Preparation method and use of a new quebrachamine boronic acid compound

Abstract

This invention relates to a novel *Bletilla striata* alkaloid boric acid compound, its preparation method, and its use in the preparation of antitumor drugs. The general chemical formula of this class of compounds is shown in structural formulas (I) and (II). In vitro antitumor activity screening results showed that compounds of formulas I and II possess broad-spectrum antitumor activity, exhibiting strong inhibitory activity against human liver cancer (HepG2), human pancreatic cancer (SW1990), human ovarian cancer (A2780), human breast cancer (MCF7), and human colon cancer (SW480) cell lines. Compounds N-3 and N-4 showed strong inhibitory effects against all five tested tumor cell lines, with IC50 values ​​exceeding 100%. 50 The values ​​were 0.26–0.89 μM and 0.22–0.91 μM, respectively; compounds N-1, N-3, and N-4 exhibited strong inhibitory effects on the human pancreatic cancer (SW1990) cell line, with IC50 values ​​of 0.26–0.89 μM and 0.22–0.91 μM, respectively. 50 The concentrations were 0.97, 0.45, and 0.63 μM, respectively, significantly superior to the control drug topotecan; simultaneously, compounds N-3 and N-4 also exhibited strong inhibitory effects on the human breast cancer (MCF7) cell line, with IC50 values ​​of 0.97, 0.45, and 0.63 μM. 50 The concentrations were 0.80 and 0.65 μM, respectively, significantly better than the control drug topotecan. Therefore, the new cypermethrin borate compound holds promise for development into a novel antitumor drug.

Description

Technical Field

[0001] This invention relates to a novel berberine-boronic acid compound, a method for preparing such a compound, and its use in antitumor applications. It belongs to the pharmaceutical field. Background Technology

[0002] Naturally derived alkaloids, as an important class of nitrogen-containing organic compounds in the biological world, possess biological activities such as antitumor, antibacterial and anti-inflammatory, antiviral, antiarrhythmic, analgesic, and antispasmodic effects. Therefore, using naturally derived alkaloids as a starting point and further modifying or altering their structure based on their structure-activity relationships is one of the important approaches to new drug development. Neo-whiteleaf alkaloid is an alkaloid isolated from the African medicinal plant *Hylocereus undatus*, possessing antimalarial, antibacterial, and antitumor activities. As an antitumor compound, neo-whiteleaf alkaloid has a narrow antitumor spectrum and relatively weak activity, requiring further structural optimization or modification to enhance its antitumor activity and broaden its antitumor spectrum. Boronic acid groups, due to their unique chemical properties, have wide applications in new drug design, chemistry, materials science, and energy research. Since 2003, the FDA has approved five boron-containing drugs, including Bortezomib. Dipeptide boric acid, a class 1 proteasome inhibitor used to treat multiple myeloma, is the first boron-containing drug on the market; Ixazomib (ethazomib) As a second-generation proteasome inhibitor, it is a dipeptidyl leucine borate that can reversibly bind to the CT-L proteolytic (β5) site of the 20S proteasome and is the first oral drug for the treatment of multiple myeloma.

[0003] Therefore, we used the natural alkaloid *Neoprothiolane* as a lead structure and introduced a boronic acid group to obtain a lead compound with high antitumor activity. In this invention, a boronic acid group was introduced at the 9-position of *Neoprothiolane*, and a series of *Neoprothiolane* boronic acid derivatives with different substitutions were designed and synthesized. Their inhibitory activity against various tumor cells was then determined. Some of these compounds exhibited good antitumor activity, and their inhibitory activity against the growth of some tumor cells was superior to that of the clinical drug topotecan, suggesting their potential for development as novel antitumor drugs. Summary of the Invention

[0004] This invention provides a novel berberine compound, along with a method for preparing this novel compound and its application in antitumor therapy.

[0005] The novel white vine alkaloid boric acid compound described in this invention refers to two types of compounds having the following general formulas (I) and (II):

[0006]

[0007] In formulas (I) and (II), R1 can be hydrogen, methyl, methoxy, fluorine, or chlorine; R2 can be hydrogen, methyl, methoxy, fluorine, or chlorine; R3 can be hydrogen, methyl, methoxy, fluorine, or chlorine; and R4 can be hydrogen, methyl, methoxy, fluorine, or chlorine.

[0008] The method for preparing a novel cypermethrin-boronic acid compound according to the present invention is carried out according to the following chemical formula 1:

[0009] Starting with quinolines with different substitutions, quinoline intermediates A1-A4 were generated by reacting with iodomethane at 90°C. These intermediates were then reacted with potassium hydroxide and hydrogen peroxide solution at room temperature for 48 hours to induce ring-opening, yielding intermediates B1-B4. Following this, intermediates C1-C4 were reacted with pinacol diboronic acid ester in a 1,4-dioxane solvent catalyzed by a palladium catalyst to obtain the target products N-1, N-3, N-5, and N-7. Finally, the products N-1, N-3, N-5, and N-7 were hydrolyzed with sodium periodate and 1M hydrochloric acid in a THF / H2O (4:1) solvent to obtain the target products N-2, N-4, N-6, and N-8.

[0010] This invention discloses a novel *Bletilla striata* boronic acid compound that can play a role in the preparation of antitumor drugs, more specifically, in the preparation of drugs for treating human liver cancer, pancreatic cancer, ovarian cancer, breast cancer, and colon cancer. In vitro antitumor activity screening results show that the novel *Bletilla striata* boronic acid compound has broad-spectrum antitumor activity, exhibiting strong inhibitory activity against human liver cancer (HepG2), human pancreatic cancer (SW1990), human ovarian cancer (A2780), human breast cancer (MCF7), and human colon cancer (SW480) cell lines. Compounds N-3 and N-4 showed strong inhibitory effects on all five tested tumor cell lines, with IC50 values ​​exceeding 100%. 50 The values ​​were 0.26-0.89 μM and 0.22-0.91 μM, respectively; compounds N-1, N-3, and N-4 exhibited strong inhibitory effects on the human pancreatic cancer (SW1990) cell line, with IC50 values ​​of 0.26-0.89 μM and 0.22-0.91 μM, respectively. 50 The concentrations were 0.97, 0.45, and 0.63 μM, respectively, significantly superior to the control drug topotecan; simultaneously, compounds N-3 and N-4 also exhibited strong inhibitory effects on the human breast cancer (MCF7) cell line, with IC50 values ​​of 0.97, 0.45, and 0.63 μM. 50 The concentrations were 0.80 and 0.65 μM, respectively, which were significantly better than the control drug topotecan.

[0011] Therefore, the novel cyperine boric acid compounds described in this invention can be used to prepare antitumor drugs. They have a novel structure, inexpensive and readily available raw materials, and high product purity. They also exhibit strong inhibitory effects on the proliferation of various tumor cell lines and have excellent application prospects.

[0012] The following detailed description of specific embodiments further illustrates the above-mentioned aspects of the present invention. However, this should not be construed as a limitation of the present invention. Detailed Implementation

[0013] Example 1: Synthesis of target compound N-1

[0014]

[0015] The synthesis of compound N-1 described in this invention is carried out according to chemical formula 2:

[0016]

[0017] Synthesis of intermediate A1: Under nitrogen protection, quinoline (0.1 mol) and iodomethane (0.15 mol) were mixed in an appropriate amount of isopropanol and heated under reflux at 90 °C for 3 hours. After the reaction was cooled to room temperature, the precipitate was separated by vacuum filtration, washed with a mixture of isopropanol / ethyl acetate (1 / 1), and then dried to obtain intermediate A1.

[0018] Synthesis of intermediate B1: First, an aqueous solution of potassium hydroxide (30 mL) and a solution of 1,2-dichloroethane (30 mL) were mixed. Then, hydrogen peroxide (30%) and 1-methylquinoline iodide (intermediate A1, 15 mmol of which was dissolved in 15 mL of water) were slowly added to the mixture at 0 °C. The resulting mixture was stirred at room temperature for 48 hours. The organic layer was then separated, and the aqueous layer was extracted multiple times with dichloromethane (30 mL). The combined organic layers were dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. The resulting mixture was purified by silica gel column chromatography to obtain intermediate B1.

[0019] Synthesis of intermediate C1: Intermediate B1 (5 mmol), 5-Br indole (5 mmol), and p-toluenesulfonic acid (p-TSA, 5 mmol) were added to anhydrous ethanol (10 mL), and the mixture was stirred and refluxed at 85 °C for 24 hours in a 50 mL round-bottom flask. After cooling to room temperature, the reaction mixture was washed with 1 M sodium hydroxide (50 mL), and the aqueous layer was extracted multiple times with dichloromethane (80 mL). The combined organic phases were then dried over anhydrous sodium sulfate, and the organic layer was concentrated under reduced pressure. The resulting mixture was purified by silica gel column chromatography. Impurities were first removed with a large amount of petroleum ether / ethyl acetate (2 / 1) mixed solvent, followed by column chromatography with a dichloromethane / methanol (50 / 1) mixed eluent to obtain intermediate C1.

[0020] Synthesis of N-1: Under nitrogen protection, intermediate C1 (0.1 mol), pinacol diborate (0.11 mol), potassium acetate (0.3 mol), and Pd(dppf)Cl2 (0.01 mol) were added to a dry solvent of 1,4-dioxane. The reaction mixture was stirred at 110 °C for 12 hours. After the reaction was complete, the solvent was removed under vacuum, and the residues were extracted with dichloromethane and saturated sodium chloride solution (15 mL × 3), respectively. The combined organic extracts were dried with anhydrous sodium sulfate, and the dried organic layer was concentrated under reduced pressure. The resulting mixture was purified by silica gel column chromatography to obtain product N-1. The detection data of the product obtained from the reaction are as follows: Yield: 56%; yellow solid; 1 H NMR(400MHz,Chloroform-d)δ:8.52(d,J=7.8Hz,2H),8.01(d,J=8.0Hz,1H),7.95(d ,J=7.9Hz,1H),7.78–7.69(m,3H),7.44(t,J=6.6Hz,1H),4.35(s,3H),1.40(s,12H). 13 C NMR(100MHz,Chloroform-d)δ:158.01,157.02,136.95,136.11,130.53,130.13,128.39,12 8.19,128.06,123.75,122.26,121.21,117.17,114.35,83.70,33.25,29.83,25.09.MS-ESI m / z:calcd for C 22 H 23 BN2O3[M+H] + :359.2480; found:359.2477.

[0021] Example 2: Synthesis of target compound N-2

[0022]

[0023] Synthesis of N-2: N-1 (0.1 mol) was dissolved in a mixed solvent of THF / H2O (4:1 10 ml). A suitable amount of NaIO4 (0.5 mol) was added to the resulting solution, followed by a suitable amount of 1.0 M HCl. The reaction mixture was then stirred at room temperature for 24 h. After the reaction was complete, the solution was concentrated under reduced pressure to remove THF, then washed with H2O and dichloromethane, and finally filtered under reduced pressure and dried to obtain the crude product. Product N-2 was obtained by column chromatography purification. The detection data of the product obtained from the reaction are as follows: Yield: 65%; yellow solid; 1HNMR(400MHz,DMSO-d6)δ:9.05(s,1H),8.62(s,1H),8.24(d,J=8.0Hz,1H),8 .08(d,J=8.6Hz,1H),8.00–7.96(m,2H),7.58(t,J=8.1Hz,2H),4.37(s,3H). 13 C NMR(100MHz,DMSO-d6)δ:159.34,158.09,136.21,135.52,131.90,131.70,130.4 3,130.27,128.04,124.74,123.84,121.73,115.90,114.21,88.45,24.80.MS-ESI m / z:calcd for C 16 H 13 BN2O2[M+H] + :277.1020; found:277.1405.

[0024] Example 3: Synthesis of target compound N-3

[0025]

[0026] Same as Example 1, except that 6-methylquinoline was used instead of quinoline. The reaction product was analyzed as follows: Yield: 43%; yellow solid; 1 H NMR(400MHz,Chloroform-d)δ:8.52(s,1H),8.47(s,1H),8.00(dd,J=8.1,1.3Hz,1H),7.72(d,J=7.8 Hz,2H),7.64(d,J=8.7Hz,1H),7.57(dd,J=8.7,2.0Hz,1H),4.35(s,3H),2.54(s,3H),1.40(s,12H). 13 C NMR(100MHz,Chloroform-d)δ:157.96,156.89,136.02,135.17,132.14,131.99,129.61,128. 22,128.13,127.97,123.75,121.29,117.08,114.24,83.70,33.30,25.10,24.79,20.9.MS-ESI m / z:calcd for C 23 H 25 BN2O2[M+H] + 373.2750; found: 373.1868.

[0027] Example 4: Synthesis of target compound N-4

[0028]

[0029] Same as Example 2, except that compound N-3 was used instead of N-1. The reaction product detection data are as follows: Yield: 65%; Red solid; 1 H NMR(400MHz,DMSO-d6)δ:8.81(s,1H),8.58(s,1H),7.97–7.92(m,1H),7.92– 7.85(m,2H),7.66(dd,J=8.8,2.1Hz,1H),7.51(d,J=7.9Hz,1H),4.29(s,3H). 13 C NMR(100MHz,DMSO-d6)δ:157.04,155.87,134.95,134.73,132.01,131.07,129.2 0,128.12,127.65,126.95,123.27,120.46,116.04,114.83,32.76,20.30.MS-ESI m / z:calcd forC 17 H 15 BN2O2[M+H] + :291.1290; found:291.1164.

[0030] Example 5: Synthesis of target compound N-5

[0031]

[0032] Same as Example 1, except that 6-chloroquinoline was used instead of quinoline. The reaction product was analyzed as follows: Yield: 76%; yellow solid; 1 H NMR(400MHz,Chloroform-d)δ:8.50(s,1H),8.39(s,1H),8.04–7.99(m,1H),7.91(d ,J=2.0Hz,1H),7.70(d,J=8.1Hz,1H),7.67–7.64(m,2H),4.33(s,3H),1.40(s,12H). 13C NMR(100MHz,Chloroform-d)δ:158.39,156.88,136.62,135.36,130.46,129.32,128.78,12 8.43,127.63,126.87,123.59,122.06,117.39,115.76,83.79,53.56,33.36,25.10.MS-ESI m / z:calcd for C 22 H 22 BClN2O2[M+H] + 393.6900; found: 393.1331.

[0033] Example 6: Synthesis of target compound N-6

[0034]

[0035] Same as Example 2, except that compound N-5 was used instead of N-1. The reaction product detection data are as follows: Yield: 67%; Red solid; 1 H NMR(400MHz, DMSO-d6)δ:8.88–8.84(m,1H),8.56(d,J=12.3Hz,1H),8.31–8.24(m,1H),7.97– 7.93(m,2H),7.84(dd,J=9.2,2.5Hz,1H),7.53(dd,J=8.1,2.0Hz,1H),4.30(d,J=2.0Hz,3H). 13 C NMR(100MHz,DMSO-d6)δ:157.24,155.82,135.51,135.15,130.06,128.42,128.1 4,127.83,127.17,125.90,123.11,121.52,117.05,116.33,48.57,32.99.MS-ESI m / z:calcd for C 16 H 12 BClN2O2[M+H] + :311.5440; found:311.0599.

[0036] Example 7: Synthesis of target compound N-7

[0037]

[0038] Same as Example 1, except that 6-fluoroquinoline was used instead of quinoline. The product analysis results are as follows: Yield: 68%; Yellow solid; 1H NMR(400MHz,Chloroform-d)δ:8.50(s,1H),8.43(s,1H),8.05–7.98(m,1H),7.70( dd,J=8.6,3.0Hz,2H),7.62(dd,1H),7.53–7.47(m,1H),4.35(s,3H),1.40(s,12H). 13 C NMR (100MHz, Chloroform-d) δ: 157.75 (d, J = 242Hz), 157.71 (d, J = 151Hz), 136.58, 133.55, 129.41, 128.44, 127.17 (d, J = 4H z),123.38,121.88(d,J=9Hz),118.85,118.61,117.25,115.96(d,J=9Hz),114.31(d,J=22Hz),83.77,33.49,25.10.MS-ESI m / z:calcdfor C 22 H 22 BFN2O2[M+H] + :377.2384; found:377.1405.

[0039] Example 8: Synthesis of target compound N-8

[0040]

[0041] Same as Example 2, except that compound N-7 was used instead of N-1. The reaction product detection data are as follows: Yield: 78%; Red solid; 1 H NMR(400MHz,DMSO-d6)δ:8.85(d,J=9.0Hz,1H),8.55(d,J=16.2Hz,1H),8.04–7.97 (m,1H),7.95(d,J=6.4Hz,2H),7.77–7.68(m,1H),7.55–7.50(m,1H),4.30(s,3H). 13 C NMR (100MHz, DMSO-d6) δ: 158.04, 155.76 (d, J = 20Hz), 135.48, 135.22, 133.28 (d, J = 2Hz), 127.84, 127.42 (d, J = 4Hz), 122.89 (d, J = 2 Hz),121.18(d,J=9Hz),118.70,118.44,117.12(d,J=7Hz),116.16(d,J=2Hz),113.93(d,J=22Hz),48.58,33.09.MS-ESIm / z:calcd for C16 H 12 BFN2O2[M+H] + :295.0924; found:295.0893.

[0042] Example 9: Test methods and results of the antitumor activity of compounds N-1 to N-8

[0043] In vitro antitumor assays were performed using the standard MTT assay. Topotecan was used as a positive control to test the antiproliferative activity of target compounds N-1 to N-8 against human hepatocellular carcinoma (HepG2), human pancreatic cancer (SW1990), human ovarian cancer (A2780), human breast cancer (MCF7), and human colon cancer (SW480) cell lines. The compounds were dissolved in DMSO to prepare a 20 mM stock solution, which was then diluted to appropriate concentrations with different culture media. The DMSO concentration in the diluent should be less than 0.01% (v / v) to reduce DMSO toxicity to cells and minimize testing errors. Tumor cells from different cell lines were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS). Log-growing cancer cells were collected, digested with trypsin / EDTA digestion solution, and prepared into appropriate cell suspensions. 100 μL of the cell suspension was added to 96-well plates (generally 5000 cells per well) and incubated at 37°C with 5% CO2 for 24 h. Then, solutions of different concentrations of the test compound were added, and after culturing for 72 h, the old culture medium was discarded, and the cells were washed twice with PBS. 20 μL of fresh MTT (5 mg / mL) was added, and the cells were cultured for another 2 h. Afterward, the culture medium was discarded, and 200 μL of LDMSO was added. The cells were shaken on a shaker for 10 min until the formazan was completely dissolved. Finally, the absorbance at 492 nm was measured using a microplate reader, and the IC50 was calculated. 50 Values. All experiments were conducted in triplicate or in triplicate. The results of the antitumor activity tests for compounds N-1 to N-8 are shown in Table 1.

[0044] Table 1. In vitro antitumor activity of compounds N-1 to N-8

[0045]

[0046] Note: (1) Screening method: standard MTT colorimetric method; (2) Action time: 72 hours; (3) Compound numbers N-1 to N-8 are the products obtained in Examples 1-8 above.

[0047] In vitro antitumor activity screening results showed that the boronic acid compounds of *Neopyrhododendron* possess broad-spectrum antitumor activity, exhibiting strong inhibitory activity against human liver cancer (HepG2), human pancreatic cancer (SW1990), human ovarian cancer (A2780), human breast cancer (MCF7), and human colon cancer (SW480) cell lines. Among them, compounds N-3 and N-4 showed strong inhibitory effects against all five tested tumor cell lines, with IC50 values ​​exceeding 100%. 50 The values ​​were 0.26-0.89 μM and 0.22-0.91 μM, respectively; compounds N-1, N-3, and N-4 exhibited strong inhibitory effects on the human pancreatic cancer (SW1990) cell line, with IC50 values ​​of 0.26-0.89 μM and 0.22-0.91 μM, respectively. 50 The concentrations were 0.97, 0.45, and 0.63 μM, respectively, significantly superior to the control drug topotecan; simultaneously, compounds N-3 and N-4 also exhibited strong inhibitory effects on the human breast cancer (MCF7) cell line, with IC50 values ​​of 0.97, 0.45, and 0.63 μM. 50 The concentrations were 0.80 and 0.65 μM, respectively, significantly better than the control drug topotecan. Therefore, the new cypermethrin borate compound holds promise for development into a novel antitumor drug.

Claims

1. A novel *Gynostemma pentaphyllum* alkaloid borate compound, as shown in formulas I and II. In formulas I and II, R1 can be hydrogen, methyl, methoxy, fluorine, or chlorine; R2 can be hydrogen, methyl, methoxy, fluorine, or chlorine; R3 can be hydrogen, methyl, methoxy, fluorine, or chlorine; and R4 can be hydrogen, methyl, methoxy, fluorine, or chlorine.

2. The synthetic route for the new white-leaved vine alkaloid boric acid compound according to claim 1, characterized in that, Includes the following steps: Synthesis of intermediates A1-A4: Under nitrogen protection, quinoline and iodomethane were mixed in an appropriate amount of isopropanol and heated under reflux at 90°C for 3 hours. After the reaction cooled to room temperature, the precipitate was obtained by vacuum filtration, washed with a mixture of isopropanol and ethyl acetate, and then the washed solid was dried to obtain intermediates A1-A4. Synthesis of intermediates B1-B4: First, an aqueous solution of potassium hydroxide and a solution of 1,2-dichloroethane were mixed. Then, hydrogen peroxide and 1-methylquinoline iodide intermediates A1 to A4 were slowly added to the mixture at 0°C. The resulting mixture was then stirred at room temperature for 48 hours. The organic layer was then separated, and the aqueous layer was extracted multiple times with dichloromethane. The combined organic layers were then dried with anhydrous sodium sulfate, and the organic layers were concentrated under reduced pressure. The resulting mixture was purified by silica gel column chromatography to obtain intermediates B1 to B4. Synthesis of intermediates C1 to C4: Intermediates B1–B4, 5-bromoindole, and p-toluenesulfonic acid were added to anhydrous ethanol, and the mixture was stirred and refluxed at 85°C for 24 hours in a 50 mL round-bottom flask. After cooling to room temperature, the reaction mixture was washed with 1 M sodium hydroxide, and the aqueous layer was extracted multiple times with dichloromethane. The combined organic phases were then dried with anhydrous sodium sulfate, and the organic layer was concentrated under reduced pressure. The resulting mixture was purified by silica gel column chromatography. First, impurities were removed with a large amount of petroleum ether / ethyl acetate mixed solvent, and then column chromatography was performed with a dichloromethane / methanol mixed eluent to obtain intermediates C1–C4. The synthesis of N-1, N-3, N-5, and N-7: Under argon protection, intermediates C1-C4, pinacol diborate, potassium acetate, and Pd(dppf)Cl2 were added to a dry solvent of 1,4-dioxane. The reaction mixture was stirred at 110°C for 12 hours. After the reaction was completed, the solvent was removed under vacuum, and the residues were extracted with dichloromethane and saturated sodium chloride solution, respectively. The combined organic extracts were dried with anhydrous sodium sulfate, and the dried organic layer was concentrated under reduced pressure. The resulting mixture was purified by silica gel column chromatography to obtain products N-1, N-3, N-5, and N-7. Synthesis of N-2, N-4, N-6, and N-8: N-1, N-3, N-5, and N-7 were dissolved in a mixed solvent of THF / H2O; an appropriate amount of NaIO4 was added to the resulting solution, followed by an appropriate amount of 1.0M HCl, and the reaction mixture was stirred at room temperature for 24 hours; after the reaction was completed, THF was removed by concentration under reduced pressure, followed by washing with H2O and dichloromethane, and then vacuum filtration and drying to obtain the crude product; the products N-2, N-4, N-6, and N-8 were obtained by column chromatography.

3. The application of the new white cyperine boric acid compound according to claim 1 in the preparation of a drug for treating human liver cancer, wherein the liver cancer cells are HepG2.

4. The use of the new white cyperine boric acid compound according to claim 1 in the preparation of a drug for treating human pancreatic cancer, wherein the pancreatic cancer cells are SW1990.

5. The use of the new white cyperine boric acid compound according to claim 1 in the preparation of a drug for treating human ovarian cancer, wherein the ovarian cancer cells are A2780.

6. The use of the new white cyperine boric acid compound according to claim 1 in the preparation of a medicament for treating human breast cancer, wherein the breast cancer cells are MCF7.

7. The use of the new white cyperine boric acid compound according to claim 1 in the preparation of a drug for treating human colon cancer, wherein the colon cancer cells are SW480.

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