Pterostilbene analogue and application thereof

Abstract

The invention provides a pterostilbene analogue and application thereof, and particularly provides a pterostilbene analogue as shown in a formula (I) and a nucleic acid-pterostilbene conjugate, the compound can be accurately delivered in a targeted manner, the pharmacological action of pterostilbene can be efficiently exerted, and the compound can be used for treating tumor diseases. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the fields of drug design and new drug research and development, and specifically relates to the structural modification and drug delivery of a natural drug, pterostilbene. Background Art

[0002] Pterostilbene is a bioactive stilbene compound that widely exists in plants such as Pterocarpus indicus, grapes, and blueberries. Pterostilbene has received extensive attention due to its significant pharmacological effects such as anti-tumor, antioxidant, anti-inflammatory, and neuroprotective effects.

[0003] However, the relatively mild activity, poor water solubility, and low bioavailability of pterostilbene also pose major challenges for clinical applications.

[0004] Currently, various methods have been used to improve the water solubility of compounds. For example, nanoparticles and liposomes are used to encapsulate pterostilbene to form a complex to increase its bioavailability. However, current nanoparticle and liposome technologies still have problems such as poor stability, high preparation costs, and limited targeting.

[0005] Therefore, there is an urgent need in the art to develop pterostilbene analogs with improved water solubility that do not affect the activity and can be precisely targeted. Summary of the Invention

[0006] The object of the present invention is to provide a pterostilbene analog and construct a drug delivery system to improve the bioavailability of pterostilbene without affecting its activity.

[0007] In the first aspect of the present invention, a pterostilbene analog is provided, which has the structure shown in formula (I):

[0008]

[0009] Wherein,

[0010] n1 is selected from 1, 2, or 3;

[0011] n2 is selected from 1, 2, or 3;

[0012] R1 and R2 are each independently selected from H, DMTr,

[0013] In another preferred example, R1 is selected from H, DMTr.

[0014] In another preferred example, R2 is selected from H, In another preferred example, R1 is H and R2 is H.

[0015] In another preferred example, R1 is DMTr and R2 is In another preferred example, n1 is 1 or 2.

[0016] In another preferred example, n2 is 1 or 2.

[0017] In another preferred example, the pterostilbene analogs are selected from the group consisting of:

[0018]

[0019] In a second aspect of the present invention, there is provided a method for preparing pterostilbene analogs, comprising the steps of: (a) reacting compound 1 with compound Z1 in an inert solvent to obtain a compound of formula 2;

[0020]

[0021] Wherein, X is selected from halogen, and the definitions of n1, n2, R1 and R2 are as described in the first aspect of the present invention.

[0022] In another preferred example, in step (a), the inert solvent is an organic solvent.

[0023] In another preferred example, in step (a), the inert solvent includes dichloromethane.

[0024] In another preferred example, in step (a), the reaction is carried out in the presence of an alkylamine and / or 4-dimethylaminopyridine (DMAP).

[0025] In another preferred example, in step (a), the temperature is 0 - 80 °C.

[0026] In another preferred example, in step (a), the time is 2 - 48 h.

[0027] In another preferred example, in step (a), in dichloromethane solvent, pterostilbene (PTE) reacts with triethylamine (Et3N) in the presence of triethylamine (Et3N) and 4-dimethylaminopyridine (DMAP) to obtain PTE-G.

[0028] In another preferred example, the method further comprises the step of:

[0029] (b) reacting compound 2 with DMTr-Cl in an inert solvent to obtain a compound of formula 3;

[0030]

[0031] Wherein, the definitions of n1 and n2 are as described in the first aspect of the present invention.

[0032] In another preferred example, in step (b), the inert solvent is an organic solvent.

[0033] In another preferred example, in step (b), the inert solvent includes pyridine.

[0034] In another preferred embodiment, in step (b), the temperature is 0 - 80 °C.

[0035] In another preferred embodiment, in step (b), the time is 0.5 - 24 h.

[0036] In another preferred embodiment, in step (b), in a pyridine solution, compound 2 reacts with DMTr-Cl to obtain compound 3.

[0037] In another preferred embodiment, the method further comprises the steps of:

[0038] (c) Under the protection of an inert gas and in an inert solvent, compound 3 reacts with 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite to obtain a compound of formula 4;

[0039]

[0040] Wherein, the definitions of n1 and n2 are as described in the first aspect of the present invention.

[0041] In another preferred embodiment, in step (c), the inert solvent is an organic solvent.

[0042] In another preferred embodiment, in step (c), the inert solvent includes dichloromethane.

[0043] In another preferred embodiment, in step (c), the inert gas includes nitrogen.

[0044] In another preferred embodiment, in step (c), it is carried out in the presence of diisopropylethylamine (DIPEA).

[0045] In another preferred embodiment, in step (c), the temperature is 0 - 80 °C.

[0046] In another preferred embodiment, in step (c), the time is 0.5 - 48 h.

[0047] In another preferred embodiment, in step (c), in a dichloromethane solvent, compound 3 reacts with 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite in the presence of diisopropylethylamine (DIPEA) to obtain compound 4.

[0048] In the third aspect of the present invention, a nucleic acid-pterostilbene conjugate is provided, and the conjugate has a structure shown in formula (II):

[0049] Z-(L-Y) m (II)

[0050] In the formula,

[0051] Z is a nucleic acid molecule;

[0052] L is a divalent linking group;

[0053] Y is a PTE module;

[0054] The said PTE module is

[0055] wherein n1 is selected from 1, 2, 3;

[0056] n2 is selected from 1, 2, 3;

[0057] R1 is selected from H, DMTr;

[0058] m is a positive integer ≥ 1.

[0059] In another preferred embodiment, the linker is

[0060] In another preferred embodiment, the said Z is an aptamer.

[0061] In another preferred embodiment, the aptamer is an aptamer targeting a pathogenic marker on the cell surface.

[0062] In another preferred embodiment, the aptamer is an aptamer targeting a tumor marker.

[0063] In another preferred embodiment, the aptamer is AS1411.

[0064] In another preferred embodiment, the said Z is a polynucleotide.

[0065] In another preferred embodiment, the said PTE module is located at the 5'-end, 3'-end and / or middle part of the polynucleotide.

[0066] In another preferred embodiment, the said PTE module is located at the 5'-end of the polynucleotide.

[0067] In another preferred embodiment, the nucleic acid molecule is selected from the group consisting of: siRNA, miRNA, mRNA, DNA.

[0068] In the fourth aspect of the present invention, there is provided a method for preparing the conjugate as described in the third aspect of the present invention, the method comprising the steps of:

[0069] (1) Providing a compound of formula 4;

[0070]

[0071] (2) Using solid-phase synthesis method, reacting the compound of formula 4 with nucleotide substrates to form the said conjugate.

[0072] In another preferred embodiment, in step (2), the compound of formula 4 is used as a module for DNA solid-phase synthesis and coupled with the solid-phase synthesized nucleotide chain, thereby forming a conjugate with the PTE module at the 5'-end of the nucleotide chain.

[0073] In another preferred embodiment, in step (2), when R1 in the PTE module is DMTr, it further includes the step of removing the DMTr group, thereby forming a PTE module with R1 being H.

[0074] In another preferred embodiment, in step (2), in the solid-phase synthesis, multiple rounds of synthesis are carried out according to the predetermined sequence of polynucleotides.

[0075] In another preferred embodiment, the solid-phase synthesis includes the solid-phase phosphoramidite triester method.

[0076] In another preferred embodiment, the solid-phase synthesis uses controlled pore glass (CPG) as the solid phase.

[0077] In another preferred embodiment, in each round of the multiple rounds of synthesis, it includes sub-steps of deprotection, coupling, and oxidation.

[0078] In another preferred embodiment, in step (2), the compound of formula 4 is synthesized at the 5'-segment of the nucleic acid molecule

[0079] In the fifth aspect of the present invention, there is provided the use of the pterostilbene analog as described in the first aspect of the present invention or the conjugate as described in the third aspect of the present invention for preparing a pharmaceutical composition for treating tumor diseases.

[0080] In the sixth aspect of the present invention, there is provided a pharmaceutical composition, which includes: 1) the pterostilbene analog as described in the first aspect of the present invention or the conjugate as described in the third aspect of the present invention; and 2) a pharmaceutically acceptable carrier.

[0081] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1 It is the preparation process of the aptamer-pterostilbene conjugate.

[0083] Figure 2 It is the 1H NMR spectrum of the pterostilbene analog, i.e., compound 2.

[0084] Figure 313C NMR spectrum of pterostilbene analogue, i.e., compound 2.

[0085] Figure 4 1H NMR spectrum of compound 3.

[0086] Figure 5 13C NMR spectrum of compound 3.

[0087] Figure 6 1H NMR spectrum of phosphoramidite module of pterostilbene, i.e., compound 4.

[0088] Figure 7 31P NMR spectrum of phosphoramidite module of pterostilbene, i.e., compound 4.

[0089] Figure 8 Mass spectrum of AS1411.

[0090] Figure 9 Mass spectrum of AS1411-Cy5.

[0091] Figure 10 Mass spectrum of AS1411-PTE.

[0092] Figure 11 Mass spectrum of AS1411-PTE-Cy5.

[0093] Figure 12 Mass spectrum of Lib-PTE.

[0094] Figure 13 Mass spectrum of Lib-PTE-Cy5.

[0095] Figure 14 Detection of specific recognition ability and internalization ability of AS1411-PTE.

[0096] Figure 15 Drug release mechanism of AS1411-PTE.

[0097] Figure 16 Study on cytotoxicity and apoptosis of AS1411-PTE.

[0098] Figure 17 In vivo distribution imaging of AS1411-PTE.

[0099] Figure 18 Effect of AS1411-PTE on growth and metastasis of xenograft tumors in zebrafish.

[0100] Figure 19 Effect of AS1411-PTE on apoptosis of xenograft tumors in zebrafish.

[0101] Figure 20Effect of AS1411-PTE on angiogenesis of xenograft tumors in zebrafish.

[0102] Figure 21 Molecular docking simulation of PTE and PTE-G binding to proteins.

[0103] Figure 22 Synthesis schematic of nucleic acid-pterostilbene conjugate.

[0104] Figure 23 Schematic of a representative solid-phase DNA synthesis method, namely solid-phase phosphoramidite triester method. Detailed implementation mode

[0105] Through extensive and in-depth research, the inventors of the present invention prepared a pterostilbene analog to improve the water solubility of pterostilbene; on the basis of a large number of screenings and tests, an aptamer-pterostilbene conjugate was further prepared. The conjugate can accurately deliver drugs to the target site, exert the anti-tumor effect of pterostilbene, and effectively improve the bioavailability of pterostilbene. On this basis, the present invention was completed.

[0106] Terms

[0107] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains.

[0108] As used herein, "aptamer" refers to a nucleic acid sequence capable of binding to a specific target molecule, including DNA aptamers, RNA aptamers, hybrid-type aptamers, or other types of aptamers. In addition, in the present invention, aptamers also include single-stranded and double-stranded forms.

[0109] As used herein, the term "room temperature" or "normal temperature" refers to a temperature of 4 - 40 °C, preferably 25 ± 5 °C.

[0110] As used herein, the term "DMTr-Cl" refers to 4,4'-dimethoxytrityl chloride, with the full name Dimethoxytrityl-Cl. It is a commonly used base protecting group that can protect nitrogen bases (such as adenine and guanine) and oxygen bases (such as thymine).

[0111] As used herein, the term "AccutaseTM cell detachment solution" refers to a cell digestion solution containing proteolytic enzyme and collagenase activities, and does not contain any components derived from animals and bacteria.

[0112] As used herein, the term "MCF-7 cells" refers to a type of human breast cancer cell, with the full name Michigan Cancer Foundation-7.

[0113] As used herein, the term "Ramos cell" refers to a human B lymphocyte tumor cell that can be used for the establishment of hybridomas.

[0114] As used herein, the term "Cy5" refers to cyanine dye 5, also known as Cyanine 5, which exhibits red fluorescence and is used to label the amino groups of peptides, proteins, and oligonucleotides, and is suitable for fluorescence microscopy or other fluorescence imaging techniques.

[0115] As used herein, the term "Hoechst 33342" refers to a blue fluorescent dye that labels DNA and has low toxicity to cells. It is commonly used for visualizing the staining of cell nuclei and mitochondria.

[0116] As used herein, the term "DPBS" refers to phosphate buffered solution, also known as Dulbecco's Phosphate Buffered Saline.

[0117] As used herein, the term "PTE" refers to pterostilbene.

[0118] As used herein, the term "PTE-G" refers to compound 2 (an analogue of pterostilbene):

[0119]

[0120] As used herein, the term "AS1411" refers to a nucleic acid aptamer sequence that can specifically bind to tumor cells overexpressing nucleolin.

[0121] As used herein, the term "Lib" refers to a random oligonucleotide sequence that serves as a negative control.

[0122] As used herein, the term "TEAA" refers to triethylammonium acetate.

[0123] As used herein, the term "MTT" refers to 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, also known as thiazolyl blue, which is a yellow dye. The MTT method, also known as the MTT colorimetric method, is a method for detecting cell survival and growth.

[0124] As used herein, the term "DiO" refers to a lipophilic fluorescent dye that can be used to label cell membranes and other hydrophobic structures and exhibits green fluorescence.

[0125] Pterostilbene analogue

[0126] As used herein, the terms "compounds of the present invention" and "pterostilbene analogs of the present invention" are used interchangeably and refer to the compounds of formula I described in the first aspect of the present invention, or pharmaceutically acceptable salts (or esters) thereof.

[0127] Preparation of Pterostilbene Analogs

[0128] The preparation methods of the pterostilbene analogs of the present invention are described in more detail below, but these specific methods do not constitute any limitation to the present invention. The compounds of the present invention can also be conveniently prepared by optionally combining various synthetic methods described in this specification or known in the art, and such combinations can be easily carried out by those skilled in the art to which the present invention pertains.

[0129] Generally, in the preparation process, each reaction is usually carried out in an inert solvent at a temperature from room temperature to reflux temperature (such as 0 °C to 80 °C, preferably 0 °C to 50 °C). The reaction time is usually from 0.1 hour to 60 hours, preferably 0.5 to 48 hours.

[0130] The following general preparation routes can be used to synthesize the pterostilbene analogs of the present invention: Compound 2 (i.e., PTE-G), Compound 3, and Compound 4.

[0131] Taking Compound 2 as an example, representative preparation methods include (but are not limited to):

[0132]

[0133] In dichloromethane solvent, pterostilbene (i.e., PTE) reacts with 3-chloro-1,2-propanediol in the presence of triethylamine (Et3N) and 4-dimethylaminopyridine (DMAP) to obtain PTE-G.

[0134] Taking Compound 3 as an example, representative preparation methods include (but are not limited to):

[0135]

[0136] In pyridine solvent, Compound 2 reacts with DMTr-Cl to obtain Compound 3.

[0137] Taking Compound 4 as an example, representative preparation methods include (but are not limited to):

[0138]

[0139] In dichloromethane solvent, Compound 3 reacts with 2-cyanoethyl N,N-diisopropyl phosphorochloridite in the presence of diisopropylethylamine (DIPEA) to obtain Compound 4.

[0140] Nucleic Acid-Pterostilbene Conjugates

[0141] The present invention also provides a conjugate formed by coupling a nucleic acid molecule with the compound of the present invention (abbreviated as "the conjugate of the present invention" or "the nucleic acid-pterostilbene conjugate of the present invention").

[0142] In the present invention, the nucleic acid molecule may be DNA, RNA, or a combination thereof. A preferred nucleic acid is a polynucleotide, especially an aptamer with a targeting function.

[0143] A particularly preferred conjugate of the present invention is an aptamer-PTE conjugate.

[0144] In the present invention, the length of the polynucleotide is not particularly limited, and generally ranges from 8 to 100 nt, preferably 10 to 80 nt.

[0145] It should be understood that in the conjugate of the present invention, one or more PTE modules of the present invention can be linked to a polynucleotide sequence.

[0146] Aptamer

[0147] Due to its high specificity and high affinity, aptamer is regarded as a key tool for targeted drug delivery.

[0148] In the present invention, the aptamer that can be used to form the conjugate of the present invention is not particularly limited. Representative aptamers include (but are not limited to): aptamers targeting tumor cell surface antigens, aptamers targeting immune cell surface antigens, etc.

[0149] A representative aptamer is AS1411. AS1411 has unique internalization ability and can specifically recognize nucleolin overexpressed on the surface of tumor cells. Therefore, in one embodiment of the present invention, it is selected as a delivery vector to construct an aptamer-pterostilbene conjugate to target target cells and efficiently exert the pharmacological effect of pterostilbene.

[0150] Preparation method of aptamer-pterostilbene conjugate

[0151] The present invention also provides a nucleic acid-pterostilbene conjugate based on Compound 4 or the PTE module of the present invention, especially a polynucleotide-pterostilbene conjugate, wherein the polynucleotide may be siRNA, miRNA, mRNA, DNA, or a combination thereof.

[0152] In the present invention, the polynucleotide may contain a natural or unnatural backbone, such as peptide nucleic acid, locked nucleic acid, morpholino nucleic acid.

[0153] In the present invention, a preferred polynucleotide is an aptamer.

[0154] In the present invention, the PTE module of the present invention can be located at the 5'-end, 3'-end, and / or middle part of the polynucleotide. Preferably, the PTE module is located at the 5'-end of the polynucleotide.

[0155] A representative solid-phase DNA synthesis is the solid-phase phosphoramidite triester method, the schematic diagram of which is as Figure 23 shown. In the solid-phase synthesis using controlled pore glass (CPG) as the solid phase and performing multiple rounds of synthesis, each round of synthesis reaction includes sub-steps of deprotection, coupling, and oxidation. In addition, a capping step can be introduced as needed.

[0156] In the present invention, compound 4 can be used as a raw material, and with existing or commercially available high-throughput DNA / RNA synthesizers and methods, compound 4 can be integrated into the aptamer sequence like a natural base. Using a phosphodiester bond as the linking bond, through deprotection, coupling, oxidation reactions, and optional capping reactions, it is attached to the solid support carrier CPG, thereby completing the preparation of the aptamer-pterostilbene conjugate. Subsequently, the aptamer-pterostilbene conjugate is purified through deprotection with concentrated ammonia water, ethanol precipitation, and high-performance liquid chromatography.

[0157] Application

[0158] In the present invention, pterostilbene analogs or aptamer-pterostilbene conjugates can be used to prepare pharmaceutical compositions for treating tumor diseases; preferably, they can be used to prepare pharmaceutical compositions for targeted treatment of tumor diseases.

[0159] In the present invention, when the pterostilbene analog or nucleic acid-pterostilbene conjugate is used to treat tumor diseases, it can be used alone or in combination with other drugs.

[0160] In the present invention, the aptamer-pterostilbene conjugate can accurately recognize target cells and can be effectively internalized into target cells.

[0161] In the present invention, the aptamer-pterostilbene conjugate can release active compounds when entering the body.

[0162] The main advantages of the present invention include:

[0163] (1) For the pterostilbene analogs of the present invention, while not changing the activity of pterostilbene, the water solubility is significantly improved;

[0164] (2) The aptamer-pterostilbene conjugates of the present invention can be effectively internalized into cells, effectively improving the bioavailability of pterostilbene;

[0165] (3) The aptamer-pterostilbene conjugate of the present invention can precisely deliver the active compound to the target site and exert its anti-tumor effect.

[0166] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions mentioned in the following embodiments are usually carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0167] Example 1 Preparation of Pterostilbene Analogue

[0168]

[0169] Under the condition of 0 °C, triethylamine (Et3N, 12 mmol), 3-chloro-1,2-propanediol (4.8 mmol) and 4-dimethylaminopyridine (DMAP, 0.4 mmol) were added to a dichloromethane solution (DCM, 10 mL) of pterostilbene (Compound 1, PTE, 4 mmol), and the mixture was stirred at room temperature for 12 hours. After the reaction was completed, the solvent was removed by rotary evaporation, and the residue was separated by silica gel column chromatography using ethyl acetate / petroleum ether = 1:3 as the eluent. The liquid containing the target product was collected and vacuum concentrated to obtain 1.1 g of colorless oily pterostilbene analogue (Compound 2, PTE-G) with a yield of 83%.

[0170] 1 H NMR (500 MHz, CDCl3) δ 7.37 (d, J = 8.6 Hz, 2H), 7.01 (d, J = 16.2 Hz, 1H), 6.84 (dd, J = 24.3, 12.4 Hz, 3H), 6.64 (d, J = 2.2 Hz, 2H), 6.36 (t, J = 2.2 Hz, 1H), 3.82 (s, 6H), 3.77 (dd, J = 11.4, 3.9 Hz, 2H), 3.71 - 3.58 (m, 5H).

[0171] 13 C NMR (101 MHz, CDCl3) δ 160.97, 158.22, 139.58, 130.51, 128.55, 127.97, 127.87, 126.92, 114.77, 104.38, 99.70, 70.41, 69.21, 63.65, 55.38.

[0172] Example 2 Preparation of Pterostilbene Phosphoramidite Module

[0173] 2.1 Preparation of Compound 3

[0174]

[0175] Under the condition of 0 °C, DMTr-Cl (1.58 mmol) was added to a pyridine (10 mL) solution of pterostilbene analog (Compound 2, 1.58 mmol), and the reaction was carried out at room temperature for 3 hours. After the reaction was completed, the solvent was removed by rotary evaporation. The residue was separated by silica gel column chromatography using ethyl acetate / petroleum ether = 1:5 as the eluent. The liquid containing the target product was collected and concentrated in vacuo to obtain 0.8 g of Compound 3 as a white solid, with a yield of 67%.

[0176] 1 H NMR(500MHz,CDCl3)δ7.41 - 7.43(m,4H),7.32 - 7.29(m,10H),6.86 - 6.81(m,8H),3.79(s,12H),3.71 - 3.63(m,3H),3.23 - 3.31(m,3H);

[0177] 13 C NMR(101MHz,Acetone)δ161.22,158.92,158.68,145.46,139.88,136.16,130.11,128.61,128.15,127.79,127.66,126.59,126.45,114.81,112.95,104.21,99.39,85.90,69.72,69.13,64.65,54.71,54.60。

[0178] 2.2 Preparation of Compound 4

[0179]

[0180] Under the condition of 0 °C, diisopropylethylamine (DIPEA, 5.9 mmol) and 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite (1.4 mmol) were added to a dichloromethane (DCM, 7.5 mL) solution of the crude product Compound 3 (1.18 mmol) from the previous step. The mixture was stirred at room temperature for 2 hours, and then the solvent was removed by rotary evaporation. The residue was separated by silica gel column chromatography using ethyl acetate / petroleum ether = 1:5 as the eluent. The liquid containing the target product was collected and concentrated in vacuo to obtain 0.7 g of the pterostilbene phosphoramidite module (Compound 4) as a white solid, with a yield of 70%.

[0181] 11H NMR (400 MHz, Acetone) δ 7.58 - 7.48 (m, 4H), 7.40 - 7.17 (m, 8H), 7.07 - 6.84 (m, 7H), 6.75 (t, J = 2.3 Hz, 2H), 6.39 (t, J = 2.3 Hz, 1H), 4.41 - 4.15 (m, 3H), 3.94 - 3.85 (m, 1H), 3.84 - 3.77 (m, 13H), 3.42 - 3.39 (m, 1H), 3.30 (t, 3H), 2.78 - 2.70 (m, 1H), 2.64 (t, J = 6.1 Hz, 1H), 1.22 - 1.17 (m, 9H), 1.09 (d, J = 6.8 Hz, 3H).

[0182] 31 31P NMR (162 MHz, Acetone) δ 149.35 (d, J = 48.4 Hz).

[0183] Preparation of the Aptamer - Pterostilbene Conjugate in Example 3

[0184] Automated synthesis provides an economical and effective method for constructing the aptamer - pterostilbene conjugate. As a phosphoramidite module of pterostilbene, compound 4 can be incorporated into the nucleic acid aptamer sequence in an automated and programmable manner by means of solid - phase automated modular synthesis technology with the aid of a DNA synthesizer, just like the natural A, T, C, G, to obtain the aptamer - pterostilbene conjugate ( Figure 22 ).

[0185] The synthetic sequence is as follows:

[0186] Table 1: Details of the Synthetic Sequence

[0187]

[0188] Note: (Y) represents Among them, R1 is H, n1 is 1, and n2 is 1.

[0189] The mass spectrometry results are as follows:

[0190] AS1411: ESI - MS: Target molecular weight 8272.36, actual molecular weight 8273.6

[0191] AS1411 - Cy5: ESI - MS: Target molecular weight 8961.16, actual molecular weight 8960.1

[0192] AS1411 - PTE: ESI - MS: Target molecular weight 9577.2, actual molecular weight 9577.2

[0193] AS1411-PTE-Cy5: ESI-MS: Target molecular weight 10266, actual molecular weight 10264.7

[0194] Lib-PTE: ESI-MS: Target molecular weight 12974.53, actual molecular weight 12974.9

[0195] Lib-PTE-Cy5: ESI-MS: Target molecular weight 13663.33, actual molecular weight 13663.7

[0196] Example 4 Evaluation of the Targeting and Internalization Abilities of Aptamer-Pterostilbene Conjugates

[0197] To analyze the specific targeting ability of AS1411-PTE to target cells, MCF-7 cells (nucleolin overexpressing cell line) and Ramos cells (non-nucleolin overexpressing negative control cell line) were treated with AccutaseTM cell dissociation solution. After three washes, the cells (3×10 5 ) were incubated with 250 nM Cy5-labeled AS411-PTE in 200 μL binding buffer at 4 °C for 30 min. At the same time, a control group was set up. Finally, the cells were suspended in 200 μL wash buffer and analyzed by flow cytometry using a flow cytometer. AS1411 was used as a positive control, and Lib-PTE was used as a negative control.

[0198] The results are as Figure 14 shown in Figure A. The results showed that compared with the negative control, the fluorescence intensity of the cells treated with AS1411-PTE increased significantly, indicating that AS1411-PTE has specific binding ability to MCF-7 cells with overexpressed nucleolin. In contrast, neither Lib-PTE nor AS1411-PTE showed positive binding to Ramos cells. These results suggest that AS1411-PTE is selective for MCF-7 cells and targets cancer cells.

[0199] To analyze the internalization ability of AS1411-PTE, MCF-7 cells (3×10 5 / well) were seeded in glass-bottom culture dishes and incubated overnight. Then, they were incubated with 500 nM Cy5-labeled AS1411-PTE at 37 °C for 2 hours, and a control group was set up at the same time. The cells were stained with Hoechst 33342 to localize the cell nucleus. After incubation, the cells were washed twice with DPBS, and then the prepared samples were observed by confocal microscopy.

[0200] In Figure 14 Figure B, obvious red fluorescence was observed in the cell membrane and cytoplasm of MCF-7 cells after treatment with AS1411-PTE. In contrast, the fluorescence intensity of the Lib-PTE group was negligible.

[0201] This observation indicates that AS1411-PTE can be effectively internalized into target cancer cells. The precise recognition and internalization ability exhibited by AS1411-PTE lay a solid foundation for specific targeted drug delivery.

[0202] Example 5 Evaluation of the in vitro release ability of the aptamer-pterostilbene conjugate

[0203] First, use PTE-G and AS1411-PTE as standard samples to measure their retention times respectively. Mix phosphodiesterase I (PDE I, 50 U / mL, 20 μL) with AS1411-PTE (50 μM, 20 μL), incubate at 37 °C for 2 hours, and perform high-performance liquid chromatography analysis with an acetonitrile gradient (5 - 95%) in a 100 mM TEAA (pH 7.0) solution to detect the release of PTE-G in the conjugate. Dual-wavelength detection is used, at 260 nm and 320 nm respectively. Assume that the bioequivalent of PTE, PTE-G, can also be released from AS1411-PTE through enzyme-mediated oligonucleotide degradation and play a role in the biological environment. To verify this hypothesis, use phosphodiesterase I (PDE I) to simulate the in vivo environment and incubate AS1411-PTE with phosphodiesterase I at 37 °C for 2 hours.

[0204] The results are as Figure 15 shown in A. The retention times of the standard sample AS1411-PTE and free PTE-G in the liquid phase are 4.528 minutes and 9.712 minutes respectively. Under the condition of 50 U / mL PDE I, Figure 15 the results in B show that AS1411-PTE is completely degraded after 2 hours, the original retention peak around 4 minutes disappears, oligonucleotide fragments after enzyme degradation appear at 2.008 minutes, and at the same time, released PTE-G is detected at around 9 minutes in the 320 nm wavelength band. This finding is confirmed by mass spectrometry data. It is verified that AS1411-PTE can release free PTE-G through the mediation of phosphodiesterase I.

[0205] Example 6 Evaluation of the in vitro activity and apoptotic effect of the aptamer-pterostilbene conjugate

[0206] The MTT method was used to evaluate the cytotoxic effect of AS1411-PTE on MCF-7 cells. MCF-7 cells were seeded in 96-well plates (2000 cells / well) and incubated for 24 hours. Then, the cells were treated with different sample groups at 0.2 μM to 25 μM for 48 hours, and 20 μL of MTT reagent was added to each well and incubated for another 4 hours. The formed formazan crystals were dissolved with DMSO. The absorbance at 570 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader to determine cell viability.

[0207] The results are asFigure 16 As shown in A, in the range of 0 - 25 μM, AS1411 - PTE showed excellent inhibitory effects on MCF - 7 cells. At low concentrations, PTE and PTE - G had no obvious inhibitory effects on tumor cells. In addition, the results of the 48 - hour cytotoxicity experiment are as Figure 16 shown in B. The IC 50 value of AS1411 - PTE was 17 μM, and the IC 50 values of PTE and PTE - G were 61.11 μM and 59.63 μM, respectively. This finding demonstrated the ability of AS1411 - PTE to significantly reduce the activity of MCF - 7 cells, and the modification of the PTE structure did not change this effect. PTE - G had similar effectiveness to PTE and became a viable biological alternative.

[0208] To evaluate the effect of AS1411 - PTE on apoptosis, MCF - 7 cells were seeded in 12 - well plates and incubated overnight. Then, the cells were treated with 20 μM of PTE and AS1411 - PTE for 48 hours, respectively, with untreated cells as the control. According to the requirements of the Annexin V - FITC apoptosis detection kit, the cells were incubated in the dark for 15 minutes at room temperature. Flow cytometry was used for analysis. To verify whether AS1411 - PTE had the same effect as PTE in promoting tumor cell apoptosis, to verify this hypothesis, PTE and AS1411 - PTE were applied to MCF - 7 cells at a consistent concentration (20 μM) for 48 hours, and apoptotic cells were detected by flow cytometry.

[0209] The results are as Figure 16 shown in C and 16D. The proportions of early and late apoptotic cells of AS1411 - PTE were significantly higher than those of PTE.

[0210] Example 7 Evaluation of the in vivo biological activity of the aptamer - pterostilbene conjugate

[0211] The zebrafish xenograft model is well - known for its easy operation and rapid experimental turnover and is an ideal platform for studying tumor development. First, MCF - 7 cells labeled with DiO cell labeling solution (green fluorescence) were injected into the yolk sac of wild - type AB strain zebrafish embryos, and about 200 cells were transplanted into each tail to establish a zebrafish - breast cancer xenograft model. Then, different doses were set to determine the maximum tolerated dose (MTD) of zebrafish.

[0212] The results are shown in Table 2. AS1411 - PTE did not cause the death of normal zebrafish within 100 μM. Therefore, 100 μM and 10 nL were selected as the subsequent dosing doses to compare the inhibitory effects of each sample group on the zebrafish breast cancer xenograft model at the same dose.

[0213] Table 2. Results of MTD experiment

[0214]

[0215] To observe the distribution of AS1411-PTE in vivo, wild-type AB zebrafish transplanted with MCF-7 cells at 2 days post-fertilization (2 dpf) were randomly selected. Subsequently, the samples were injected intravenously, with Lib-PTE as the negative control group, and cultured at 35 °C for 12 h. Ten zebrafish were randomly selected from each group for fluorescence microscopy imaging to observe the distribution of the samples.

[0216] The results are as Figure 4 shown. AS1411-PTE mainly accumulated in the intestine, blood vessels, and tumor cells, confirming the effectiveness of its targeting of tumor cells. In contrast, Lib-PTE was mainly distributed in the intestine and blood vessels.

[0217] To evaluate the effect of AS1411-PTE on tumor suppression and metastasis, wild-type AB zebrafish transplanted with MCF-7 cells at 3 days post-fertilization (3 dpf) were randomly assigned to 6-well plates (30 zebrafish per well), and then the samples were injected intravenously. After treatment at 35 °C for 48 h, ten zebrafish were randomly selected from each group and imaged under a fluorescence microscope. The data were collected and analyzed using NIS-Elements D 3.20 and Image J software to analyze the fluorescence intensity of tumor cells and the metastatic distance of tumor cells.

[0218] The results are as Figure 18 shown in A. After 48 h of exposure to the samples, the green fluorescence intensity in the AS1411-PTE group was significantly reduced compared with the control group. In addition, Figure 18 C shows that in the AS1411-PTE group, the metastatic distance of tumor cells to the tail of the zebrafish decreased. Quantitative analysis using Image J software found that both the total density of tumor cells and the metastatic distance to the tail were significantly reduced after treatment with AS1411-PTE ( Figure 18 B and Figure 18 D). It is worth noting that the anti-tumor effect of AS1411-PTE is better than that of PTE, while Lib-PTE, as the negative control, has the least inhibitory effect on tumor growth and metastasis. This emphasizes the potential of AS1411-PTE in targeted drug delivery and treatment.

[0219] Subsequently, wild-type AB zebrafish transplanted with MCF-7 cells at 3 days post-fertilization (3 dpf) were selected, the samples were injected intravenously, incubated at 35 °C for 48 h, and stained with acridine orange. Ten zebrafish were randomly selected from each group for fluorescence microscopy imaging, and data analysis was performed using NIS-Elements D 3.20 software to analyze the fluorescence intensity of apoptotic tumor cells. The statistical analysis results of this index were used to evaluate the effect of the samples in promoting tumor cell apoptosis. The yellow frame line indicates the analysis area.

[0220] The results are as Figure 19 shown in A. After treatment with AS1411-PTE, the fluorescence intensity of apoptotic cells increased significantly, and the effect of PTE was slightly weaker. In contrast, Lib-PTE had little effect on promoting tumor cell apoptosis. The data histogram is as Figure 19 shown in B.

[0221] To evaluate the effect of AS1411-PTE on tumor neovascularization, human breast cancer (MCF-7) cells were microinjected into the yolk sac of transgenic vascular green fluorescent Flk1 strain zebrafish eggs at 2 days post-fertilization (2 dpf) to establish a transgenic zebrafish tumor transplantation model. Zebrafish with relatively consistent transplanted tumor cells were selected under a microscope and randomly assigned to 6-well plates, with 30 tails in each well (experimental group). The samples were administered by intravenous injection, and a normal control group and a model control group were set up simultaneously, with a volume of 3 mL per well. After treatment at 35 °C for 48 hours, 10 zebrafish were randomly selected from each experimental group and placed under a fluorescence microscope for photographing, and data were analyzed and collected using NIS-Elements D 3.20 advanced image processing software to analyze the number of subintestinal vessel sprouts. The statistical analysis results of this index were used to evaluate the efficacy of the samples in inhibiting tumor angiogenesis.

[0222] The results are as Figure 20 shown. After administration of AS1411-PTE, the number of subintestinal vessel (SIV) sprouts decreased significantly, demonstrating that AS1411-PTE has an effective inhibitory effect on tumor occurrence and development in the in vivo environment.

[0223] Discussion

[0224] Aptamer-based macromolecules have become a unique and promising platform for biological and medical research. Nucleic acid aptamers, derived from oligonucleotide libraries, are selected for their high specificity and affinity for specific molecular targets and have been widely recognized as specific ligands. These ligands contribute to the construction of targeted therapies, including aptamer-drug conjugates, aptamer-RNA conjugates, aptamer-liposome complexes, and aptamer-nanoparticle complexes. The versatility and precision of aptamers in molecular recognition make them a valuable component in the development of innovative therapeutic strategies.

[0225] The synthesis of oligonucleotides typically employs solid-phase synthesis techniques. This method offers efficiency and reproducibility in the construction of aptamers and facilitates the expansion of the applications of these macromolecules in targeted drug delivery. Automated and programmable synthesis has opened up new possibilities for the design and creation of specific ligand-drug conjugates, making the process more efficient and economical. However, there are still some challenges to overcome in the automated conjugation of new drug molecules with oligonucleotides. The first challenge is the conversion of the molecules into the corresponding phosphoramidites, which is crucial for their integration into the aptamer sequence. This key step ensures that the drug can be precisely and programmably incorporated into the oligonucleotide structure. The second challenge involves the controlled release of the drug molecule from the aptamer-drug conjugate in a biological environment. An effective drug delivery system should not only target specific cells or tissues but also ensure the timely and controlled release of the drug payload. Achieving a balance between stability during delivery and release after the drug reaches the target is a fundamental consideration.

[0226] In the present invention, these challenges are addressed through organic synthesis and biological studies. PTE is a phenolic compound that can be converted into the corresponding phosphoramidite in a simple one-step reaction. However, it should be noted that this phosphoramidite only allows the incorporation of PTE at the 5'-end of the oligonucleotide and is limited to one unit during the automated synthesis process. To address this limitation, the inventors of the present invention designed a PTE analogue, forming the PTE-G molecule by adding specific structural fragments. Molecular docking analysis was performed to elucidate the binding interactions of PTE and PTE-G with proteins.

[0227] As Figure 21 shown by the results, the predicted binding mode of PTE-G ( Figure 21 B) is very similar to the orientation observed for PTE ( Figure 21 A and 21C). The similarity in the binding interactions of the core structures of PTE and PTE-G with the target protein further supports the feasibility of PTE-G as a functional analogue. The synthesis of compound 4 is a key step that enables the smooth integration of PTE as a phosphoramidite module into the oligonucleotide sequence.

[0228] The present invention provides a general method for incorporating PTE into oligonucleotides, expanding the potential of aptamer-based drug delivery systems. Based on the present invention, the PTE of the present invention can be further combined with other drug elements, such as aptamer-multidrug conjugates. This innovative approach will contribute to the study of synergistic effects and further improve the accuracy and efficiency of targeted drug delivery strategies.

[0229] In summary, the combination of aptamer-based macromolecular, organic synthesis, and biological research has opened up new horizons in the field of targeted drug delivery. The present invention paves the way for more efficient and adaptable drug delivery systems, and is expected to provide innovative solutions for the precise and effective delivery of therapeutic agents.

[0230] All documents mentioned in this invention are cited herein by reference as if each individual document was cited by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A pterostilbene analogue, characterized in that, It has the structure shown in formula (I): Wherein, n1 is selected from 1, 2, or 3; n2 is selected from 1, 2, or 3; R1 and R2 are each independently selected from H, DMTr, 2. The pterostilbene analog according to claim 1, wherein Selected from the following group:

3. A method for preparing pterostilbene analogs, characterized in that, Comprising the steps of: (a) In an inert solvent, compound 1 reacts with compound Z1 to obtain a compound of formula 2; Wherein, X is selected from halogen, and the definitions of n1, n2, R1 and R2 are as described in claim 1.

4. The method according to claim 3, wherein The method further comprises the step of: (b) In an inert solvent, compound 2 reacts with DMTr-Cl to obtain a compound of formula 3; Wherein, the definitions of n1 and n2 are as described in claim 1.

5. The method according to claim 4, wherein The method further comprises the step of: (c) Under the protection of an inert gas and in an inert solvent, compound 3 reacts with 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite to obtain a compound of formula 4; Wherein, the definitions of n1 and n2 are as described in claim 1.

6. A nucleic acid-pterostilbene conjugate, characterized in that, The conjugate has the structure shown in formula (II): Z-(L-Y) m (II) In the formula, Z is a nucleic acid molecule; L is a divalent linking group; Y is a PTE module; The PTE module is Wherein n1 is selected from 1, 2, 3; n2 is selected from 1, 2, 3; R1 is selected from H, DMTr; m is a positive integer ≥ 1.

7. The conjugate according to claim 6, wherein, The nucleic acid molecule is selected from the following group: siRNA, miRNA, mRNA, DNA.

8. The method for preparing the conjugate according to claim 6, wherein, The method comprises the steps of: (1) Provide a compound of formula 4; (2) Using solid-phase synthesis, the compound of formula 4 reacts with nucleotide substrates to form the conjugate.

9. Use of the pterostilbene analogues as claimed in claim 1 or the conjugate as claimed in claim 6, characterized in that, For the preparation of a pharmaceutical composition for treating tumor diseases.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: 1) a pterostilbene analogue as described in claim 1 or a conjugate as described in claim 6; and 2) a pharmaceutically acceptable carrier.

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