Synthesis and anti-tumor application of cycloicariin dihydroflavonoid derivatives

Abstract

The present application discloses a cycloicariin dihydroflavonoid derivative (3) in the field of medicinal chemistry, the structural formula of which is shown below: #imgabs0# wherein: R is H or C 1‑3 Unsaturated hydrocarbon group. Experiments show that a cycloicariin dihydroflavonoid derivative (3) of the present invention has strong inhibitory activity against lung cancer and liver cancer, and its activity is stronger than that of its parent icariin, and its cytotoxic activity against normal cells LO2 is relatively low.

Description

Technical Field

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

[0002] As scientific research on traditional Chinese herbal medicine continues to deepen, more and more researchers have begun to pay attention to the anti-tumor efficacy of Chinese herbal medicine. 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, spermatorrhea, kidney yang deficiency, rheumatic pain, 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, essential fatty acids, trace elements and other bioactive ingredients and nutrients. 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-chromen-4-one, and its molecular formula is C 21 H 20 O6, with a molecular weight of 368.3799. Icaritin can also be produced by hydrolysis of icariin, the main active ingredient of epimedium. Icaritin is already available 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 below.

[0003]

[0004] Structures of icariin and cycloicariin

[0005] Studies have shown that icariin possesses a wide range of biological activities, including anti-tumor, anti-hepatic fibrosis, anti-osteoporosis, prostate function improvement, neuroprotection, immunosuppression, anti-inflammatory, and antioxidant properties. Like many natural products, icariin has several limitations, attracting pharmaceutical researchers to conduct in-depth research on its structure modification and activity.

[0006] Copper(II) chloride can be used as a chlorination agent, oxidation reagent, and Lewis acid reagent in organic synthesis. In particular, copper chloride is an effective chlorination agent for various functional group compounds, enabling α-chlorination of carbonyl compounds and chlorination of aromatic compounds. For example, using carbon tetrachloride as the reaction solvent, copper(II) chloride reacts with phenol or alkoxybenzene compounds to produce ortho- or para-chlorinated products of the benzene ring.

[0007] Considering the chlorinating effect of copper chloride, the inventors hoped to use CuCl2 to introduce a halogen atom into the benzene ring of cycloicariin (2) during their research. 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 instead unexpectedly produced 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

[0008] 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.

[0009] One of the purposes of the present invention is to provide a cycloicariin dihydroflavonoid derivative, the structural formula of which is shown below:

[0010]

[0011] Formula (3)

[0012] Wherein: R is selected from H, C 1-3 saturated hydrocarbon group.

[0013] A second object of the present invention is to provide a method for synthesizing cycloicariin dihydroflavonoid derivatives, comprising the following steps:

[0014] Step 1: Icaritin (1) is heated under reflux in formic acid to obtain cycloicariin (2).

[0015] Step 2: Cycloicariin (2) is reacted with alcohol in the presence of a specific catalyst under specific conditions to obtain cycloicariin dihydroflavonoid derivatives (3); wherein: R is selected from H, C 1-3 The reaction scheme is as follows:

[0016] .

[0017] Preferably, the catalyst in step 2 is selected from CuCl2, CuBr2, CuI and their corresponding hydrates.

[0018] 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.

[0019] For further optimization, the solvent is preferably dichloromethane.

[0020] Preferably, the reaction temperature is 20-60°C.

[0021] Preferably, the catalyst is selected from CuCl2.2H2O or CuBr2.

[0022] 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.

[0023] The results of the activity experiment showed that the dihydroflavonoid derivatives of cycloicariin (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.

[0024] The third object of the present invention is to provide the use of cycloicariin dihydroflavonoid derivatives (3) in the preparation of anti-tumor drugs.

[0025] Specifically, the anti-tumor drug is a drug for liver cancer or lung cancer. DETAILED DESCRIPTION

[0026] The present invention will be further illustrated by specific examples below, but these examples are not intended to limit the scope of protection of the present invention. Without departing from the scope of the present invention, those skilled in the art may make improvements to the preparation method and the apparatus used, and such improvements should also be considered within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be based on the appended claims.

[0027] In the following examples, unless otherwise stated, the experimental methods are generally carried out according to conventional conditions or conditions recommended by the manufacturer; the raw materials and reagents shown can be obtained through commercial purchase.

[0028] Example 1: Preparation of cycloicariin (2)

[0029]

[0030] Icaritin (50 mg, 0.136 mmol) was weighed into a round-bottom flask, and 4 mL of formic acid was added. The reaction flask was then placed in an 85°C oil bath and refluxed. The reaction solution became clear at this point. The reaction was continued, and a yellow solid gradually precipitated from the solution. The reaction was allowed to react for 4 hours, and the reaction was determined to be terminated by the absence of icariin on the TLC plate using a 4:1 volume ratio of petroleum ether to ethyl acetate as the developing solvent. After the reaction was complete, the reaction flask was placed in a 4°C refrigerator for 10 minutes, filtered, and dried to obtain cycloicariin (2, 45 mg, 89.7%) as a yellow powder. The product was a light yellow solid; R f =0.23 (eluent: petroleum ether: ethyl acetate = 6:1). 1 H NMR (300 MHz, Chloroform- d ) δ11.47 (s, 1H), 8.15 (d, J = 8.6 Hz, 2H), 7.01 (d, J = 8.7 Hz, 2H), 6.70 (s, 1H), 6.23 (s, 1H), 3.87 (s, 3H), 2.87 (t, J = 6.8 Hz, 2H), 1.87 (t, J = 6.8 Hz, 2H),1.37 (s, 6H). 13 C NMR (101 MHz, Chloroform- d ) δ 177.62, 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. The compound was identified as cycloicariin by comparison with the literature.

[0031] Example 2: Preparation of cycloicariin dihydroflavonoid derivatives (3)

[0032] Cycloicariin (2, 50 mg, 0.136 mmol), CuCl2·2H2O (115 mg, 0.675 mmol), and ROH alcohol (1–4 mL, excess) were weighed and placed in a reaction flask and stirred 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. The mixture was concentrated in vacuo, and the residue was chromatographed using a 3:1 ratio of petroleum ether to ethyl acetate to afford the desired product, cycloicariin dihydroflavonoid derivative (3, 50.6 mg, 90%), as a yellow powdery solid. This reaction can be performed without a solvent; the ROH alcohol solvent used can also function as a reaction solvent. Alternatively, an additional reaction solvent such as dichloromethane can be added.

[0033] 3

[0035] Table 1 Synthesis yield of cycloicariin dihydroflavonoid derivatives (3)

[0036]

[0037]

[0038] 3a

[0039] To a dry round-bottom flask, cycloicariin (2,50 mg, 0.135 mmol), CuCl2·2H2O (115 mg, 0.675 mmol), and 2 mL of methanol were added and reacted at room temperature for 3 h. Ethyl acetate and water were added to the reaction mixture for extraction and separation. The resulting organic layer was washed twice with water, dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and purified by column chromatography (eluent: petroleum ether:ethyl acetate = 3:1) to afford the desired product 3a as a light yellow solid (50.6 mg, 90% yield). Mp: 125.5-127°C, R f = 0.3 (PE: EA = 3: 1), 1 H NMR (300 MHz, CDCl3)δ 10.58 (s, 1H), 7.72 (d, J = 8.5 Hz, 2H), 6.98 (dd, J = 21.4, 8.6 Hz, 2H), 6.07(d, J = 8.5 Hz, 1H), 4.83 (s, 1H), 3.84 (d, J = 11.2 Hz, 3H), 3.27 (d, J= 1.0 Hz,1H), 3.07 (s, 2H), 2.83 – 2.66 (m, 2H), 2.65 (d, J = 6.3 Hz, 1H), 1.90 – 1.77(m, 2H), 1.38 (dd, J = 12.8, 2.7 Hz, 6H). 13 C NMR (10,1 MHz, CDCl3) δ 190.92,164.32, 161.40, 160.84, 155.09, 129.99, 113.80, 106.15, 101.99, 98.53, 89.74,76.52, 55.35, 50.95, 31.75, 27.28, 26.30, 16.16, 16.08. HRMS-ESI (m / z): calcdfor C 22 H 24 O8Na [M+Na] + : 439.1369, found 439.1367.

[0040]

[0041] 3b

[0042] To a dry round-bottom flask, cycloicariin (2,50 mg, 0.135 mmol), CuCl2·2H2O (115 mg, 0.675 mmol), and 2 mL of ethanol were added and reacted at room temperature for 3 h. Ethyl acetate and water were added to the reaction mixture for extraction and separation. The resulting organic layer was washed twice with water, dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and purified by column chromatography (eluent: petroleum ether:ethyl acetate = 3:1) to afford the desired product 3b as a light yellow solid (53.2 mg, 92% yield). Mp: 119.5-121.5°C, R f = 0.24 (PE: EA = 3: 1), 1 H NMR (300 MHz, CDCl3) δ 10.60 (s, 1H), 7.74 – 7.69 (m, 2H), 7.01 – 6.93 (m, 2H), 6.06 (d, J =10.6 Hz, 1H), 4.81 (s, 1H), 3.83 (d, J= 9.9 Hz, 3H), 3.55 – 3.19 (m, 2H), 2.86 – 2.64 (m, 2H), 2.60 (dt, J = 16.9, 6.3 Hz, 1H), 1.88 – 1.73 (m, 2H), 1.37 (d, J = 15.2 Hz, 6H), 1.13 (t, J = 7.1 Hz, 1H), 0.99 (t, J = 7.1 Hz, 2H). 13 C NMR (101MHz, CDCl3) δ 191.13, 164.17, 161.28, 160.69, 155.39, 129.83, 128.88, 125.10,113.98, 113.67, 106.13, 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.

[0043]

[0044] 3c

[0045] To a dry round-bottom flask, cycloicariin (2,50 mg, 0.135 mmol), CuCl2·2H2O (115 mg, 0.675 mmol), and 2 mL of n-propanol were added and reacted at room temperature for 3 h. Ethyl acetate and water were added to the reaction mixture for extraction and separation. The resulting organic layer was washed twice with water, dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and purified by column chromatography (eluent: petroleum ether:ethyl acetate = 3:1) to afford the desired product 3c as a light yellow solid (50.8 mg, 85% yield). Mp: 107.3-109.3°C, R f = 0.28 (PE: EA = 3: 1), 1H NMR (300MHz, CDCl3) δ 11.37 (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 = 11.8 Hz, 2H), 4.82 (s, 1H), 3.84 (d, J = 8.6 Hz, 6H), 3.47 – 3.40 (m, 1H), 3.40 – 3.27 (m, 2H), 3.10 (dt, J = 9.4,6.8 Hz, 1H), 2.84 – 2.68 (m, 4H), 2.67 – 2.55 (m, 2H), 1.89 – 1.76 (m, 4H),1.49 (p, J = 7.0 Hz, 2H), 1.37 (dd, J = 13.1, 3.6 Hz, 13H), 0.70 (dt, J = 52.4, 7.4Hz, 6H). 13 C NMR (101 MHz, CDCl3) δ 191.21, 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, 27.12, 26.84, 26.75, 26.37, 22.68,22.57, 16.23, 16.18, 10.50, 10.42. HRMS-ESI (m / z): calcd for C 24 H 28 O8Na [M+Na] + : 467.1676, found 467.1677.

[0046]

[0047] 3d

[0048] To a dry round-bottom flask, cycloicariin (2,50 mg, 0.135 mmol), CuCl2·2H2O (115 mg, 0.675 mmol), and 2 mL of isopropanol were added and reacted at room temperature for 3 h. Ethyl acetate and water were added to the reaction mixture for extraction and separation. The resulting organic layer was washed twice with water, dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and purified by column chromatography (eluent: petroleum ether:ethyl acetate = 3:1) to afford the desired product 3d as a light yellow solid (52.9 mg, 88% yield). Mp: 176.7-177.8°C, R f = 0.28 (PE: EA = 3: 1), 1 H NMR (300MHz, CDCl3) δ 11.37 (s, 1H), 7.82 – 7.42 (m, 2H), 6.95 (dd, J = 21.4, 8.4 Hz,2H), 6.04 (d, J = 6.2 Hz, 1H), 4.89 (s, 1H), 3.99 (p, J = 6.2 Hz, 1H), 3.83 (d, J =13.2 Hz, 3H), 2.82 (q, J = 8.2, 6.8 Hz, 1H), 2.70 (p, J = 6.8 Hz, 1H), 1.87 (s,2H), 1.38 (d, J = 14.0 Hz, 6H), 1.09 (d, J = 6.6 Hz, 6H). 13C NMR (101 MHz, CDCl3)δ 191.07, 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, 105.95,105.66, 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.

[0049] Example 3: Single crystal diffraction of cycloicariin dihydroflavonoid derivative (3a)

[0050] 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.

[0051]

[0052] Single crystal diffraction structure of cycloicariin dihydroflavonoid derivative 3a

[0053] Example 4: Tumor Growth Inhibitory Activity Test

[0054] Human lung cancer A549 and human liver cancer SMMC-7721 cells growing in logarithmic growth were added to a 96-well plate, with approximately 3000 cells per well in 100 μL. Compound (3) obtained in Example 1 above was then added, along with icariin (1) and cycloicariin (2) as positive controls. Three parallel wells were set up in each group, and the cells were cultured at 37°C in a carbon dioxide incubator for 48 hours. Four hours before the experiment was terminated, MTT (20 mg / mL) solution was added at 20 μL / well, 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 dissolved, the OD value of each well was measured at a wavelength of 490 nm on a microplate reader. The half-maximal inhibitory concentration (IC50) was then calculated using GraphPadPrism software. 50The results are shown in Table 2. Compared to the positive control icariin, compounds (3a-l) exhibited higher inhibition rates than icariin at a concentration of 50 μM / L for 48 hours. All synthesized compounds exhibited inhibition rates exceeding 90% against human lung cancer A549 and human liver cancer SMMC-7721 cells. Furthermore, the IC values ​​of the synthesized cycloicariin dihydroflavonoid derivatives against human lung cancer A549 and human liver cancer SMMC-7721 cells were significantly higher than those of the icariin derivatives. 50 The values ​​ranged from 9.95 to 27.39 μMol / L and 12.39 to 31.47 μMol / L, respectively, indicating that these cycloicariin dihydroflavonoid derivatives (3) had excellent inhibitory activities against the growth of lung cancer and liver cancer.

[0055] Table 2. Inhibitory activity of compound 3 against lung cancer A549 and liver cancer SMMC-7721 a

[0056]

[0057] a: The test solvent was DMSO, b: The test drug concentration was 50 μMol / L, *: P < 0.01.

[0058] The above is only an embodiment of the present invention, and the common knowledge such as the specific structure and characteristics of the scheme is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the structure of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection 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 Wherein: R is selected from H, C 1-3 saturated hydrocarbon group.

2. The method for synthesizing cycloicariin dihydroflavonoid derivatives according to claim 1, wherein: The following steps are involved: Step 1: Icaritin (1) is heated in formic acid for reflux reaction to obtain cycloicariin (2). Step 2: Cycloicariin (2) is reacted with alcohol in the presence of a specific catalyst under specific conditions 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, wherein: The catalyst in step 2 is selected from CuCl2, CuBr2, CuI and their corresponding hydrates.

4. The synthesis method according to claim 3, wherein: 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, wherein: 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 liver cancer or lung cancer.

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