Artemisinin biotin marker and preparation method thereof

Abstract

The present invention discloses an artemisinin biotin marker, characterized in that the artemisinin biotin marker has the following structure: #imgabs0# or; #imgabs1# The present invention first reduces the ester carbonyl of artemisinin without separating the product, and adds the biotin fragment in a "one-pot" manner to react with the hemiacetal hydroxyl group to obtain the target molecule. The present invention realizes the synthesis of a class of artemisinin biotin markers (ATT-P1 and ATT-P2) for the first time by chemical methods, providing new ideas and approaches for the synthesis of artemisinin biotin markers and the study of artemisinin targets.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to an artemisinin biotin marker and a preparation method thereof. Background Art

[0002] Artemisinin is an endoperoxide closely related to artemisinin, with a structure very similar to artemisinin. It can be extracted and chemically synthesized from the stems and leaves of the composite inflorescence plant Artemisia annua. Artemisinin has good biological activity, can activate Nrf-2-dependent antioxidant response, treat lung injury induced by chemotherapy drug bleomycin (Faseb Journal, 2016, 30(7): 2500-2510; Biomed Res Int, 2019, 2019: 5198138.), and has a killing effect on a variety of tumor cells (Phytomedicine, 2011, 18(11): 959-969; Phytomedicine, 2015, 22(11): 1045-1054; Journal of Practical Medicine, 2016, 32(9): 1384-387.). Chen et al. also confirmed that artemisinin exhibits broad-spectrum and highly effective anti-tumor effects at both the cell and animal levels, and has no obvious toxic effects on normal tissues (Oncogene. 2018, 37(37): 5079-5087), but the mechanism of action has not yet been elucidated. In addition, Chen et al. also confirmed that artemisinin has the potential to treat rheumatoid arthritis (Clin Transl Med. 2022, 12(12): e1148.), but the target of action is still unclear.

[0003] Targets are the connection points and hubs for the interaction between drug chemical components and human biological systems. Target discovery is of great significance for the optimization of drug chemical structure and the interpretation of effect mechanisms. Today, the confirmation of drug targets is gradually becoming a bottleneck problem that hinders the development of medicinal chemistry. What's more, the mechanism of action of drugs is gradually revealed after they are used in clinical practice. Although artemisinin has good biological activity and broad clinical application prospects, the unclear target protein is one of the most critical bottlenecks affecting the clinical application of artemisinin. Clarifying the target protein of artemisinin will help in the study of artemisinin's structural optimization, structure-activity relationship and molecular action mechanism, laying the foundation for its clinical application. It can also provide a scientific basis for the application of related targets in clinical translational medicine.

[0004] The biotin-avidin system is a signal amplification labeling technology with the advantages of high affinity, high sensitivity, strong specificity and good stability. At present, the most common and effective means to find drug target proteins is to design molecular probes of synthetic natural products, and use the high affinity between biotin and avidin and protein spectrum technology to obtain candidate target proteins. Due to the instability of the artemisinin structure, the synthesis of its biotin label has not been reported in the literature so far. Summary of the invention

[0005] In order to solve the problems of the prior art, the present invention provides an artemisinin biotin marker and a preparation method thereof. The technical solution is as follows:

[0006] In a first aspect, an artemisinin biotin marker is provided, wherein the artemisinin biotin marker has the following structure:

[0007]

[0008] In a second aspect, a method for preparing an artemisinin biotin marker is provided, comprising:

[0009] (1) Compound 1 and D-biotin coenzyme R were dissolved in acetonitrile, and 4-dimethylaminopyridine and dicyclohexylcarbodiimide were added to react. After the reaction was completed, water was added to quench the reaction solution, and the reaction solution was extracted with ethyl acetate. The organic phases were collected and combined, dried, filtered, concentrated, and separated by silica gel column to obtain compound 2;

[0010] (2) Artemisinin was dissolved in methanol, sodium borohydride was added, and after one hour of reaction, a tetrahydrofuran solution of compound 2 was added, followed by potassium hydroxide and 18-crown ether-6, and the reaction was continued. After the reaction was completed, water was added to quench the reaction, and the reaction solution was extracted with ethyl acetate. The organic phases were collected and combined, dried, filtered, concentrated, and separated by silica gel column to obtain compound ATT-P1;

[0011]

[0012] Furthermore, in the step (1), the molar ratio of compound 1: D-biotin coenzyme R: 4-dimethylaminopyridine: dicyclohexylcarbodiimide is 1.5:1.5:0.15:2.2.

[0013] Furthermore, in the step (2), the molar ratio of artemisinin: sodium borohydride: compound 2: potassium hydroxide: 18-crown ether-6 is 1.2:1.2:2:1.2:1.

[0014] Furthermore, the step (1) is reacted at room temperature for 3 hours; and the step (2) is reacted for 12 hours and then quenched with water.

[0015] In a third aspect, a method for preparing an artemisinin biotin marker is provided, comprising:

[0016] Artemisinene was dissolved in methanol, sodium borohydride was added, and after reacting for 1 hour, an acetonitrile solution of compound 4 was added, and potassium carbonate was added to continue the reaction. After the reaction was completed, water was added to quench the reaction, and the reaction solution was extracted with ethyl acetate. The organic phases were collected and combined, dried, filtered, concentrated, and separated by silica gel column to obtain compound ATT-P2;

[0017]

[0018] Furthermore, in terms of molar ratio, artemisinin: sodium borohydride: compound 4: potassium carbonate = 1.2: 1.2: 2: 1.2.

[0019] Furthermore, water was added to quench the reaction after 15 hours.

[0020] The technical solution provided by the embodiment of the present invention has the following beneficial effects: the present invention first reduces the ester carbonyl of artemisinin, does not separate the product, adds the biotin fragment in one pot, and reacts with the hemiacetal hydroxyl group to obtain the target molecule. The present invention realizes the synthesis of a class of artemisinin biotin markers (ATT-P1 and ATT-P2) for the first time by chemical methods. The design idea of ​​such molecules provides new ideas and approaches for the synthesis of artemisinin biotin markers and the study of artemisinin targets. DETAILED DESCRIPTION

[0021] In order to make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be described in further detail below.

[0022] (I) Synthesis of artemisinin biotin marker

[0023] (1) Synthesis of ATT-P1

[0024]

[0025] ① Take compound 11.5mmol and D-biotin coenzyme R (D-biotin) 1.5mmol and dissolve them in 15ml acetonitrile, then add 4-dimethylaminopyridine (DMAP) 0.15mmol and dicyclohexylcarbodiimide (DCC) 2.2mmol; react at room temperature for 3 hours, then add water to quench, and then extract the reaction solution with ethyl acetate (EA), collect and combine the organic phases, dry the organic phases with anhydrous sodium sulfate, filter and concentrate, and separate on a silica gel column to obtain compound 2 (product 70%).

[0026] Compound 2 1H NMR(500MHz,Chloroform-d)δ5.59(d,J=7.8Hz,1H),5.19(d,J=7.5Hz,1H),4.46–4.35(m,2H),4.2 6(dt,J=7.7,3.1Hz,1H),4.05(dt,J=12.4,6.2Hz,1H),3.94–3.81(m,2H),3.72–3.58(m,7H),3.58 –3.49(m,1H),3.09(td,J=4.6,2.8Hz,1H),2.99(dd,J=14.3,3.1Hz,1H),2.81(dd,J=14.3,2.2Hz, 1H),2.41–2.24(m,2H),1.81(dtd,J=13.2,6.5,4.6Hz,1H),1.69–1.49(m,4H),1.47–1.35(m,1H).

[0027] ② Take 1.2 mmol of artemisinene (ATT) and dissolve it in 10 ml of methanol. Add 1.2 mmol of sodium borohydride in batches. After reacting for 1 hour, add 3 ml of tetrahydrofuran solution of compound 2 (content of compound 2 2 mmol), then add 1.2 mmol of potassium hydroxide and 1 mmol of 18-crowen-6, react for 12 hours, then quench with water, extract the reaction solution with ethyl acetate (EA), collect and combine the organic phases, dry the organic phases with anhydrous sodium sulfate, filter and concentrate, and separate on a silica gel column to obtain compound ATT-P1 (product 61%).

[0028] Compound ATT-P1 11H NMR (500 MHz, Chloroform-d) δ 5.66 (d, J = 7.8 Hz, 1H), 5.42 (d, J = 7.5 Hz, 1H), 5.16 (t, J = 1.0 Hz, 1H), 4.98 (dt, J = 2.0, 0.9 Hz, 1H), 4.94–4.89 (m, 2H), 4.43 (dtd, J = 7.9, 3.2, 2.3 Hz, 1H), 4.29–4.21 (m, 3H), 3.79–3.73 (m, 2H), 3.73–3.63 (m, 8H), 3.12 (td, J = 4.6, 2.8 Hz, 1H), 2.98 (dd, J = 14.3, 3.1 Hz, 1H), 2.80 (dd, J = 14.4, 2.3 Hz, 1H), 2.73 (ddt, J = 6.6, 4.4, 1.0 Hz, 1H), 2.32 (d, J = 6.9 Hz, 1H), 2.31–2.22 (m, 2H), 2.08–1.96 (m, 2H), 1.92 (dddd, J = 12.6, 8.1, 5.9, 4.4 Hz, 1H), 1.83–1.64 (m, 5H), 1.64–1.40 (m, 9H), 1.22 (dddd, J = 12.8, 8.2, 5.9, 4.2 Hz, 1H), 0.97 (d, J = 7.5 Hz, 3H).

[0029] 13 13C NMR (125 MHz, Chloroform-d) δ 173.34, 163.71, 144.42, 112.82, 104.58, 98.80, 91.12, 80.67, 70.69, 70.68, 69.29, 68.32, 67.79, 64.30, 63.56, 60.06, 55.77, 49.84, 42.73, 39.01, 36.51, 35.75, 34.43, 31.98, 30.04, 26.06, 25.52, 24.88, 24.03, 22.89, 20.26.

[0030] High resolution mass spectrometry (ESI): M+H: 641.3111.

[0031] ③ Take 1.2 mmol of artemisinene (ATT) and dissolve it in 10 ml of methanol. Add 1.2 mmol of sodium borohydride in batches. After reacting for 1 hour, add 3 ml of tetrahydrofuran solution of compound 2 (content of compound 2 2 mmol), then add 1.2 mmol of potassium carbonate and 1 mmol of 18-crowen-6, react for 12 hours, then quench with water, extract the reaction solution with ethyl acetate (EA), collect and combine the organic phases, dry the organic phases with anhydrous sodium sulfate, filter and concentrate, and separate on a silica gel column to obtain compound ATT-P1 (product 5%).

[0032] ④ Take 1.2 mmol of artemisinene (ATT) and dissolve it in 10 ml of methanol. Add 1.2 mmol of sodium borohydride in batches. After reacting for 1 hour, add 3 ml of tetrahydrofuran solution of compound 2 (compound 2 content 2 mmol), then add 1.2 mmol of cesium carbonate and 1 mmol of 18-crowen-6, react for 12 hours, then quench with water, extract the reaction solution with ethyl acetate (EA), collect and combine the organic phases, dry the organic phases with anhydrous sodium sulfate, filter and concentrate, and separate on a silica gel column to obtain compound ATT-P1 (product 6%).

[0033] (2) Synthesis of ATT-P2

[0034]

[0035] ① Take 1.2 mmol of artemisinene (ATT) and dissolve it in 10 ml of methanol. Add 1.2 mmol of sodium borohydride in batches. After reacting for 1 hour, add 2 ml (1 mmol / ml) of acetonitrile solution of compound 4, and then add 1.2 mmol of potassium carbonate. React for 15 hours, then quench with water, extract the reaction solution with ethyl acetate (EA), collect and combine the organic phases, dry the organic phases with anhydrous sodium sulfate, filter and concentrate, and separate on a silica gel column to obtain compound ATT-P2 (yield 56%).

[0036] Compound ATT-P2 1H NMR(500MHz,Chloroform-d)δ5.76(d,J=7.9Hz,1H),5.41(d,J=7.5Hz,1H),5.13–5.04(m,2H),4.99(dt,J=2.0,0.9Hz,1H),4.91(s,1H),4.43(dtd,J =7.9,3.2,2.3Hz,1H),4.29–4.21(m,3H),4.15(dt,J=7.9,3.8Hz,1H),3.7 3–3.67(m,3H),3.67–3.59(m,3H),3.54(td,J=5.3,0.8Hz,2H),3.09(td,J =4.6,2.7Hz,1H),2.98(dd,J=14.3,3.1Hz,1H),2.91(td,J=5.3,3.8Hz,2H ),2.80(dd,J=14.4,2.3Hz,1H),2.64(ddt,J=6.6,4.4,1.1Hz,1H),2.35–2 .22(m,3H),2.09–1.96(m,2H),1.92–1.63(m,6H),1.63–1.54(m,2H),1.55 –1.51(m,1H),1.51–1.38(m,6H),1.27–1.17(m,1H),0.97(d,J=7.5Hz,3H).

[0037] 13 C NMR(125MHz,Chloroform-d)δ173.34,163.71,143.67,113.84,104.70,90.92,83.67,81.01,70.50,69.72,69.37,68.32,64.30, 63.56,60.06,55.77,49.85,45.12,41.57,39.01,36.51,35.75,34.43,31.90,30.04,26.06,25.52,24.88,24.54,24.03,20.26.

[0038] High resolution mass spectrometry (ESI): M+H: 640.3261.

[0039] ② Take 1.2 mmol of artemisinene (ATT) and dissolve it in 10 ml of methanol. Add 1.2 mmol of sodium borohydride in batches. After reacting for 1 hour, add 2 ml of acetonitrile solution (1 mmol / ml) of compound 4, and then add 1.2 mmol of cesium carbonate. React for 15 hours, then quench with water, extract the reaction solution with ethyl acetate (EA), collect and combine the organic phases, dry the organic phases with anhydrous sodium sulfate, filter and concentrate, and separate on a silica gel column to obtain compound ATT-P2 (yield 8%).

[0040] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An artemisinin biotin marker, characterized in that: The artemisinin biotin marker has the following structure:

2. A method for preparing an artemisinin biotin marker, characterized in that: include: (1) Compound 1 and D-biotin coenzyme R were dissolved in acetonitrile, and 4-dimethylaminopyridine and dicyclohexylcarbodiimide were added to react. After the reaction was completed, water was added to quench the reaction solution, and the reaction solution was extracted with ethyl acetate. The organic phases were collected and combined, dried, filtered, concentrated, and separated by silica gel column to obtain compound 2; (2) Artemisinin was dissolved in methanol, sodium borohydride was added, and after one hour of reaction, a tetrahydrofuran solution of compound 2 was added, followed by potassium hydroxide and 18-crown ether-6, and the reaction was continued. After the reaction was completed, water was added to quench the reaction, and the reaction solution was extracted with ethyl acetate. The organic phases were collected and combined, dried, filtered, concentrated, and separated by silica gel column to obtain compound ATT-P1; 3. The preparation method according to claim 2, characterized in that: In the step (1), the molar ratio of compound 1: D-biotin coenzyme R: 4-dimethylaminopyridine: dicyclohexylcarbodiimide is 1.5:1.5:0.15:2.

2.

4. The preparation method according to claim 2, characterized in that: In the step (2), the molar ratio of artemisinin: sodium borohydride: compound 2: potassium hydroxide: 18-crown ether-6 is 1.2:1.2:2:1.2:

1.

5. The preparation method according to claim 2, characterized in that: The step (1) is reacted at room temperature for 3 hours; the step (2) is reacted for 12 hours and then quenched with water.

6. A method for preparing an artemisinin biotin marker, characterized in that: include: Artemisinene was dissolved in methanol, sodium borohydride was added, and after reacting for 1 hour, an acetonitrile solution of compound 4 was added, and potassium carbonate was added to continue the reaction. After the reaction was completed, water was added to quench the reaction, and the reaction solution was extracted with ethyl acetate. The organic phases were collected and combined, dried, filtered, concentrated, and separated by silica gel column to obtain compound ATT-P2; 7. The preparation method according to claim 6, characterized in that: In terms of molar ratio, artemisinin: sodium borohydride: compound 4: potassium carbonate = 1.2: 1.2: 2: 1.

2.

8. The preparation method according to claim 6, characterized in that: After the reaction was continued for 15 hours, water was added to quench the reaction.

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