressed as mean values and statistical analysis was performed using Prism v.6. The enzyme activity inhibition ratio of the objective compound was calculated as follows.

CYP1a2 enzyme activity inhibition rate (positive control group or experimental group-blank group)/(negative control group-blank group) × 100%

3. Results of the experiment

The experimental results are shown in table 3, and the results show that the inhibition rate of the positive control alpha-naphthoflavone is 50.13%, the inhibition rate of the compound I (inhibition rate-2.507%), the inhibition rate of the compound III (inhibition rate-3.50%), and the inhibition rate of the compound V (inhibition rate 6.84%) on CYP1A2 enzyme is obviously lower than that on CYP1B1 enzyme, which indicates that the compounds I, III and V have higher targeting property on CYP1B1 and can selectively inhibit CYP1B1 enzyme.

TABLE 3 inhibition of CYP1B1, CYP1A1, CYP1A2 enzyme Activity by Compounds