Synthesis of cycloicaritin flavanone derivative and antitumor application of cycloicaritin flavanone derivative

Abstract

The invention discloses a cycloicaritin flavanone derivative (3) in the field of medicinal chemistry. The structural formula of the cycloicaritin flavanone derivative (3) is as follows: # imgabs0 #, wherein R is H or C1-3 unsaturated alkyl. Experiments show that the cycloicaritin flavanone derivative (3) has high inhibitory activity on lung cancer and liver cancer, the activity of the cycloicaritin flavanone derivative (3) is higher than that of parent icaritin of the cycloicaritin flavanone derivative (3), and the cytotoxic activity of the cycloicaritin flavanone derivative (3) on normal cells LO2 is relatively low.

Description

Technical Field

[0001] The invention relates to the field of pharmaceutical chemistry, and in particular to the synthesis of cycloicariin dihydroflavonoid derivatives and their anti-tumor application. Background Art

[0002] In recent years, the incidence and mortality of malignant tumors in my country have increased year by year, and it has become the main killer threatening the health of the Chinese people. The development of new anti-tumor drugs is therefore crucial. With the deepening of scientific research on traditional Chinese herbal medicines in my country, more and more researchers have begun to pay attention to the anti-tumor efficacy of Chinese herbal medicines. Research results show that many natural medicines can kill tumor cells at multiple targets, and have the advantages of relatively low cost and low toxicity. Epimedium is a traditional Chinese medicine, belonging to the genus Epimedium of the Berberidaceae family. It is pungent, sweet, and warm in nature. Its dried leaves are used as medicine to treat impotence and spermatorrhea, kidney yang deficiency, rheumatism, muscle and bone weakness, numbness and cramps, etc. Pharmaceutical studies have shown that the main active ingredients of epimedium extracts include icariin, icariin, epimedium flavonoid derivatives, alkaloids, anthraquinones, lignans, phytosterols, anthocyanins, terpenoids, chlorogenic acid and other biologically active ingredients and nutrients such as essential fatty acids and trace elements. Modern pharmacological research has shown that Epimedium and its active ingredients not only have the functions of dilating coronary arteries, inhibiting microorganisms, anti-aging, and promoting bone cell growth, but also have multiple biological activities such as inhibiting tumors. Icaritin (1) is an active ingredient present in Epimedium medicinal materials. It is a flavonoid substance and is a yellow solid at room temperature. Its chemical name is 3,5,7-trihydroxy-2-(4-methoxyphenyl)-8-(3-methylbut-2-enyl)-4H-chromene-4-one, and its molecular formula is C 21 H 20 O6, molecular weight 368.3799. Icaritin can also be hydrolyzed from icariin, the main active ingredient of epimedium. Icaritin has been marketed in my country for the treatment of advanced liver cancer. Cycloicariin (2) can be synthesized from icariin (1). The structures of icariin and cycloicariin are shown in the following formula.

[0003]

[0004] Studies have shown that icariin has rich biological activities such as anti-tumor, anti-liver fibrosis, anti-osteoporosis, improvement of prostate function, neuroprotection, immunosuppression, anti-inflammatory, antioxidant, etc. Similar to many natural products, icariin still has many shortcomings, which attracts pharmaceutical researchers to conduct in-depth drug structure modification and activity research.

[0005] Copper(II) chloride can be used as a chlorination agent, oxidation agent and Lewis acid agent in organic synthesis. In particular, copper(II) chloride is an effective chlorination agent for various functional group compounds, and can achieve α-chlorination of carbonyl compounds and chlorination of aromatic compounds. For example, using carbon tetrachloride as a reaction solvent, copper(II) chloride reacts with phenol or alkoxybenzene compounds to obtain ortho- or para-chlorinated products of the benzene ring.

[0006] Considering the chlorination effect of copper chloride, the inventors hoped to use CuCl2 to introduce halogen atoms into the benzene ring of cycloicariin (2) in the research project. However, unexpectedly, the reaction of cycloicariin (2) with copper (II) chloride dihydrate (CuCl2·2H2O) in an alcohol solvent did not form the corresponding chloride, but unexpectedly obtained a cycloicariin dihydroflavonoid derivative (3) with a completely new structure. Activity studies confirmed that compound 3 has strong inhibitory activity against lung cancer and liver cancer. Summary of the invention

[0007] In view of the deficiencies of the prior art, the present invention transforms the structure of icariin and provides a new cycloicariin dihydroflavonoid derivative for preventing or treating tumors.

[0008] One of the purposes of the present invention is to provide a cycloicariin dihydroflavonoid derivative, the structural formula of which is shown below:

[0009]

[0010] Where: R is selected from H, C 1-3 of saturated hydrocarbon groups.

[0011] The second object of the present invention is to provide a method for synthesizing cycloicariin dihydroflavonoid derivatives, comprising the following steps:

[0012] Step 1: heating icariin (1) in formic acid for reflux reaction to obtain cycloicariin (2),

[0013] Step 2: reacting cycloicariin (2) with alcohol under specific conditions in the presence of a specific catalyst to obtain cycloicariin dihydroflavonoid derivatives (3); wherein: R is selected from H, C 1-3 The reaction route is as follows:

[0014]

[0015] Preferably, the catalyst in step 2 is selected from CuCl2, CuBr2, CuI and their corresponding hydrates.

[0016] The specific conditions of step 2 refer to: reacting at 10-100°C without a solvent, or reacting at 10-200°C with acetonitrile, dichloromethane, chloroform, tetrahydrofuran, DMF or DMSO as a solvent.

[0017] Further optimized, the solvent is preferably dichloromethane.

[0018] Preferably, the reaction condition is 20-60°C.

[0019] Preferably, the catalyst is selected from CuCl2.2H2O or CuBr2.

[0020] Preferably, the alcohol in step 2 is an alcohol having a substituent corresponding to R, specifically C 1-3 Saturated alcohol, for example, when R is methyl, the alcohol is methanol.

[0021] The results of the activity experiment showed that the cycloicariin dihydroflavonoid derivative (3) could significantly inhibit the growth of two tumor cells - lung cancer A549 and liver cancer SMMC-7721, and its activity was stronger than that of its parent structure icariin and cycloicariin.

[0022] The third object of the present invention is to provide the use of cycloicariin dihydroflavonoid derivatives (3) in the preparation of anti-tumor drugs.

[0023] Specifically, the anti-tumor drug is a drug against liver cancer or lung cancer. DETAILED DESCRIPTION

[0024] The present invention will be further described below by specific examples, but they are not intended to limit the scope of protection of the present invention. Without departing from the concept of the present invention, those skilled in the art may make improvements to the preparation method and the apparatus used within the scope of the claims, and these improvements should also be regarded as the scope of protection of the present invention. Therefore, the scope of protection of the patent of the present invention shall be subject to the attached claims.

[0025] In the following examples, unless otherwise specified, the experimental methods are usually carried out according to conventional conditions or conditions recommended by the manufacturer; the raw materials and reagents shown can be obtained by commercial purchase.

[0026] Example 1: Preparation of cycloicariin (2)

[0027]

[0028] Weigh icariin (50 mg, 0.136 mmol) in a round-bottom flask, add 4 mL of formic acid, and place the reaction flask in an 85°C oil bath for reflux reaction. At this time, the reaction solution becomes clear. Continue the reaction, and a yellow solid gradually precipitates from the reaction solution. React for 4 hours, and use thin layer chromatography to detect with a volume ratio of 4:1 petroleum ether-ethyl acetate developing agent. The reaction endpoint is determined when no icariin is detected on the thin layer chromatography plate. After the reaction is completed, place the reaction flask in a 4°C refrigerator for 10 minutes, filter and dry to obtain yellow powder solid cycloicariin (2, 45 mg, 89.7%). The product is a light yellow solid; R f =0.23 (eluent: petroleum ether: ethyl acetate = 6:1). 1 H NMR(300MHz,Chloroform-d)δ11.47(s,1H),8.15(d,J=8.6Hz,2H),7.01(d,J=8.7Hz,2H),6.7 0(s,1H),6.23(s,1H),3.87(s,3H),2.87(t,J=6.8Hz,2H),1.87(t,J=6.8Hz,2H),1.37(s,6H). 13 C NMR (101MHz, Chloroform-d)δ177.62,161.25,160.24,158.54,155.17,153.26,136.46,129.88,123.20,114.28,104.82,103.71,99.09,75.19,55.32,28.16,26.78,26.71. By comparing the NMR data with the literature, the compound was determined to be cycloicariin.

[0029] Example 2: Preparation of cycloicariin dihydroflavonoid derivatives (3)

[0030] Weigh cycloicariin (2, 50 mg, 0.136 mmol), CuCl2·2H2O (115 mg, 0.675 mmol) and ROH alcohol solvent (1-4 mL, excess), put them in a reaction bottle, and stir at room temperature for 3 h. The reaction mixture was extracted twice with ethyl acetate and water. The organic layer was separated and dried over MgSO4. Concentrate in vacuo, and the residue was chromatographed with petroleum ether: ethyl acetate = 3:1 as the eluent to obtain the target product, cycloicariin dihydroflavonoid derivative (3, 50.6 mg, 90%), as a yellow powdery solid. This reaction can be done without a solvent, and the ROH alcohol solvent used also has the function of a reaction solvent. Alternatively, an additional reaction solvent such as dichloromethane can be added.

[0031]

[0032] Table 1 Synthesis yield of cycloicariin dihydroflavonoid derivatives (3)

[0033]

[0034] Cycloicariin (2, 50 mg, 0.135 mmol), CuCl2·2H2O (115 mg, 0.675 mmol) and 2 mL of methanol were added to a dry round-bottom flask and reacted at room temperature for 3 h. Ethyl acetate and water were added to the reaction mixture for extraction and separation. The obtained organic layer was washed with water twice, dried over anhydrous magnesium sulfate, concentrated under vacuum, and purified by column chromatography (eluent: petroleum ether: ethyl acetate = 3:1) to obtain the target product 3a as a light yellow solid, 50.6 mg, with a yield of 90%. Mp125.5-127℃, R f =0.3(PE:EA=3:1), 1 H NMR (300MHz, CDCl3) δ10.58(s,1H),7.72(d,J=8.5Hz,2H),6.98(dd,J=21.4,8.6Hz,2H),6.07(d,J=8.5Hz,1H),4.83(s,1H),3.84(d,J=11 .2Hz,3H),3.27(d,J=1.0Hz,1H),3.07(s,2H),2.83–2.66(m,2H),2.65(d,J=6.3Hz,1H),1.90–1.77(m,2H),1.38(dd,J=12.8,2.7Hz,6H). 13 C NMR(10,1MHz,CDCl3)δ190.92,164.32,161.40,160.84,155.09,129.99,113.80,106.15,101.9 9,98.53,89.74,76.52,55.35,50.95,31.75,27.28,26.30,16.16,16.08.HRMS-ESI(m / z):calcd forC 22 H 24 O8Na[M+Na] + :439.1369,found 439.1367.

[0035]

[0036] Cycloicariin (2, 50 mg, 0.135 mmol), CuCl2·2H2O (115 mg, 0.675 mmol) and 2 mL of ethanol were added to a dry round-bottom flask and reacted at room temperature for 3 h. Ethyl acetate and water were added to the reaction mixture for extraction and separation. The obtained organic layer was washed with water twice, dried over anhydrous magnesium sulfate, concentrated under vacuum, and purified by column chromatography (eluent: petroleum ether: ethyl acetate = 3:1) to obtain the target product 3b as a light yellow solid, 53.2 mg, with a yield of 92%. Mp119.5-121.5℃, R f =0.24(PE:EA=3:1), 1 H NMR (300MHz, CDCl3) δ10.60(s,1H),7.74–7.69(m,2H),7.01–6.93(m,2H),6.06(d,J=10.6Hz,1H),4.81(s,1H),3.83(d,J=9.9Hz,3H),3.55–3.19 (m,2H),2.86–2.64(m,2H),2.60(dt,J=16.9,6.3Hz,1H),1.88–1.73(m,2 H), 1.37 (d, J = 15.2Hz, 6H), 1.13 (t, J = 7.1Hz, 1H), 0.99 (t, J = 7.1Hz, 2H). 13 CNMR(101MHz, CDCl3)δ191.13,164.17,161.28,160.69,155.39,129.83,128.88,125.10,113.98,113.67,10 6.13,101.91,99.49,98.37,89.77,59.60,55.34,31.74,27.25,26.30,16.14,15.02.HRMS-ESI(m / z):calcd for C 23 H 26 O8Na[M+Na] + :453.1520, found 453.1515.

[0037]

[0038] Cycloicariin (2, 50 mg, 0.135 mmol), CuCl2·2H2O (115 mg, 0.675 mmol) and 2 mL of n-propanol were added to a dry round-bottom flask and reacted at room temperature for 3 h. Ethyl acetate and water were added to the reaction mixture for extraction and separation. The obtained organic layer was washed with water twice, dried over anhydrous magnesium sulfate, concentrated under vacuum, and purified by column chromatography (eluent: petroleum ether: ethyl acetate = 3:1) to obtain the target product 3c as a light yellow solid, 50.8 mg, with a yield of 85%. Mp107.3-109.3℃, R f =0.28(PE:EA=3:1), 1 H NMR (300MHz, CDCl3) δ11.37(s,1H),10.60(s,1H),7.75–7.70(m,2H),7.48–7.41(m,2H),7.02 –6.93(m,4H),6.06(d,J=11.8Hz,2H),4.82(s,1H),3.84(d,J=8.6Hz,6H),3.47–3.40(m,1H),3 .40–3.27(m,2H),3.10(dt,J=9.4,6.8Hz,1H),2.84–2.68(m,4H),2.67–2.55(m,2H),1.89–1. 76(m,4H),1.49(p,J=7.0Hz,2H),1.37(dd,J=13.1,3.6Hz,13H),0.70(dt,J=52.4,7.4Hz,6H). 13 C NMR (101MHz, CDCl3) δ191.21,187.32,178.67,165.70,164.14,163.17,161.29,160.69, 156.07,155.39,129.91,128.95,124.98,124.51,113.92,113.65,105.91,104.95,102. 68,102.00,99.54,99.19,98.36,89.85,76.44,66.04,65.42,55.32,31.78,31.70,27.1 2,26.84,26.75,26.37,22.68,22.57,16.23,16.18,10.50,10.42.HRMS-ESI(m / z):calcd forC 24 H 28 O8Na[M+Na] + :467.1676, found 467.1677.

[0039]

[0040] Cycloicariin (2, 50 mg, 0.135 mmol), CuCl2·2H2O (115 mg, 0.675 mmol) and 2 mL of isopropanol were added to a dry round-bottom flask and reacted at room temperature for 3 h. Ethyl acetate and water were added to the reaction mixture for extraction and separation. The obtained organic layer was washed with water twice, dried over anhydrous magnesium sulfate, concentrated under vacuum, and purified by column chromatography (eluent: petroleum ether: ethyl acetate = 3:1) to obtain the target product 3d as a light yellow solid, 52.9 mg, with a yield of 88%. Mp176.7-177.8℃, R f =0.28(PE:EA=3:1), 1 H NMR (300MHz, CDCl3) δ11.37(s,1H),7.82–7.42(m,2H),6.95(dd,J=21.4,8.4Hz,2H),6.04(d,J=6.2Hz,1H),4.89(s,1H),3.99(p,J=6.2Hz, 1H),3.83(d,J=13.2Hz,3H),2.82(q,J=8.2,6.8Hz,1H),2.70(p,J=6.8Hz,1H),1.87(s,2H),1.38(d,J=14.0Hz,6H),1.09(d,J=6.6Hz,6H). 13 C NMR (101MHz, CDCl3) δ191.07,187.66,178.75,165.77,164.18,163.12,161.21,160.69 ,156.60,155.84,130.27,129.17,125.68,124.76,113.84,113.39,106.41,105.95,10 5.66,102.22,101.69,98.97,98.16,89.88,77.23,69.67,67.99,55.33,31.76,31.69, 26.99,26.79,26.65,26.57,23.96,23.76,23.41,22.76,16.54.HRMS-ESI(m / z):calcd for C 24 H 28 O8Na[M+Na] + :467.1676, found 467.1668.

[0041] Example 3: Single crystal diffraction of cycloicariin dihydroflavonoid derivatives (3a)

[0042] The cycloicariin dihydroflavonoid derivative compound synthesized and prepared by the present invention is solid and has a stable structure. The single crystal diffraction structure of compound 3a is shown below.

[0043]

[0044] Example 4: Tumor Growth Inhibitory Activity Test

[0045] Human lung cancer A549 and human liver cancer SMMC-7721 cells growing in logarithmic growth were added to a 96-well plate, with each well containing approximately 3000 cells in 100 μL. Compound (3) obtained in Example 1 above was then added, and icariin (1) and cycloicariin (2) were added as positive controls. Three parallel wells were set up in each group and cultured at 37°C in a carbon dioxide incubator for 48 hours. MTT (20 mg / mL) solution was added at 20 μL / well 4 hours before the experiment was terminated, and the cells were cultured for another 4 hours. The culture medium was discarded, and DMSO was added at 150 μL / well. After the crystals were dissolved, the OD value of each well was measured at a wavelength of 490 nm on an ELISA reader. The half inhibition concentration (IC50) was then calculated using GraphPad Prism software. 50 The results are shown in Table 2. Compared with the positive control icariin, the inhibition rate of compound (3a-1) was higher than that of icariin at a concentration of 50 μMol / L for 48 hours. The inhibition rates of all synthesized compounds on human lung cancer A549 and human liver cancer SMMC-7721 cells exceeded 90%. At the same time, the IC values ​​of the synthesized cycloicariin dihydroflavonoid derivatives on human lung cancer A549 and human liver cancer SMMC-7721 were 50 The values ​​were 9.95 to 27.39 μMol / L and 12.39 to 31.47 μMol / L, respectively, indicating that these cycloicariin dihydroflavonoid derivatives (3) have excellent inhibitory activity against the growth of lung cancer and liver cancer.

[0046] Table 2. Inhibitory activity of compound 3 against lung cancer A549 and liver cancer SMMC-7721 a

[0047]

[0048]

[0049] a: The test solvent was DMSO, b: The test drug concentration was 50 μMol / L, *: P<0.01.

[0050] The above is only an embodiment of the present invention, and the common knowledge such as the known specific structure and characteristics in the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several deformations and improvements can be made without departing from the structure of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A cycloicariin dihydroflavonoid derivative, characterized in that: The structural formula is shown below Where: R is selected from H, C 1-3 of saturated hydrocarbon groups.

2. The method for synthesizing cycloicariin dihydroflavonoid derivatives according to claim 1, characterized in that: The following steps are involved: Step 1: heating icariin (1) in formic acid for reflux reaction to obtain cycloicariin (2), Step 2: reacting cycloicariin (2) with alcohol under specific conditions in the presence of a specific catalyst to obtain cycloicariin dihydroflavonoid derivatives (3); wherein: R is selected from H, C 1-3 The reaction scheme is as follows:

3. The synthesis method according to claim 2, characterized in that: The catalyst in step 2 is selected from CuCl2, CuBr2, CuI and their corresponding hydrates.

4. The synthesis method according to claim 3, characterized in that: The specific conditions of step 2 refer to: reacting at 10-100°C without a solvent, or reacting at 10-200°C with acetonitrile, dichloromethane, chloroform, tetrahydrofuran, DMF or DMSO as a solvent.

5. The synthesis method according to claim 4, characterized in that: The solvent is preferably dichloromethane.

6. The synthesis method according to claim 5, characterized in that: The reaction conditions are 20-60°C.

7. The synthesis method according to any one of claims 4 to 6, characterized in that: The catalyst is selected from CuCl2.2H2O or CuBr2.

8. The synthesis method according to claim 7, characterized in that: The alcohol in step 2 is: C 1-3 Saturated alcohol.

9. Use of the cycloicariin dihydroflavonoid derivatives according to claim 1 in the preparation of anti-tumor drugs.

10. The use according to claim 9, characterized in that: The anti-tumor drug is a drug for preventing liver cancer or lung cancer.

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