A polymethoxyflavone derivative, and a preparation method and application thereof

Abstract

The application discloses a polymethoxyflavone derivative, a preparation method and application thereof, and belongs to the field of medicinal chemistry, and particularly relates to a kind of polymethoxyflavone derivatives with anti-hepatitis B virus activity, and the polymethoxyflavone derivative has the structure shown in formula (I) or (II). By applying visual drug screening model to carry out antiviral drug screening on hepatitis B virus replication, it is found that the polymethoxyflavone derivative has good anti-hepatitis B virus activity, which provides a good foundation for subsequent development of hepatitis B treatment drugs.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry, specifically relating to a class of polymethoxyflavonoid derivatives with anti-hepatitis B virus activity, their preparation methods, and applications. Background Technology

[0002] Pithecellobium clypearia (Jack.) Benth, a plant belonging to the genus Pithecellobium in the subfamily Mimosoideae of the Fabaceae family, is derived from the dried young branches and leaves of the plant. It is also known as "Luodi Sanqian," "Jisanshu," and "Jiaolongmu." Propagation is primarily by seed, and its main production areas are Zhejiang, Taiwan, and Fujian provinces. The main components isolated from Pithecellobium clypearia are flavonoids, including dihydroflavonoids, flavans, flavonoids, and chalcones. Modern pharmacological studies have shown that Pithecellobium clypearia has strong anti-inflammatory activity and is often used to treat upper respiratory tract infections, pharyngitis, and tonsillitis. It also exhibits significant antiviral effects. Leung et al., in screening 59 traditional Chinese medicines, found that the ethanol extract of Pithecellobium clypearia had significant anti-duck hepatitis B virus (DHBV) activity (Phytotherapy Research, 2006, 20: 911-914). Roschek et al. described its ability to attach to the surface proteins of viruses and inhibit viral entry into host cells. Some flavonoids act as transcription blockers, affecting the replication process, while others inhibit later stages of viral assembly, packaging, and release. Flavonoids can also modulate the immune system and reduce viral load (Biomedicine & Pharmacotherapy 140 (2021)111596).

[0003] Hepatitis B (HBV) is caused by infection with the hepatitis B virus (HBV). HBV is a hepatotropic DNA virus that primarily infects liver cells. The main routes of transmission include blood transmission, mother-to-child transmission, and sexual transmission. HBV infection can cause acute or chronic hepatitis B, and some patients may gradually develop cirrhosis, liver failure, or liver cancer. Currently, the treatment of chronic HBV infection mainly relies on pegylated interferon-α (PEGIFNα) and nucleoside(t) analogues. Long-term use of nucleoside(t) analogues for anti-HBV treatment may lead to drug resistance and side effects. Therefore, there is an urgent need to develop safe and effective anti-HBV drugs with novel mechanisms of action. Summary of the Invention

[0004] The polyhydroxyflavan 7-O-galloyltricetiflavan (GTF), extracted and isolated from monkey earrings, has a structure similar to epigallocatechin gallate (EGCG), a compound with anti-hepatitis virus activity. Thiazolidinediones exhibit significant anti-hepatitis virus activity.

[0005] Based on this, the present invention provides a polymethoxyflavonoid derivative, its preparation method, and its application. First, using GTF as a lead compound, an intermediate GTF-1 is designed and synthesized. Then, this intermediate is linked to a thiazolidinedione fragment via a linker arm, thereby obtaining a flavonoid thiazolidinedione compound with good activity, high bioavailability, and low toxicity. Experiments using this invention have demonstrated that this type of compound has significant efficacy against HBV.

[0006] The above-mentioned objective is achieved through the following specific technical solutions:

[0007] Polymethoxyflavonoid derivatives or pharmaceutically acceptable salts thereof, as shown in general formula (I) or (II):

[0008]

[0009] In equations (I) and (II), n is an integer from 1 to 10, preferably an integer from 1 to 5.

[0010] Preferably, the polymethoxyflavonoid derivatives of the present invention are selected from any one of the following compounds or their pharmaceutically acceptable salts:

[0011]

[0012] The "pharmaceutically acceptable salt" as described in this invention refers to salts formed by polymethoxyflavonoid derivatives and inorganic / organic acids or inorganic / organic bases within the scope of reliable pharmaceutical evaluation, including sulfates, hydrochlorides, hydrobroms, phosphates, acetates, methanesulfonates, benzenesulfonates, methylbenzenesulfonates, tartrates, maleates, benzoates, succinates, fumarates, fumarates, tartrates, gallates, citrates, as well as ammonium salts, sodium salts, potassium salts, etc.

[0013] The present invention also provides a pharmaceutical composition comprising a polymethoxyflavonoid derivative of formula (I) or (II) of the present invention or a pharmaceutically acceptable salt thereof, and a conventional pharmaceutical carrier or excipient, wherein the pharmaceutical composition may be administered orally or via parenteral routes (e.g., injection). The pharmaceutical compositions of the present invention can be prepared into various dosage forms, including capsules, tablets, solutions, suspensions, granules, and injections, according to conventional methods in the art.

[0014] The present invention also provides a method for preparing polymethoxyflavonoid derivatives of general formula (I) or (II):

[0015] The synthesis route is as follows:

[0016]

[0017] Step 1: 2,4,6-Trihydroxyacetophenone (A) reacts selectively with chloromethyl methyl ether to give compound B;

[0018] Step 2: Compound B reacts with dimethyl sulfate to give compound C;

[0019] Step 3: Compound C is condensed with 3,4,5-trimethoxybenzaldehyde to obtain compound D;

[0020] Step 4: Compound D undergoes a deprotection reaction to yield compound E;

[0021] Step 5: Compound E reacts selectively with chloromethyl methyl ether to give compound F;

[0022] Step 6: Compound F undergoes cyclization in a sodium acetate-ethanol system to yield compound G;

[0023] Step 7: Compound G undergoes a deprotection reaction to yield intermediate GTF-1;

[0024] Step 8: The intermediate GTF-1 reacts with a dibromide or a thiazolidinedione under basic conditions to give the compound shown in general formula (I) or (II).

[0025] Preferably, the dibromide mentioned in step 8 is selected from any one of 1,2-dibromoethane, 1,3-dibromopropane, 1,4-dibromobutane, 1,5-dibromopentane, 1,6-dibromohexane, 2,2′-dibromodiethyl ether, brominated-polyethylene glycol-bromination, and brominated-tetraethylene glycol-bromination.

[0026] The present invention also provides the use of the polymethoxyflavonoid derivatives thereof or pharmaceutically acceptable salts thereof or the pharmaceutical compositions thereof in the preparation of drugs for treating hepatitis B virus infection.

[0027] The present invention also provides the use of the polymethoxyflavonoid derivatives or pharmaceutically acceptable salts thereof, or the pharmaceutical compositions thereof, in the preparation of medicaments for the prevention and / or treatment of hepatitis B. Preferably, the hepatitis B is severe hepatitis B.

[0028] The beneficial effects of this invention are:

[0029] This invention provides a novel polymethoxyflavonoid derivative, which features a thiazolidinedione group on the flavonoid skeleton, resulting in a unique structure. Using a visual drug screening model to screen antiviral drugs against hepatitis B virus replication, the polymethoxyflavonoid derivative provided by this invention exhibits good anti-hepatitis B virus activity, providing a solid foundation for the subsequent development of drugs to treat hepatitis B. Attached Figure Description

[0030] Figure 1 A schematic diagram of the experimental procedure for anti-HBV activity and cytotoxicity;

[0031] Figure 2 The effects of compounds Y1-Y7 and control drugs on HBsAg secretion levels and cell viability in HBV-infected cells are shown in Figure 1. A represents the ELISA results of HBsAg detection, and B represents the cell viability detection results. Detailed Implementation

[0032] The present invention will be further described below through specific embodiments. Typical compounds of the present invention include, but are not limited to, the following compounds or pharmaceutically acceptable salts thereof:

[0033] Example 1: Preparation of intermediate compound GTF-1

[0034]

[0035] (1) Under reaction conditions with acetone as solvent and potassium carbonate as acid-binding agent, 2,4,6-trihydroxyacetophenone (A) as starting material reacts selectively with chloromethyl methyl ether to obtain compound B;

[0036] (2) Under the reaction conditions of acetone as solvent and potassium carbonate as acid-binding agent, compound B reacts with dimethyl sulfate to obtain compound C;

[0037] (3) Under ice bath conditions, compound C condenses with 3,4,5-trimethoxybenzaldehyde in an alkaline environment to give compound D;

[0038] (4) Compound D undergoes a deprotection reaction under acidic conditions to give compound E;

[0039] (5) Compound E reacts selectively with chloromethyl methyl ether to give compound F;

[0040] (6) Compound F undergoes cyclization in a sodium acetate-ethanol system to give compound G;

[0041] (7) Compound G undergoes a deprotection reaction under acidic conditions to give intermediate GTF-1.

[0042] Example 2: Preparation of compound Y1

[0043]

[0044] Compound GTF-1 (20.00 mg, 0.056 mmol) was dissolved in anhydrous acetonitrile, and anhydrous potassium carbonate (38.35 mg, 0.28 mmol) was added. After stirring for 30 min, 1,2-dibromoethane (10.43 mg, 0.056 mmol) was added, and the mixture was refluxed at 70 °C for 8 h until thin-layer chromatography (TLC) showed complete reaction of the starting material. Subsequently, anhydrous potassium carbonate (38.35 mg, 0.28 mmol) and potassium iodide (9.21 mg, 0.056 mmol) were added to the reaction system, and after stirring for 30 min, 2,4-thiazolidinedione (32.50 mg, 0.28 mmol) was added. The mixture was then reacted at 70 °C for 8 h until thin-layer chromatography (TLC) showed complete reaction of the starting material. The extract was taken three times with ethyl acetate. The combined organic phases were washed successively with distilled water, saturated brine, and dried over anhydrous sodium sulfate. The mixture was filtered, concentrated under reduced pressure, filtered through a 0.45 μm microporous membrane, and separated by high-performance liquid chromatography (HPLC) with MeCN-H₂O (MeCN:H₂O = 70:30) as the mobile phase. After separation and drying, the solid was weighed to obtain the yellow solid, which was compound Y1, with a yield of 50.7%. The structural identification data are as follows: HR-ESI-MS m / z: 504.1313 [M+H] + , (calcd for C 24 H 26 NO9S, 504.1323); 1 H-NMR (600 MHz, DMSO-d6) δ 6.01 (s, 2H), 5.40 (d, J = 2.2 Hz, 1H), 5.32 (d, J= 2.4 Hz, 1H), 4.60 (dd, J = 13.1, 2.8 Hz, 1H), 3.40 (s, 2H), 3.38 (t, J =5.8 Hz, 2H), 3.04 (t, J = 5.7 Hz, 2H), 2.96 (d, J = 6.7 Hz, 9H), 2.84 (s,3H), 2.28 (dd, J = 16.4, 13.1 Hz, 1H), 1.82-1.77 (m, 1H); 13C NMR (150 MHz, DMSO-d6) δ 188.33, 172.72, 172.39(×2), 164.64, 164.41, 162.28, 153.36,137.85, 134.95, 130.13, 106.04, 104.48(×2), 94.84, 93.58, 79.04, 64.71,60.47, 56.43(×2), 56.40, 45.28, 34.36.

[0045] Example 3: Preparation of compound Y2

[0046]

[0047] The procedure was the same as in Example 2, except that 1,2-dibromoethane was replaced with 1,3-dibromobutane to obtain the monomer compound Y2 in 55.4% yield. The structural identification data are as follows: HR-ESI-MS m / z: 518.1469 [M+H] + ,(calcd for C 25 H 28 NO9S, 518.1479); 1 H NMR (600 MHz, DMSO-d6) δ 6.84 (s, 2H), 6.19 (d, J = 2.2 Hz, 1H), 6.17 (d, J = 2.2 Hz, 1H), 5.42 (dd, J = 13.0, 2.7Hz, 1H), 4.18 (s, 2H), 4.07-4.01 (m, 2H), 3.79 (d, J = 6.7 Hz, 9H), 3.66 (d,J = 2.7 Hz, 5H), 3.12 (dd, J = 16.4, 13.1 Hz, 1H), 2.60 (dd, J = 16.4, 2.8Hz, 1H), 1.96 (p, J = 6.4 Hz, 2H); 13 C-NMR (150 MHz, DMSO-d6) δ 188.31,172.94, 172.56, 164.94, 164.70, 162.16, 153.36(×2), 137.87, 134.96, 105.84,104.54(×2), 94.47, 93.77, 79.10, 66.51, 60.47, 56.43(×3), 56.35, 45.28,34.42, 26.95.

[0048] Example 4: Preparation of compound Y3

[0049]

[0050] The procedure was the same as in Example 2, except that 1,2-dibromoethane was replaced with 1,4-dibromobutane to obtain the monomer compound Y3 in 55.9% yield. The structural identification data are as follows: HR-ESI-MS m / z: 532.1625 [M+H] + ,(calcd for C26H30NO9S, 532.1636); 1 H NMR (600 MHz, Chloroform-d) δ 6.67 (s,2H), 6.14 (d, J = 2.1 Hz, 1H), 6.10 (d, J = 2.1 Hz, 1H), 5.32 (dd, J = 13.3,2.6 Hz, 1H), 4.00 (s, 2H), 3.95 (s, 2H), 3.90 (d, J = 5.7 Hz, 9H), 3.86 (s,3H), 3.70 (t, J = 6.5 Hz, 2H), 3.05-2.98 (m, 1H), 2.80-2.76 (m, 1H), 1.80 (p,J = 3.1 Hz, 4H); 13 C NMR (150 MHz, Chloroform-d) δ 189.30, 171.94, 171.59,165.36, 164.98, 162.45, 153.67(×2), 138.30, 134.46, 106.11, 103.33(×2),94.12, 93.77, 79.57, 67.58, 61.01, 56.38, 56.36(×2), 45.90, 41.69, 33.92,26.34, 24.47.

[0051] Example 5: Preparation of compound Y4

[0052]

[0053] The procedure was the same as in Example 2, except that 1,2-dibromoethane was replaced with 1,6-dibromohexane to obtain the monomer compound Y4 in 55.8% yield. The structural identification data are as follows: HR-ESI-MS m / z: 560.1934 [M+H] + ,(calcd for C 28 H34 NO9S, 560.1937); 1 H NMR (600 MHz, Chloroform-d) δ 6.67 (s,2H), 6.14 (d, J = 2.2 Hz, 1H), 6.10 (d, J = 2.2 Hz, 1H), 5.32 (dd, J = 13.3,2.7 Hz, 1H), 3.96 (t, J = 6.4 Hz, 2H), 3.93 (s, 2H), 3.90 (d, J = 8.5 Hz,9H), 3.85 (s, 3H), 3.65-3.61 (m, 2H), 3.01 (dd, J = 16.5, 13.4 Hz, 1H), 2.78(dd, J = 16.5, 2.8 Hz, 1H), 1.78 (dt, J = 14.2, 6.4 Hz, 2H), 1.62 (q, J = 7.6Hz, 2H), 1.48 (q, J = 7.7 Hz, 2H), 1.37 (t, J = 7.8 Hz, 2H); 13 C NMR (150 MHz, Chloroform-d) δ 189.29, 171.92, 171.59, 165.60, 164.96, 162.43, 153.64(×2),138.26, 134.50, 105.97, 103.31(×2), 94.13, 93.74, 79.53, 68.29, 60.99, 56.35(×3), 45.89, 42.05, 33.87, 28.88, 27.58, 26.48, 25.66.

[0054] Example 6: Preparation of compound Y5

[0055]

[0056] Compound GTF-1 (20.00 mg, 0.056 mmol) was dissolved in anhydrous acetonitrile, and anhydrous potassium carbonate (38.35 mg, 0.28 mmol) was added. After stirring for 30 min, 2,2'-dibromodiethyl ether (12.87 mg, 0.056 mmol) was added, and the mixture was refluxed at 70 °C for 8 h until thin-layer chromatography (TLC) showed complete reaction of the starting material. Then, anhydrous potassium carbonate (38.35 mg, 0.28 mmol) and potassium iodide (9.21 mg, 0.056 mmol) were added to a round-bottom flask, and after stirring for 30 min, thiazolidinedione (32.50 mg, 0.28 mmol) was added. The mixture was then reacted at 70 °C for 8 h until thin-layer chromatography (TLC) showed complete reaction of the starting material. The extract was taken three times with ethyl acetate. The combined organic phases were washed successively with distilled water, saturated brine, and dried over anhydrous sodium sulfate. The mixture was filtered, concentrated under reduced pressure, filtered through a 0.45 μm microporous membrane, and separated by high-performance liquid chromatography (HPLC) with MeCN-H₂O (MeCN:H₂O = 70:30) as the mobile phase. After separation and drying, the solid was weighed to obtain the yellow solid, which was compound Y5, with a yield of 50.7%. The structural identification data are as follows: HR-ESI-MS m / z: 548.1517 [M+H] + , (calcd for C 26 H 29 NO 10 S, 548.1585); 1 HNMR (600 MHz, Chloroform-d) δ 6.67 (s, 2H), 6.19-6.15 (m, 2H), 5.33 (dd, J =13.3, 2.7 Hz, 1H), 4.19-4.12 (m, 2H), 3.95 (m, 2H), 3.90 (d, J = 6.5 Hz, 9H), 3.86 (s, 5H), 3.82 (t, J = 6.8 Hz, 2H), 3.28 (t, J = 6.8 Hz, 2H), 3.05-2.99(m, 1H), 2.79 (dd, J = 16.5, 2.8 Hz, 1H). 13C NMR (150 MHz, Chloroform-d) δ189.29, 165.14, 164.92, 162.44, 153.67(×2), 138.31, 134.42, 106.26, 103.33(×2), 94.24, 93.95, 79.59, 77.37, 77.16, 76.95, 72.29, 69.04, 67.82, 61.01,56.41, 56.36(×3), 45.89.

[0057] Example 7: Preparation of compound Y6

[0058]

[0059] The procedure was the same as in Example 6, except that the 2,2'-dibromodiethyl ether in Example 6 was replaced with brominated-polyethylene glycol-brominated monomer compound Y6, with a yield of 53.8%. The structural identification data are as follows: HR-ESI-MS m / z: 592.1838 [M+H] + , (calcd for C 28 H 34 NO 11 S, 592.1847); 1 H NMR (600 MHz, Chloroform-d) δ 6.67(s, 2H), 6.16 (s, 2H), 5.33 (dd, J = 13.3, 2.7 Hz, 1H), 4.16- 4.09 (m, 2H),3.93 (s, 2H), 3.90 (d, J = 4.2 Hz, 9H), 3.86 (s, 3H), 3.83 (t, J = 5.4 Hz,4H), 3.69-3.63 (m, 6H), 3.02 (dd, J = 16.5, 13.4 Hz, 1H), 2.78 (dd, J = 16.5,2.8 Hz, 1H); 13C NMR (150 MHz, Chloroform-d) δ 189.52, 172.04, 171.72, 165.50,165.13, 162.62, 153.87(×2), 138.50, 134.65, 106.39, 103.55(×2), 94.42,94.19, 79.78, 71.17, 70.45, 69.74, 68.11, 67.35, 61.21, 56.61, 56.57(×2),46.09, 41.30, 34.04.

[0060] Example 8: Preparation of compound Y7

[0061]

[0062] The procedure was the same as in Example 6, except that the 2,2'-dibromodiethyl ether in Example 6 was replaced with brominated-tetraethylene glycol-brominated monomer compound Y7, with a yield of 53.1%. The structural identification data are as follows: HR-ESI-MS m / z: 636.2264 [M+H] + , (calcd for C 30 H 38 NO 12 S, 636.2266); 1 H NMR (600 MHz, Chloroform-d) δ 6.67(s, 2H), 6.16 (q, J = 2.2 Hz, 2H), 5.32 (dd, J = 13.3, 2.7 Hz, 1H), 4.18-4.12 (m, 2H), 3.94 (s, 2H), 3.89 (d, J = 2.2 Hz, 9H), 3.86 (s, 5H), 3.82 (t,J = 5.7 Hz, 2H), 3.70 (dd, J = 5.8, 3.4 Hz, 2H), 3.68- 3.64 (m, 5H), 3.60 (s,3H), 3.02 (dd, J = 16.5, 13.4 Hz, 1H), 2.78 (dd, J = 16.5, 2.8 Hz, 1H); 13CNMR (150 MHz, Chloroform-d) δ 189.30, 171.85, 171.53, 165.29, 164.92, 162.40, 153.65(×2), 138.28, 134.44, 106.16, 103.34(×2), 94.21, 93.95, 79.57, 71.00,70.82, 70.75, 70.22, 69.49, 67.90, 67.04, 61.00, 56.39, 56.36(×2), 45.88,41.10, 33.82.

[0063] Example 9: Screening experiment for anti-HBV activity

[0064] 1. Experimental Materials

[0065] (1) HepG2-hNTCP-C4 was derived from the National Institute of Infectious Diseases in Tokyo, Japan.

[0066] (2) Cell proliferation kit II (XTT Assay)

[0067] (3) Test drug: polymethoxyflavonoid derivatives (Y1-Y7) synthesized in this invention.

[0068] (4) Positive control drugs: myrcludex-B and ciglitazone

[0069] 2. Experimental apparatus

[0070] Fluorescence microscope, LD4-2A low-speed centrifuge, Varioskan Flash microplate reader, electric thermostatic water bath, ML104 analytical electronic balance, clean bench, ultra-low temperature freezer, micropipette.

[0071] 3. Experimental Methods

[0072] like Figure 1 As shown, HepG2-hNTCP-C4 cells were first pretreated with the test compound for 2 hours, and then inoculated with HBV for 16 hours in the presence of the test compound. After washing away free HBV and the test compound, the cells were cultured for 12 days in the absence of the test compound, and HBsAg antigen was quantified by enzyme-linked immunosorbent assay (ELISA). Myrcludex-B and ciglitazone were used as positive controls to determine the inhibitory effect of compounds Y1-Y7 on viral production. Simultaneously, cell proliferation and cell viability were measured using the XTT Assay to confirm the cytotoxicity of the tested compounds.

[0073] The results of the tests on the inhibitory activity and cytotoxicity of each compound against HBV are as follows: Figure 2 As shown in the figure. The experimental results show that the designed and synthesized derivatives Y1-Y7 are all non-cytotoxic, among which Y3 exhibits better anti-HBV activity, providing a good foundation for the subsequent development of drugs for the treatment of hepatitis B.

Claims

1. A polymethoxyflavonoid derivative of formula (I) or (II) or a pharmaceutically acceptable salt thereof, characterized in that, The structural formula of the derivative is as follows: Where n is an integer from 1 to 10.

2. The polymethoxyflavonoid derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, Selected from any of the following compounds or their pharmaceutically acceptable salts: 。 3. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the polymethoxyflavonoid derivative of claim 1 or 2 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier; the dosage form of the pharmaceutical composition includes capsules, tablets, solutions, suspensions, granules and injections.

4. A method for preparing the polymethoxyflavonoid derivative of claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, Includes the following steps: Where n is an integer from 1 to 10; Step 1: 2,4,6-Trihydroxyacetophenone reacts selectively with chloromethyl methyl ether to give compound B; Step 2: Compound B reacts with dimethyl sulfate to give compound C; Step 3: Compound C is condensed with 3,4,5-trimethoxybenzaldehyde to obtain compound D; Step 4: Compound D undergoes a deprotection reaction to yield compound E; Step 5: Compound E reacts selectively with chloromethyl methyl ether to give compound F; Step 6: Compound F undergoes cyclization in a sodium acetate-ethanol system to yield compound G; Step 7: Compound G undergoes a deprotection reaction to yield intermediate GTF-1; Step 8: The intermediate GTF-1 reacts with a dibromide or a thiazolidinedione under basic conditions to give the compound shown in general formula (I) or (II).

5. The use of the polymethoxyflavonoid derivative of claim 1 or 2 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 3, in the preparation of an anti-hepatitis B virus infection drug.

6. The use of the polymethoxyflavonoid derivative of claim 1 or 2 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 3 in the preparation of a medicament for the prevention and / or treatment of hepatitis B.

7. The application according to claim 6, characterized in that, The hepatitis B mentioned is severe hepatitis B.

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