Novel artesunate-amino acid-targeting group derivative, NANO formulation and use

Abstract

Disclosed are a novel artesunate-amino acid-targeting group derivative, a nano formulation and a use. The structure of the compound is represented by formula (I), wherein R is as described in the claims and the description. Further disclosed is a nano formulation prepared by means of nanoprecipitation. The target compound has anti-tumor cell proliferation activity superior to that of artesunate, dihydroartemisinin and oxaliplatin, has good therapeutic effects against tumors, and can be used for the preparation of anti-tumor drugs.

Description

Novel Artesunate-Amino Acid-Targeting Group Derivatives and Nanoformulations and Applications Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically involving novel artesunate-amino acid-targeting group derivatives and nano-formulations and their applications. Background Technology

[0002] Artesunate (ART) is a succinate monoester derivative of the natural product artemisinin. It is a potent inducer of ferroptosis, and its peroxide bridges can bind to intracellular Fe. 2+ The Fenton reaction occurs, generating a large amount of reactive oxygen species (ROS), which promote ferroptosis in tumor cells. ART can also effectively stimulate the tumor immune response and inhibit the proliferation, invasion, and metastasis of cancer cells. ART not only shows good activity against various solid tumors but also exhibits low cytotoxicity to normal cells. Although it shows many advantages in anti-tumor activity, its potential targets and mechanisms of action remain unclear. The in vivo activity of ART... 1 / 2 Its short cell length, environmental sensitivity, and tendency to convert into dihydroartemisinin (DHA) and subsequently degrade into peroxide-free products are all challenges. Furthermore, ART monotherapy exhibits weak activity, and high doses carry risks of embryotoxicity, neurotoxicity, and immunotoxicity. Therefore, researchers are exploring various methods to develop multiple ART derivatives to enhance its activity, tumor targeting, and reduce its toxicity. Summary of the Invention

[0003] The purpose of this invention is to provide artesunate-amino acid-targeting group derivatives or pharmaceutically acceptable salts thereof, as shown in formula I.

[0004] Another object of the present invention is to provide a method for preparing the artesunate-amino acid-targeting group derivative or a pharmaceutically acceptable salt thereof.

[0005] Another object of the present invention is to provide the use of artesunate-amino acid-targeting group derivatives or pharmaceutically acceptable salts thereof.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] An artesunate-amino acid-targeting group derivative or a pharmaceutically acceptable salt thereof, as shown in formula I:

[0008] Wherein, R is selected from, but not limited to: -H, -CH3, -CH2CF3, -CH2CH(CH3)2, -CHCH3CH2CH3, -CHCH3CH2CF3, -CH(CH3)2, -(CH2)2SCH3, -CH2SH, -CH2OH, -CHCH3OH, -(CH2)2CONH2, -(CH2)2COOH, -CH2CONH2, -CH2COOH, -(CH2)4NH2, -(CH2)3NHCNHNH2,

[0009] The amino acid configuration is selected from: D-type or L-type;

[0010] The targeting groups are selected from, but are not limited to, phenylboronic acid (PBS), folic acid, hyaluronic acid, triphenylphosphine, biotin, transferrin, etc.

[0011] The preferred form of R in this invention is -CH3, -CH2CF3, -(CH2)2SCH3, -CH2SH, -(CH2)2CONH2, -(CH2)2COOH, -CH2CONH2, -CH2COOH.

[0012] The preferred configuration of the amino acid is L-type;

[0013] The target group is preferably phenylboronic acid (PBS), folic acid, or hyaluronic acid.

[0014] The R in this invention is more preferably: -CH3;

[0015] The targeting group is more preferably phenylboronic acid (PBS).

[0016] The preferred artesunate-amino acid-targeting group derivatives or pharmaceutically acceptable salts thereof of the present invention include, but are not limited to:

[0017] The following synthetic route is the synthetic route for the compound of general formula (I) of this invention. Artesunate reacts with an amino acid such as alanine (Ala) to form an amide under condensation conditions; the resulting product then reacts with a targeting group to form an amide or ester under condensation conditions. The synthetic methods described in this route are all well known to those skilled in the art of organic chemistry. The synthetic method of this invention is simple, and the prepared compound exhibits good antitumor activity.

[0018] The artesunate-amino acid-targeting group derivative and its pharmaceutically acceptable salt nanoformulations of this invention are prepared by a nanoprecipitation method: the artesunate-amino acid-targeting group derivative and its pharmaceutically acceptable salt are dissolved in an organic solvent such as ethyl acetate, acetonitrile, and acetone, and then added dropwise to water containing a surfactant or emulsifier. The nanoformulations are then prepared by nanoprecipitation. The surfactant is a hydrophilic or lipophilic surfactant, selected from any one or more of polyvinyl alcohol, polysorbate 80, polysorbate 20, sorbitan oleate, and dextran.

[0019] This invention includes pharmaceutical compositions containing an artesunate-amino acid-targeting group derivative of general formula (I) and its pharmaceutically acceptable salt, as the main active ingredient, prepared by mixing with a pharmaceutically acceptable excipient. Examples include tablets, capsules, suspensions, or injections. Pharmaceutically acceptable excipients refer to pharmaceutically acceptable drug carriers, adjuvants, or diluents.

[0020] The artesunate-amino acid-targeting group derivatives and their pharmaceutically acceptable salts involved in this invention can be used alone or in combination with clinical antitumor drugs for the treatment of tumors.

[0021] The tumors mentioned are preferably solid tumors and hematologic malignancies that highly express sialic acid molecules.

[0022] Furthermore, the tumors mentioned may be, but are not limited to, solid tumors and hematological malignancies that highly express sialic acid molecules, such as multiple myeloma, liver cancer, colorectal cancer, lung cancer, stomach cancer, ovarian cancer, and breast cancer.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] 1. This invention provides a novel artesunate-amino acid-targeting group derivative with a targeting group. These compounds possess antitumor activity and can be used to prepare antitumor drugs. In vitro antitumor cell proliferation activity test data show that these compounds have varying degrees of inhibitory effects on CT26 cells (mouse colon cancer cells), HCT116 cells (human colon cancer cells), and HuH7 cells (human liver cancer cells). The antiproliferative activity of some compounds is superior to that of ART, DHA, and oxaliplatin.

[0025] 2. The compounds with antitumor activity provided by this invention are synthesized from inexpensive and readily available raw materials, and the synthesis process is simple and easy to implement, making them suitable for large-scale production. Furthermore, the resulting compounds exhibit superior structural stability compared to ART, facilitating transportation and storage. Attached Figure Description

[0026] Figure 1. In vitro antitumor cell proliferation activity of compound ART-Ala-3-PBS. (A) CT26 cells; (B) SW480 cells; (C) HCT116 cells; (D) HuH7 cells;

[0027] Figure 2. Particle size distribution of the ART-Ala-3-PBS nanoformation and the Tyndall effect of the nanoformation.

[0028] Figure 3. Changes in tumor volume and body weight in tumor-bearing mice.

[0029] Figure 4. Detection of serum biochemical indicators in tumor-bearing mice Detailed Implementation

[0030] The present invention will now be described in detail with reference to the embodiments and accompanying drawings. The following embodiments are merely further illustrations of the present invention and do not limit the scope of application of the present invention in any way.

[0031] Unless otherwise specified, all reagents used in the synthesis experiments were commercially available analytical grade reagents and were used directly without purification. The silica gel used in the experiments was column chromatography silica gel (H, 200-300 mesh) from Qingdao Ocean Chemical Co., Ltd. 1 ¹H NMR spectra were determined using a Bruker AVIII-HD-400 NMR analyzer. TMS was used as an internal standard. Peak shapes are represented as s (singleton), d (doublet), t (triplet), q (quartet), dd (doublet), dt (doubletuplet), and m (multiplet). Mass spectrometry was performed using an Agilent 1100 series SL mass spectrometer with an electrospray ionization (ESI) source. Melting points (mp) of the compounds were determined using a Büchi B-450 melting point apparatus, and the temperature was not corrected.

[0032] Example 1: Preparation of the intermediate artesunate-glycine (ART-Gly)

[0033] Artesunate (0.2 g, 0.52 mmol, 1 eq), NHS (0.06 g, 0.52 mmol, 1 eq), EDCI (0.12 g, 0.57 mmol, 1.1 eq), and 10 mL THF were added to a 25 mL two-necked flask and stirred at room temperature for 1 h to activate the mixture. The THF solution was then slowly added dropwise to 2 mL of a THF solution of glycine (0.04 g, 0.57 mmol, 1.1 eq). After the addition was complete, the mixture was stirred at room temperature for 10 h, and then the reaction was stopped. The insoluble solids were removed by filtration, and the THF in the filtrate was removed by vacuum distillation. The residue was adjusted to pH 4 with 50% citric acid aqueous solution, and then extracted twice with 30 mL of dichloromethane each time. The combined organic layers were washed once with 60 mL of saturated brine, and the organic layer was dried over anhydrous MgSO4. The MgSO4 was removed by filtration, and the filtrate was concentrated under reduced pressure to give 0.26 g of an off-white solid. The above-mentioned off-white solid was separated by column chromatography (eluent: DCM:MeOH:HAc = 150:3:1) to obtain 0.21 g of white solid, with a yield of 73.4%. 1 H NMR (400MHz, CDCl3) δ5.83 (d, J = 9.2Hz, 1H), 5.39 (s, 1H), 4.06 (s, 2H), 2.89-2.80 (m, 2H), 2 .78-2.69(m,2H),2.62-2.57(m,1H),2.41-2.33(m,1H),2.06-2.00(m,1H),1.96-1.89(m,1 H),1.79-1.68(m,2H),1.65-1.57(m,1H),1.49-1.43(m,1H),1.42(s,3H),1.41-1.26(m,3H ),1.14-1.03(m,1H),0.95(d,J=6.2Hz,3H),0.88(d,J=7.3Hz,3H); ESI-MS(m / z):440.32[MH + Example 2: Preparation of the intermediate artesunate-isoleucine (ART-Ile) The title compound was prepared by replacing glycine in Example 1 with isoleucine, following the method of Example 1.

[0034] The eluent DCM:MeOH:HAc = 150:4:1, a white solid of 0.17 g, yielded 65.7%. 1H NMR (400MHz, CDCl3) δ6.41(d,J=8.6Hz,1H),5.77(d,J=9.8Hz,1H),5.44(s,1H),4.57(dd,J=8.6, 4.9Hz,1H),2.85-2.70(m,2H),2.65-2.50(m,3H),2.37(td,J=14.0,3.9Hz,1H),2.05-2.00(m,1H) ,1.97-1.85(m,2H),1.80-1.69(m,2H),1.65-1.59(m,1H),1.54-1.44(m,2H),1.43(s,3H),1.41-1 .14(m,6H),1.06-0.99(m,1H),0.97-0.91(m,8H),0.84(d,J=7.1Hz,3H); ESI-MS(m / z):496.41[MH + ].

[0035] Example 3: Preparation of intermediate artesunate-alanine (ART-Ala) The title compound was prepared by replacing glycine in Example 1 with alanine as the raw material, according to the method of Example 1.

[0036] The eluent DCM:MeOH:HAc = 150:4:1, a white solid of 0.20 g, yielded 84.4%. 1 H NMR (400MHz, CDCl3) δ6.64(d,J=7.0Hz,1H),5.77(d,J=9.8Hz,1H),5.44(s,1H),4.54(p,J=7.1Hz,1H), 2.77(t,J=7.2Hz,2H),2.65-2.52(m,3H),2.37(td,J=13.8,3.9Hz,1H),2.05-1.99(m,1H),1.92-1.85(m ,1H),1.81-1.69(m,2H),1.64-1.59(m,1H),1.53-1.47(m,1H),1.44(d,J=7.2Hz,3H),1.42(s,3H),1.39 -1.29(m,3H),1.07-0.99(m,1H),0.96(d,J=5.8Hz,3H),0.85(d,J=7.1Hz,3H); ESI-MS(m / z):454.50[MH + ].

[0037] Example 4: Preparation of the intermediate artesunate-methionine (ART-Met)

[0038] The title compound was prepared by replacing glycine in Example 1 with methionine and following the method of Example 1.

[0039] The eluent DCM:MeOH:HAc = 150:5:1, a white solid of 0.12 g, yielded 44.7%. 1 H NMR (400MHz, CDCl3) δ6.82(s,1H),5.77(d,J=9.8Hz,1H),5.45(s,1H),4.64(q,J=7.0Hz,1H),2.79- 2.75(m,2H),2.67-2.52(m,6H),2.42-2.32(m,1H),2.21-2.15(m,1H),2.11(s,3H),2.03-2.00(m,1H ),1.93-1.86(m,1H),1.80-1.69(m,2H),1.65-1.59(m,1H),1.54-1.45(m,1H),1.42(s,3H),1.37-1. 27(m,3H),1.07-0.99(m,1H),0.96(d,J=5.9Hz,3H),0.85(d,J=7.1Hz,3H); ESI-MS(m / z):514.37[MH + ].

[0040] Example 5: Preparation of the intermediate artesunate-phenylalanine (ART-Phe)

[0041] The title compound was prepared by replacing glycine in Example 1 with phenylalanine, following the method of Example 1.

[0042] The eluent DCM:MeOH:HAc = 150:5:1, a white solid of 0.20 g, yielded 72.3%. 1H NMR (400MHz, CDCl3) δ7.29(d,J=7.4Hz,2H),7.26-7.21(m,1H),7.17(d,J=7.0Hz,2H),6.37(d,J=7.5Hz,1H),5.75(d,J=9.9Hz ,1H),5.43(s,1H),4.83(q,J=6.6Hz,1H),3.21(dd,J=14.0,5.5Hz,1H),3.08(dd,J=14.0,6.6Hz,1H),2.76-2.65(m,2H),2.58 -2.46(m,3H),2.46-2.30(m,2H),2.04-1.99(m,1H),1.92-1.85(m,1H),1.78-1.67(m,2H),1.64-1.58(m,1H),1.52-1.44(m,1 H),1.42(s,3H),1.38-1.28(m,3H),1.05-0.98(m,1H),0.96(d,J=5.7Hz,3H),0.83(d,J=7.1Hz,3H); ESI-MS(m / z):530.40[MH + ].

[0043] Example 6: Preparation of the intermediate artesunate-glutamic acid (ART-Glu)

[0044] The title compound was prepared by replacing glycine in Example 1 with glutamic acid, following the method of Example 1.

[0045] The eluent DCM:MeOH:HAc = 150:10:1, a white solid of 0.16 g, yielded 60.0%. 1H NMR (400MHz, CDCl3) δ7.23(d,J=7.4Hz,1H),5.75(d,J=9.7Hz,1H),5.47(s,1H),4.56(q,J=7.4,6.9Hz,1H ),2.82-2.71(m,2H),2.62-2.56(m,2H),2.57-2.51(m,1H),2.48-2.44(m,2H),2.23-2.18(m,1H),2.08-2 .01(m,2H),1.92-1.86(m,1H),1.80-1.70(m,2H),1.66-1.58(m,1H),1.51-1.44(m,1H),1.40(s,3H),1.3 7-1.26(m,3H),1.05-0.98(m,1H),0.96(d,J=5.6Hz,3H),0.85(d,J=7.1Hz,3H); ESI-MS(m / z):512.22[MH + ].

[0046] Example 7: Preparation of the intermediate artesunate-aspartic acid (ART-Asp)

[0047] The title compound was prepared by replacing glycine in Example 1 with aspartic acid, following the method of Example 1.

[0048] The eluent DCM:MeOH:HAc = 150:10:1, a white solid of 0.17 g, yielded 65.4%. 1 H NMR (400MHz, CDCl3) δ7.42(d,J=7.9Hz,1H),5.74(d,J=9.7Hz,1H),5.46(s,1H),4.83(s,1H),3.05-2. 83(m,2H),2.76-2.71(m,2H),2.62-2.59(m,2H),2.57-2.51(m,1H),2.40-2.31(m,1H),2.04-1.98(m, 1H),1.92-1.85(m,1H),1.79-1.70(m,2H),1.64-1.58(m,1H),1.47-1.43(m,1H),1.40(s,3H),1.30-1 .24(m,3H),1.04-1.01(m,1H),0.96(d,J=6.0Hz,3H),0.84(d,J=7.0Hz,3H); ESI-MS(m / z):498.27[MH + ].

[0049] Example 8: Preparation of the intermediate artesunate-glutamine (ART-Gln)

[0050] The title compound was prepared by replacing glycine in Example 1 with glutamine, following the method of Example 1.

[0051] The eluent DCM:MeOH:HAc = 150:10:1, white solid 0.17 g, yield 63.6%. ESI-MS (m / z): 511.23 [MH] + ].

[0052] Example 9: Preparation of the intermediate artesunate-asparagine (ART-Asn)

[0053] The title compound was prepared by replacing glycine in Example 1 with asparagine, following the method of Example 1.

[0054] The eluent DCM:MeOH:HAc = 150:10:1, white solid 0.19 g, yield 73.1%. ESI-MS (m / z): 497.31 [MH] + ].

[0055] Example 10: Preparation of the intermediate artesunate-leucine (ART-Leu)

[0056] The title compound was prepared by replacing glycine in Example 1 with leucine, following the method of Example 1.

[0057] The eluent DCM:MeOH:HAc = 150:4:1, 0.13 g of white solid, yield 50.2%. ESI-MS (m / z): 482.11 [MH] + ].

[0058] Example 11: Preparation of the intermediate artesunate-valine (ART-Val)

[0059] The title compound was prepared by replacing glycine in Example 1 with valine and following the method of Example 1.

[0060] The eluent DCM:MeOH:HAc = 150:4:1, 0.16 g of white solid, yield 63.6%. ESI-MS (m / z): 482.12 [MH] + ].

[0061] Example 12: Preparation of the intermediate artesunate-proline (ART-Pro)

[0062] The title compound was prepared by replacing glycine in Example 1 with proline, following the method of Example 1.

[0063] The eluent DCM:MeOH:HAc = 150:4:1, 0.15 g white solid, yield 59.8%. ESI-MS (m / z): 480.56 [MH] + ].

[0064] Example 13: Preparation of the intermediate artesunate-cysteine ​​(ART-Cys)

[0065] The title compound was prepared by replacing glycine in Example 1 with cysteine, following the method of Example 1.

[0066] The eluent DCM:MeOH:HAc = 150:7:1, 0.11 g white solid, yield 43.4%. ESI-MS (m / z): 486.16 [MH] + ].

[0067] Example 14: Preparation of the intermediate artesunate-serine (ART-Ser)

[0068] The title compound was prepared by replacing glycine in Example 1 with serine, following the method of Example 1.

[0069] Eluent DCM:MeOH:HAc = 150:7:1, 0.13 g white solid, yield 53.0%. ESI-MS (m / z): 470.18 [MH] + ].

[0070] Example 15: Preparation of the intermediate artesunate-threonine (ART-Thr)

[0071] The title compound was prepared by replacing glycine in Example 1 with threonine, following the method of Example 1.

[0072] The eluent DCM:MeOH:HAc = 150:7:1, 0.14 g white solid, yield 55.4%. ESI-MS (m / z): 484.33 [MH] + ].

[0073] Example 16: Preparation of the intermediate artesunate-tyrosine (ART-Tyr)

[0074] The title compound was prepared by replacing glycine in Example 1 with tyrosine, following the method of Example 1.

[0075] The eluent DCM:MeOH:HAc = 150:7:1, 0.21 g of white solid, yield 73.7%. ESI-MS (m / z): 546.08 [MH] + ].

[0076] Example 17: Preparation of the intermediate artesunate-tryptophan (ART-Trp)

[0077] The title compound was prepared by replacing glycine in Example 1 with tryptophan, following the method of Example 1.

[0078] The eluent DCM:MeOH:HAc = 150:7:1, 0.19 g white solid, yield 64.0%. ESI-MS (m / z): 569.17 [MH] + ].

[0079] Example 18: Preparation of the intermediate artesunate-lysine (ART-Lys)

[0080] Artesunate (0.2 g, 0.52 mmol, 1 eq), NHS (0.06 g, 0.52 mmol, 1 eq), DCC (0.12 g, 0.57 mmol, 1.1 eq), and 10 mL of THF were added to a 25 mL two-necked flask and stirred at room temperature for 1 h to activate the mixture. 3 mL of a THF solution of N'-Fmoc-L-lysine (0.19 g, 0.57 mmol, 1.1 eq) was slowly added dropwise to the above system. After the addition was complete, the mixture was stirred at room temperature for 9 h, and then the reaction was stopped. The insoluble solids were removed by filtration, and the THF in the filtrate was removed by vacuum distillation. The residue was adjusted to pH 4 with 50% citric acid aqueous solution, and then extracted twice with 30 mL of dichloromethane each time. The combined organic layers were washed once with 60 mL of saturated brine, and the organic layer was dried over anhydrous MgSO4. The MgSO4 was removed by filtration, and the filtrate was concentrated under reduced pressure to give 0.33 g of a white solid. 0.33 g of a white solid was dissolved in 6 mL of THF, followed by the addition of 1.5 mL of piperidine. The mixture was stirred at room temperature for 12 h, and the THF was removed under reduced pressure. The residue was dissolved in 30 mL of dichloromethane and washed once each with 30 mL of water and 30 mL of saturated brine. The organic layer was dried over anhydrous MgSO4. The mixture was filtered to remove MgSO4, and the filtrate was concentrated under reduced pressure to give 0.43 g of a pale yellow solid. The pale yellow solid was separated by column chromatography with DCM:MeOH:TEA as the eluent (150:8:1). The yield was 0.13 g of a white solid, with a yield of 48.8%. ESI-MS (m / z): 511.28 [MH] + ].

[0081] Example 19: Preparation of the intermediate artesunate-arginine (ART-Arg)

[0082] The title compound was prepared by replacing glycine in Example 1 with arginine and following the method of Example 1.

[0083] Eluent DCM:MeOH:TEA = 150:12:1, white solid 0.09 g, yield 32.0%. ESI-MS (m / z): 539.32 [MH] + ].

[0084] Example 20: Preparation of the intermediate artesunate-histidine (ART-His)

[0085] The title compound was prepared by replacing glycine in Example 1 with histidine, following the method of Example 1.

[0086] The eluent DCM:MeOH:HAc = 150:8:1, 0.15 g white solid, yield 55.3%. ESI-MS (m / z): 520.40 [MH] + ].

[0087] Example 21: Intermediate Artesunate - D -Alanine (ART- D Preparation of -Ala)

[0088] Raw materials D The title compound was prepared by replacing glycine with alanine in Example 1, following the method of Example 1.

[0089] The eluent DCM:MeOH:HAc = 150:4:1, 0.17 g white solid, yield 71.7%. ESI-MS (m / z): 454.50 [MH] + ].

[0090] Example 22: Intermediate Artesunate - D -Methionine (ART- D Preparation of -Met)

[0091] Raw materials D The title compound was prepared by replacing glycine with methionine according to the method of Example 1.

[0092] The eluent DCM:MeOH:HAc = 150:5:1 yielded 0.11 g of a white solid, with a yield of 41.0%. ESI-MS (m / z): 514.37 [MH]. +].

[0093] Example 23: Preparation of compound ART-Ala-2-PBS

[0094] Artesunate-alanine (0.2 g, 0.44 mmol, 1 eq) and 2-aminophenylboronic acid (0.06 g, 0.44 mmol, 1 eq) were dissolved in 5 mL of dry THF. Then, HOBt (0.07 g, 0.53 mmol, 1.2 eq) and EDCI (0.13 g, 0.66 mmol, 1.5 eq) were added to the reaction system, and the mixture was stirred at room temperature. The reaction was stopped after TLC detection showed complete reaction. The solvent was evaporated under reduced pressure, and the residue was dissolved in 10 mL of ethyl acetate. The residue was washed once with 10 mL of 50% citric acid aqueous solution and once with 10 mL of saturated NaCl aqueous solution. The organic layer was dried over anhydrous MgSO4. The MgSO4 solid was removed by filtration, and the filtrate was concentrated to dryness under reduced pressure. The residue was separated by column chromatography with DCM:MeOH:HAc = 120:9:1 as the eluent, yielding 0.16 g of a white solid, with a yield of 63.4%. mp 151-153℃; 1 H NMR (400MHz, CDCl3) δ9.49(s,1H),8.11(dd,J=7.5,2.0Hz,1H),7.81(dd,J=7.4,2.0Hz,1H),7.50(s,1H),7.41(td,J=7.5,2.0H z,1H),7.31(d,J=7.5Hz,1H),6.58(d,J=7.1Hz,1H),5.82(d,J=9.6Hz,1H),5.44(s,1H),4.92-4.81(m,1H),2.91-2.81(m,2H),2 .73-2.62(m,3H),2.41-2.32(m,1H),2.16(s,1H),1.92-1.75(m,1H),1.72-1.63(m,2H),1.60-1.56(m,1H),1.42(d,J=6.5Hz,4 H),1.39(s,3H),1.31-1.25(m,3H),1.15-1.03(m,1H),0.99(d,J=5.6Hz,3H),0.85(d,J=7.4Hz,3H); ESI-MS(m / z):597.33[M+Na + ].

[0095] Example 24: Preparation of compound ART-Ala-3-PBS

[0096] The title compound was prepared according to the method of Example 23, using 3-aminophenylboronic acid instead of 2-aminophenylboronic acid in Example 23.

[0097] The eluent DCM:MeOH:HAc = 120:3:1, 0.21 g of white solid, yield 83.3%. mp 139-141℃; 1 H NMR (400MHz, CDCl3) δ9.39 (s, 1H), 7.98 (d, J = 8.4Hz, 1H), 7.68 (s, 1H), 7.52 (d, J = 7.4Hz, 1H), 7.30 (d, J = 7.7Hz, 1H) ,7.08(s,1H),5.74(d,J=9.9Hz,1H),5.41(s,1H),4.89-4.77(m,1H),2.82-2.71(m,2H),2.66-2.50(m,3H),2.39-2 .32(m,1H),2.01(s,1H),1.90-1.84(m,1H),1.74-1.67(m,2H),1.63-1.56(m,1H),1.49(d,J=6.7Hz,4H),1.41(s,3 H),1.33-1.27(m,3H),1.06-0.98(m,1H),0.95(d,J=5.1Hz,3H),0.80(d,J=7.1Hz,3H); ESI-MS(m / z):597.30[M+Na + ].

[0098] Example 25: Preparation of compound ART-Ala-4-PBS

[0099] The title compound was prepared according to the method of Example 23, using 4-aminophenylboronic acid instead of 2-aminophenylboronic acid in Example 23.

[0100] The eluent DCM:MeOH:HAc = 120:3:1 yielded 0.19 g of an off-white solid, with a yield of 75.3%. The eluent temperature was 157-160℃. 1H NMR (400MHz, CDCl3) δ8.99(s,1H),7.74(d,J=8.1Hz,2H),7.40(d,J=8.7Hz,2H),5.73(d,J=9.2Hz,1H),5. 36(s,1H),4.69(s,1H),2.84-2.74(m,2H),2.57-2.49(m,3H),2.39-2.32(m,1H),2.01(s,1H),1.83-1.79( m,1H),1.73-1.66(m,2H),1.59-1.55(m,1H),1.48-1.45(m,1H),1.40(d,J=5.4Hz,3H),1.26(s,3H),1.24 -1.18(m,3H),0.98-0.95(m,1H),0.90(d,J=5.6Hz,3H),0.79(d,J=7.8Hz,3H); ESI-MS(m / z):597.32[M+Na + ].

[0101] Example 26: Preparation of compound ART-Ala-3-OMPBS

[0102] Artesunate-alanine (0.2 g, 0.44 mmol, 1 eq) and 3-hydroxymethylphenylboronic acid (0.07 g, 0.44 mmol, 1 eq) were dissolved in 5 mL of dry THF. Then, DMAP (0.05 g, 0.53 mmol, 1 eq) and EDCI (0.13 g, 0.66 mmol, 1.5 eq) were added to the reaction system, and the mixture was stirred at room temperature. The reaction was stopped after TLC detection showed complete reaction. The solvent was evaporated under reduced pressure, and the residue was dissolved in 10 mL of ethyl acetate. The residue was washed once with 10 mL of 50% citric acid aqueous solution and once with 10 mL of saturated NaCl aqueous solution. The organic layer was dried over anhydrous MgSO4. The MgSO4 solid was removed by filtration, and the filtrate was concentrated to dryness under reduced pressure. The residue was separated by column chromatography with DCM:MeOH:HAc = 120:2:1. 0.22 g of white solid was obtained, with a yield of 87.1%. mp 136-138℃; 1H NMR (400MHz, CDCl3) δ8.79(s,1H),7.92(d,J=7.9Hz,1H),7.67(s,1H),7.47(d,J=7.4Hz,1H),7.27(d,J=7.8Hz,1H),7.1 1(s,1H),5.64(d,J=9.5Hz,1H),5.43(s,1H),4.81-4.79(m,1H),2.79-2.69(m,2H),2.62-2.50(m,3H),2.42-2.31(m,1H) ,2.05(s,2H),2.04-2.01(m,1H),1.95-1.85(m,1H),1.81-1.68(m,2H),1.65-1.56(m,1H),1.49-1.45(m,1H),1.44-1.4 1(m,3H),1.33-1.27(m,3H),1.05-0.98(m,1H),0.95(d,J=5.5Hz,3H),0.83(d,J=7.3Hz,3H); ESI-MS(m / z):612.29[M+Na + ].

[0103] Example 27: Preparation of compound ART-Ala-3-OPBS

[0104] The title compound was prepared according to the method of Example 26, using 3-hydroxyphenylboronic acid instead of 3-hydroxymethylphenylboronic acid in Example 26.

[0105] Eluent DCM:MeOH:HAc = 120:2:1, white solid 0.18 g, yield 71.4%. mp 148-150℃; 1H NMR (400MHz, CDCl3) δ8.08(s,1H),7.71(d,J=6.7Hz,1H),7.54(t,J=2.9Hz,1H),7.39-7.35(m,1H),7.13(dd,J=8.2,2 .4Hz,1H),6.66(d,J=7.4Hz,1H),5.75(d,J=5.5Hz,1H),5.47(s,1H),4.78(q,J=7.2Hz,1H),2.82-2.73(m,2H),2.62-2 .51(m,3H),2.43-2.37(m,1H),1.91-1.85(m,1H),1.74-1.67(m,2H),1.63-1.58(m,2H),1.53(t,J=7.6Hz,3H),1.42( s,3H),1.33-1.28(m,3H),1.03-0.97(m,1H),0.94(d,J=5.2Hz,3H),0.83(t,J=7.2Hz,3H); ESI-MS(m / z):598.28[M+Na + ].

[0106] Example 28: Preparation of compound ART-3-PBS

[0107] The title compound was prepared by replacing glycine in Example 1 with 3-aminophenylboronic acid, according to the method of Example 1.

[0108] The eluent DCM:MeOH:HAc = 120:3:1 yielded 0.23 g of a white solid, with a yield of 60.2%. mp 117-119℃; 1H NMR (400MHz, CDCl3) δ9.44(s,1H),7.99(d,J=8.1Hz,1H),7.71(s,1H),7.59(d,J=7.6Hz,1H),7.34(d,J=7.7Hz,1H) ,7.11(s,1H),5.91(d,J=9.6Hz,1H),5.44(s,1H),2.92-2.84(m,2H),2.80-2.72(m,2H),2.70-2.60(m,1H),2.46-2 .32(m,1H),2.11-2.02(m,1H),1.96-1.85(m,1H),1.83-1.70(m,2H),1.66-1.59(m,1H),1.51-1.43(m,1H),1.42(s ,3H),1.46-1.22(m,3H),1.18-1.04(m,1H),0.94(d,J=6.0Hz,3H),0.85(d,J=7.1Hz,3H); ESI-MS(m / z):504.41[M+H + ].

[0109] Example 29: In vitro screening assay for anti-tumor cell proliferation activity

[0110] 1. Screening of in vitro antitumor activity of the intermediate artesunate-amino acid derivative of the present invention

[0111] 1.1 Cell resuscitation

[0112] Remove the required cell cryovials from the -80℃ freezer and thaw them rapidly by shaking in a 37℃ water bath. After complete thawing, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add 1 mL of cell culture medium, gently pipette, and transfer the cell suspension to a culture dish containing 8 mL of culture medium. Incubate in a constant temperature and humidity incubator at 37℃ and 5% CO2, changing the medium every 24 hours.

[0113] 1.2 Cell Culture and Passaging

[0114] All cell culture media used in this experiment contained 1% penicillin / streptomycin solution and 10% fetal bovine serum. CT26 cells (mouse colon cancer cells) were cultured in RPMI-1640 medium; HCT116 cells (human colon cancer cells) were cultured in McCoy's 5A medium; and HuH7 cells (human liver cancer cells) were cultured in DMEM medium. All three cell lines were cultured in a 37°C, 5% CO2 incubator. When the cell density reached 80%-90%, passage culture was performed. The culture medium in the culture dish was aspirated, and the cells were washed once with 1 mL of PBS. Then, 1 mL of trypsin was added for digestion. Under a microscope, when the cells became rounded, 2 mL of complete culture medium was added to stop digestion. The cells were gently pipetted to transfer the cell suspension to centrifuge tubes, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, 2 mL of cell culture medium was added, and the mixture was gently pipetted to mix. The cell suspension was then aspirated and centrifuged at 2 × 10⁻⁶ cm⁻¹. 5 Inoculation and passage were performed at a density of cells / mL.

[0115] 1.3 Determination of the in vitro antitumor activity of artesunate-amino acid derivatives by CCK-8 assay

[0116] The anti-proliferative effect of the product of this invention on tumor cells was determined using the CCK-8 assay. The CCK-8 kit can sensitively and rapidly detect the cytotoxicity and anti-tumor proliferative activity of the drug. The working principle is as follows: In the presence of an electron carrier, WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazole monosodium salt) in the kit is reduced by dehydrogenases in the cell mitochondria to generate a water-soluble orange-yellow dye, formazan, which is soluble in the culture medium. The intensity of the color is directly proportional to cell proliferation and inversely proportional to cytotoxicity. The absorbance of each well was measured at 450 nm using a microplate reader.

[0117] Cells in the logarithmic growth phase were taken, and the three cell lines were respectively inoculated at 5 × 10⁻⁶. 3 Cells were seeded evenly in 96-well plates at a uniform density. After 24 hours of culture and observation of cell adhesion, drug administration began. 100 μL of different concentrations of drug solution was added to each well. A blank control group (containing culture medium but no cells) was set up, with no drug added as a negative control. ART and DHA served as positive control groups. After drug administration, the plates were incubated for 48 hours. Then, 10 μL of CCK-8 solution was added to each well for CCK-8 cell viability assay, and the plates were incubated for 0.5 hours. The absorbance (OD value) of each well was measured at 450 nm using a microplate reader, and the IC50 was calculated. 50 The values ​​are shown in Table 1. Six parallel samples were set up for each sample in the experiment.

[0118] As shown in Table 1, although amino acid-modified antitumor drugs can increase tumor targeting and enhance antitumor activity, the antitumor activity of ART varies greatly depending on the amino acid modification. Compounds ART-Ala, ART-Trp, and ART-Glu exhibited the best antitumor cell proliferation activity. Among them, compound ART-Ala showed the best activity, being twice that of ART and 2.5 times that of DHA. Therefore, compound ART-Ala was selected for further research.

[0119] Table 1. Antitumor cell proliferation activity of artesunate-amino acid derivatives

[0120] Note: All of the above amino acids are L-type amino acids.

[0121] 2. The intermediate artesunate of this invention - D -In vitro antitumor activity of amino acid derivatives

[0122] Select ART- D -Ala and ART- D -Met was used to investigate the effect of amino acid configuration on antitumor activity. The results are shown in Table 2. As can be seen from Table 2, the antitumor effect of ART linked to D-configuration amino acids was basically not improved compared with ART; at the same time, the antitumor cell proliferation effect was worse than that of L-configuration amino acids.

[0123] Table 2. Effects of amino acid configuration on anti-tumor cell proliferation activity

[0124] 3. In vitro antitumor activity test of the artesunate-amino acid-targeting group derivative of the present invention

[0125] Cells in the logarithmic growth phase were taken, and the three cell lines were respectively inoculated at 5 × 10⁻⁶. 3 Cells were seeded evenly in 96-well plates at a uniform density. After 24 hours of culture and observation of cell adhesion, drug administration began. 100 μL of different concentrations of drug solution was added to each well. A blank control group (containing culture medium but no cells) and a negative control group (no drug administration) were set up. ART, DHA, and oxaliplatin served as positive control groups. After drug administration, the plates were incubated for 48 hours. Then, 10 μL of CCK-8 cell viability assay reagent was added to each well, and the plates were incubated for 0.5 hours. The absorbance (OD value) of each well was measured at 450 nm using a microplate reader, and the IC50 was calculated. 50 The values ​​are shown in Table 3 and Figure 1. Six parallel samples were set up for each sample in the experiment.

[0126] The results showed that when aminophenylboronic acid (PBS) was linked, the inhibitory effect on tumor growth was superior to that of hydroxyphenylboronic acid; the anti-tumor cell activity of 3-aminophenylboronic acid was superior to that of 2-aminophenylboronic acid and 4-aminophenylboronic acid. The compound ART-ALA-3-PBS exhibited the strongest activity, superior to ART-Ala and ART-3-PBS, and its killing effect on HCT116 cells was superior to that of Oxaliplatin, indicating that the introduction of amino acid fragments and phenylboronic acid structures can increase the anti-tumor activity of ART.

[0127] Table 3. Antitumor cell proliferation activity of the compounds

[0128] Example 30: Structural stability of compound ART-Ala-3-PBS

[0129] 5 mg of compounds ART-Ala-3-PBS and ART were weighed and placed in 2 mL of 5% NaHCO3 aqueous solution, respectively. After standing at room temperature for 36 h, the pH was adjusted to approximately 5 with 20% citric acid aqueous solution, followed by extraction. TLC analysis showed that ART underwent complete hydrolysis, while ART-Ala-3-PBS showed only minor hydrolysis. Similarly, 5 mg of ART-Ala-3-PBS and ART were placed at 50 °C for 36 h. TLC analysis again showed that ART underwent complete hydrolysis, while ART-Ala-3-PBS showed only minor hydrolysis. Therefore, the compound exhibits better structural stability than ART.

[0130] Example 31: Preparation of Nanoparticle Formulations

[0131] The compound ART-Ala-3-PBS was dissolved in acetone and slowly added dropwise to an aqueous solution with continuous stirring to form nanoparticles through self-assembly. The resulting nanoparticles were then subjected to particle size analysis, as shown in Figure 2. The figure shows that the prepared nanomicelles exhibited relatively stable particle size (162.1 ± 13.2 nm), a PDI of 0.044, and a significant Tyndall effect.

[0132] Example 32: Study on the inhibition of tumor growth in tumor-bearing mice in vivo

[0133] Thirty-two female BALB / c mice (CT26 mouse colorectal cancer model), weighing 18-22g, were used to evaluate the efficacy of ART derivative nanoformulation. Each mouse was subcutaneously inoculated with CT26 cells (1×10⁻⁶). 6To model tumors in mice, once the tumors reached a certain size (7-9 days), these mice were randomly divided into four groups (n=8) and injected via tail vein: saline (NaCl), ART NPs, ART-Ala NPs, and ART-Ala-3-PBS NPs. The mice were administered 20 mg / kg every other day for 14 days, and tumor size and body weight were monitored daily to evaluate the anticancer effects and toxicity of the compounds in mice, as shown in Figure 3.

[0134] Tumors in the Control and ART NPs groups grew rapidly and continuously, with no significant difference between the two groups. However, tumor growth in the ART-Ala NPs group was inhibited to some extent compared to the Control and ART NPs groups; the ART-Ala-3-PBS NPs group showed a significant difference compared to the ART-Ala NPs and ART NPs groups, with stronger inhibition of tumor growth in mice. During the treatment period, there were no significant changes in the body weight of mice in any group, indicating that the nano-formulation has good biocompatibility.

[0135] Example 33: Detection of serum biochemical indicators in tumor-bearing mice

[0136] After the extracted mouse heart blood was allowed to stand for 1 hour and stratification occurred, the blood sample was centrifuged at 3000 rpm for 15 min. The upper serum layer was transferred to a new 1.5 mL centrifuge tube and centrifuged at 12000 rpm for 3 min to further separate the remaining red blood cells in the serum. The serum was then placed in a 1.5 mL centrifuge tube, and the levels of ALT (alanine aminotransferase), AST (aspartate aminotransferase), BUN (blood urea nitrogen), and CREA (creatinine) in the serum samples of each group of mice were detected (n=3), as shown in Figure 4.

[0137] The results showed that the ALT and AST levels in the ART NPs group were higher than those in the Control group, suggesting that ART may cause liver damage; while the levels of each indicator in the ART-Ala-3-PBS NPs group were basically the same as those in the Control group, with no significant difference, which also proves that the nano-formulation has good biosafety.

[0138] It should be noted that the above description is merely an embodiment for the purpose of understanding the present invention and does not limit the present invention. For those skilled in the art, any modifications, improvements, or equivalent substitutions made without departing from the spirit and principles of the present invention are included within the scope of protection of this application.

Claims

1. An artesunate-amino acid-targeting group derivative or a pharmaceutically acceptable salt thereof, as shown in formula I: in, R is selected from, but not limited to: -H, -CH3, -CH2CF3, -CH2CH(CH3)2, -CHCH3CH2CH3, -CHCH3CH2CF3, -CH(CH3)2, -(CH2)2SCH3, -CH2SH, -CH2OH, -CHCH3OH, -(CH2)2CONH2, -(CH2)2COOH, -CH2CONH2, -CH2COOH, -(CH2)4NH2, -(CH2)3NHCNHNH2, The amino acid configuration is selected from either the D or L form; The targeting group is selected from, but is not limited to, phenylboronic acid, folic acid, hyaluronic acid, triphenylphosphine, biotin, and transferrin.

2. The artesunate-amino acid-targeting group derivative or its pharmaceutically acceptable salt according to claim 1, characterized in that, The R is selected from -CH3, -CH2CF3, -(CH2)2SCH3, -CH2SH, -(CH2)2CONH2, -(CH2)2COOH, -CH2CONH2, -CH2COOH. Any one of them; The amino acids in this amino acid configuration are L-type. The targeting group is selected from phenylboronic acid, folic acid, or hyaluronic acid.

3. The artesunate-amino acid-targeting group derivative or its pharmaceutically acceptable salt according to claim 2, characterized in that, R is -CH3, and the targeting group is phenylboronic acid.

4. The artesunate-amino acid-targeting group derivative or its pharmaceutically acceptable salt according to claim 1, characterized in that... Choose from, but not limited to, any of the following: PBS represents the target group phenylboronic acid.

5. The method for preparing the artesunate-amino acid-targeting group derivative or its pharmaceutically acceptable salt according to claim 1, characterized in that, Includes the following steps: Artesunate reacts with amino acids to form amides under the condensing agent NHS / EDCI conditions; the resulting product then reacts with a targeting group to form amides or esters under the condensing agents HOBt / EDCI or DMAP / EDCI conditions. The definition of R is the same as in claim 1.

6. The nanoformulation of artesunate-amino acid-targeting group derivative or its pharmaceutically acceptable salt as described in claim 1, characterized in that, The preparation is mainly achieved through the following steps: dissolving the artesunate-amino acid-targeting group derivative and its pharmaceutically acceptable salt as described in claim 1 in an organic solvent, adding it dropwise to water containing an emulsifier or surfactant, and preparing the nano-formulation by nanoprecipitation; the organic solvent is preferably acetonitrile, tetrahydrofuran, DMF, DMSO and acetone; the surfactant is a hydrophilic or lipophilic surfactant, selected from any one or more of polyvinyl alcohol, polysorbate 80, polysorbate 20, sorbitan oleate and dextran.

7. A pharmaceutical composition, characterized in that, The active ingredient comprises the artesunate-amino acid-targeting group derivative and its pharmaceutically acceptable salt as described in any one of claims 1-5, and a pharmaceutically acceptable excipient.

8. Use of the artesunate-amino acid-targeting group derivative and its pharmaceutically acceptable salt as described in any one of claims 1-5, and the nanoformulation as described in claim 6, in the preparation of antitumor drugs.

9. The application according to claim 8, characterized in that, The tumors mentioned are solid tumors and hematologic malignancies that highly express sialic acid molecules.

10. The application according to claim 9, characterized in that, The tumors mentioned are multiple myeloma, liver cancer, colorectal cancer, lung cancer, stomach cancer, ovarian cancer, or breast cancer.

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