Functionalized sialic acid compound as well as preparation method and application thereof

Abstract

The invention discloses a functional sialic acid compound as well as a preparation method and application thereof, the structural formula of the functional sialic acid compound is # imgabs0 #, after the functional sialic acid compound is taken by tumor cells, the functional sialic acid compound is catalyzed by esterase and carboxypeptidase which are highly expressed in tumors, and butyric acid and PBA-Sia are released respectively; wherein the butyric acid, as a known tumor growth inhibitor, can play a role in regulating and controlling a cell cycle and inducing cell apoptosis, and has an inhibiting effect on various tumors; pBA-Sia of a high-binding-force ligand of site B cell membrane protein CD22 introduced to the surface of cancer cells through glycometabolism can enhance the tumor recognition and attack ability of B immune cells; pBA-Sia on the surfaces of cancer cells and butyric acid released in the cells have a synergistic effect, and tumor cell proliferation is inhibited.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical compounds, and in particular relates to a functionalized sialic acid compound and a preparation method and application thereof. Background Art

[0002] The occurrence and development of tumors is a complex multifactorial process involving tumor cell proliferation, immune escape, and dynamic regulation of the microenvironment. The chemical prodrug strategy designed based on tumor-specific characteristics provides a new direction for effectively inhibiting tumor growth and reducing systemic toxicity. Through the catalytic action of tumor cell-specific enzymes, the functional molecules are selectively released to achieve targeted treatment of tumors, which is expected to reduce damage to normal tissues.

[0003] Sialic acid (Sia) is a type of 9-carbon monosaccharide derivative that is often located at the end of the cell surface glycocalyx. Many tumor cell glycocalyx overexpress sialic acid. This overexpression contributes to tumor immune escape, and the overexpression of sialic acid on the surface of tumor membranes also indicates that tumor cells require a large amount of sialic acid due to their own metabolism. In previous studies, C-2 N-acylated mannose was used as an exogenous sialic acid precursor to generate the corresponding C-5 N-acylated sialic acid through anabolism in cells. The C-5 N-acylated sialic acid generated intracellularly is expressed on the cell surface glycocalyx through metabolic pathways, thereby introducing non-natural sialic acid or sialic acid derivatives on the cell membrane surface. However, N-acyl mannosamine has no cell and tissue specificity, resulting in the expression of N-acylated sialic acid on the cell surface of various tissues of animals, making it difficult to achieve tumor-specific sialic acid metabolic labeling. In contrast, N-acylated mannosamine modified with a hydroxyl group at the C-1 or C-6 position is activated by specific biological species in cancer cells to release N-acylated mannose for tumor-specific cell membrane sugar metabolism labeling. These strategies can introduce non-natural C-5-position sialic acids with different N-acylated C-9 hydroxyl groups on the cell membrane surface. Phosphorylation of the C-6 hydroxyl group of N-acylated mannose is an essential intermediate in the bioconversion of N-acylated mannose to sialic acid precursors, which limits the feasibility of introducing C-9 functionalized sialic acid on the cell membrane surface by using N-acylated mannose as an exogenous sialic acid precursor.

[0004] James C. Paulson's research group reported a variety of high-binding sugar ligands for Siglec lectin hemoglobin, which are characterized by containing different substituents at the C-9 position of sialic acid, such as PBA-Sia recognizes CD22 (Siglec-2) of B cells, and TCC-Sia recognizes CD169 (Siglec-1) of macrophages. B cells are an important type of immune cells, and their functions include the production and secretion of antibodies, antigen presentation, and the secretion of cytokines. In addition, B cells can activate a variety of immune cells, such as DC cells and NK cells. Tumor tissues also contain tumor-associated B cells. However, to date, there are relatively few small molecule tumor immunoregulatory methods based on B cells. Summary of the invention

[0005] The purpose of the present invention is to overcome the defects of the prior art and provide a functionalized sialic acid compound which can be uncaged in specific cells to release functionalized sialic acid molecules and is suitable for glycosylation modification of the membrane surface of specific cells.

[0006] Another object of the present invention is to provide a method for preparing the functionalized sialic acid compound.

[0007] Another object of the present invention is to provide applications of the functionalized sialic acid compound.

[0008] The technical solution of the present invention is as follows:

[0009] A functionalized sialic acid compound (Pro-PBA-Sia) having the structural formula Among them, R is

[0011] The method for preparing the functionalized sialic acid compound is characterized in that: its synthesis route is as follows:

[0012]

[0013] Use of the functionalized sialic acid compound in preparing an anti-tumor composition.

[0014] In a preferred embodiment of the present invention, the anti-tumor composition has a growth inhibitory effect on melanoma.

[0015] An anti-tumor composition, the effective component of which includes the functionalized sialic acid compound.

[0016] In a preferred embodiment of the present invention, the effective ingredient is the functionalized sialic acid compound.

[0017] In a preferred embodiment of the invention, it has a growth inhibitory effect on melanoma.

[0018] The beneficial effects of the present invention are:

[0019] 1. After being taken up by tumor cells, the present invention is catalyzed by esterase and carboxypeptidase highly expressed in the tumor to release butyrate and PBA-Sia ( Figure 1 ); among them, butyrate, as a known tumor growth inhibitor, can play a role by regulating the cell cycle and inducing cell apoptosis. Butyrate has an inhibitory effect on a variety of tumors; PBA-Sia, a high-binding ligand of the B cell membrane protein CD22 introduced into the surface of cancer cells through sugar metabolism, can enhance the recognition and attack ability of B immune cells against tumors ( Figure 1 ); PBA-Sia on the surface of cancer cells works synergistically with butyrate released inside the cells to inhibit tumor cell proliferation.

[0020] 2. The functionalized sialic acid compound of the present invention can be activated by specific enzymes in cells to decage the carboxylic acid at the C-1 position in cells, thereby releasing the functionalized sialic acid molecule, wherein the carboxylic acid used to encapsulate the C-1 position is L-lysine, and L-lysine can also be replaced by other molecules within an understandable range for the membrane surface glycosylation modification of specific cell types. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The functionalized sialic acid compound of the present invention is catalyzed by carboxypeptidase in melanoma to release PBA-Sia and butyric acid. It can be seen that the released PBA-Sia is introduced into the tumor cell membrane through the sugar metabolism pathway, used to recognize the CD22 protein that binds to B cells, and inhibit tumor growth.

[0022] Figure 2 This is a marker of tumor cell membrane sugar metabolism in which carboxypeptidase in melanoma cells triggers Az-Sia-Lys-OH to detach from the cage in Example 3 of the present invention. Compared with cells incubated with Az-Sia-Glu-OH and Az-Sia-Phe-OH treated with DBCO-FITC, the cell surface incubated with Az-Sia-K treated with DBCO-FITC exhibits obvious fluorescence, indicating that carboxypeptidase B triggers lysine removal, causing Az-Sia to enter the cell membrane through the sugar metabolism pathway.

[0023] Figure 3The schematic diagram of the chemical reaction for confirming the selective sugar metabolism modification of cancer cells based on masked sialic acid and enzyme detachment by the C-1 fluorescence imaging method in Example 3 of the present invention. Among them: the C-1 masking group prevents 9-azido sialic acid from entering the cell membrane through the sugar metabolism pathway; carboxypeptidase catalyzes the removal of the C-1 masking group to generate 9-azido sialic acid, and 9-azido sialic acid is introduced into the cell membrane surface through the sugar metabolism pathway; DBCO-FITC is used to detect the content of azide on the cell membrane surface, which can confirm that the strategy based on enzyme-catalyzed removal of C-1 masked sialic acid is used for selective cancer cell sugar metabolism modification.

[0024] Figure 4 The results show that Pro-PBA-Sia and its structural analogs in Example 4 of the present invention have an inhibitory effect on B16F10 tumors. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further illustrated and described below through specific implementation modes in combination with the accompanying drawings.

[0026] Example 1

[0027] The synthetic routes of Az-Sia-Lys-OH, Az-Sia-Phe-OH and Az-Sia-Glu-OH in this embodiment are as follows:

[0028]

[0029] The specific steps include:

[0030] (1) 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 1.05 g, 5.46 mmol) was added to a pyridine (40.00 mL) solution containing S1 (1.20 g, 2.73 mmol) and N6-(tert-butyloxycarbonyl)-L-lysine tert-butyl ester (989 mg, 3.28 mmol). The reaction was stirred at room temperature overnight until the reaction product was completely converted, and then concentrated. The crude product was further separated and purified by silica gel column chromatography (eluent: dichloromethane / methanol, 50:1) to obtain a white solid compound S2 (70%, 1.38 g).

[0031] (2) Compound S2 (1.00 g, 1.38 mmol) was dissolved in a mixed solution of tetrahydrofuran (THF, 20 mL) and water (2 mL), and N-iodosuccinimide (NIS, 932 mg, 4.14 mmol) was added, and the mixture was reacted at room temperature for 10 min, and then concentrated. The crude product was further separated and purified by silica gel column chromatography (eluent: dichloromethane / methanol, 10:1) to obtain a white solid compound S3 (85%, 723 mg).

[0032] (3) S3 (70 mg, 0.11 mmol) was dissolved in trifluoroacetic acid (5 mL). The reaction was completed after 10 min at room temperature. After concentration under reduced pressure, the mixture was redissolved in water several times and then pumped dry with an oil pump to obtain a light yellow solid compound Az-Sia-Lys-OH (94%, 49 mg).

[0033] (4) 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 349 mg, 1.82 mmol) was added to a pyridine (20.00 mL) solution containing S1 (400 mg, 0.91 mmol) and L-phenylalanine tert-butyl ester (241 mg, 1.09 mmol). The reaction was stirred at room temperature overnight until the reaction product was completely converted. After concentration under reduced pressure, the organic phase was washed with 1M hydrochloric acid aqueous solution (40 mL), and then the organic phase was separated and dehydrated with anhydrous sodium sulfate. The above crude product was further separated and purified by silica gel column chromatography (eluent: dichloromethane / methanol, 30:1) to obtain a white solid compound S4 (85%, 497 mg).

[0034] (5) S4 (200 mg, 0.31 mmol) was dissolved in a mixed solution of tetrahydrofuran (THF, 20 mL) and water (2 mL), and N-iodosuccinimide (NIS, 209 mg, 0.93 mmol) was added. The mixture was reacted at room temperature for 10 min, and then concentrated. The crude product was further separated and purified by silica gel column chromatography (eluent: dichloromethane / methanol, 5:1) to obtain a white solid compound (93%, 157 mg). The obtained solid compound (100 mg, 0.19 mmol) was dissolved in trifluoroacetic acid (5 mL), and the reaction was completed after 10 min at room temperature. After reduced pressure concentration, the mixture was redissolved in water several times and then pumped dry with an oil pump to obtain a light yellow solid compound Az-Sia-Phe-OH (94%, 49 mg).

[0035] (6) 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 349 mg, 1.82 mmol) was added to a pyridine (20.00 mL) solution containing S1 (400 mg, 0.91 mmol) and L-glutamic acid di-tert-butyl ester hydrochloride (322 mg, 1.09 mmol). The reaction was stirred at room temperature overnight until the reaction product was completely converted. After concentration under reduced pressure, the organic phase was washed with 1M hydrochloric acid aqueous solution (40 mL), and then the organic phase was separated and dehydrated with anhydrous sodium sulfate. The above crude product was further separated and purified by silica gel column chromatography (eluent: dichloromethane / methanol, 25:1) to obtain a white solid compound S5 (88%, 544 mg).

[0036] (7) S5 (400 mg, 0.59 mmol) was dissolved in a mixed solution of tetrahydrofuran (THF, 20 mL) and water (2 mL), and N-iodosuccinimide (NIS, 398 mg, 1.77 mmol) was added. The mixture was reacted at room temperature for 10 min, and then concentrated. The crude product was further separated and purified by silica gel column chromatography (eluent: dichloromethane / methanol, 25:1) to obtain a white solid compound (65%, 220 mg). The obtained solid compound (100 mg, 0.17 mmol) was dissolved in trifluoroacetic acid (5 mL), and the reaction was completed after 10 min at room temperature. After reduced pressure concentration, the mixture was redissolved in water several times and then pumped dry with an oil pump to obtain a light yellow solid compound Az-Sia-Glu-OH (94%, 76 mg).

[0037] Example 2

[0038] The synthetic routes of Pro-PBA-Sia, Pro-PBA-Sia-OH and Pro-BPC-Sia in this embodiment are as follows:

[0039]

[0040] The specific steps include:

[0041] (1) Compound S3 (600 mg, 0.97 mmol) was added to a methanol (10 mL) solution containing a palladium carbon catalyst (Pd / C, 60 mg, 10%). Hydrogen was introduced at room temperature and the reaction was stirred overnight. After the reaction was completed, the catalyst was removed by diatomaceous earth filtration to obtain a reduced compound S4 (93%, 534 mg).

[0042] (2) N-hydroxysuccinimide (NHS, 1.89 g, 18.68 mmol) was added to a solution of 3-phenoxybenzoic acid (2.00 g, 9.34 mmol) and EDC (3.59 g, 18.68 mmol) dissolved in dichloromethane (80 mL). The reaction was completed after stirring at room temperature for 3 h. The organic phase was washed with 1 M hydrochloric acid aqueous solution (100 mL) and saturated brine (100 mL) in turn, and then the organic phase was separated and dehydrated with anhydrous sodium sulfate to obtain PBA-NHS for standby use. PBA-NHS (422 mg, 1.36 mmol) was added to a solution of S4 (400 mg, 0.68 mmol) dissolved in methanol (5 mL), and then N,N-diisopropylethylamine (DIPEA, 262 mg, 2.04 mmol) was added. The reaction was completed after overnight reaction at room temperature. The organic phase was concentrated under reduced pressure and purified by silica gel column chromatography (eluent: dichloromethane / methanol, 25:1) to give the product S7 (50%, 266 mg).

[0043] (5) S7 (70 mg, 0.09 mmol) was dissolved in trifluoroacetic acid (5 mL). The reaction was completed after 10 min at room temperature. After concentration under reduced pressure, the mixture was purified using a Sep-Pak C18 column to obtain a light yellow solid compound Pro-PBA-Sia-OH (95%, 53 mg).

[0044] (6) S7 (140 mg, 0.18 mmol) was dissolved in pyridine (5 mL), and butyric anhydride (284 mg, 1.8 mmol) and 4-dimethylaminopyridine (DMAP, 11 mg, 0.09 mmol) were added in sequence. The reaction was completed after 24 hours at room temperature. After being pumped dry by an oil pump, it was redissolved in ethyl acetate (30 mL), and the organic phase was washed with saturated sodium bicarbonate solution (30 mL), 1M hydrochloric acid aqueous solution (30 mL), and saturated brine (30 mL) in sequence. The organic phase was then separated and dehydrated with anhydrous sodium sulfate. After the organic phase was concentrated under reduced pressure, it was separated and purified by silica gel column chromatography (eluent: dichloromethane / methanol, 50:1) to obtain a light yellow solid compound (76%, 144 mg). The obtained solid compound (100 mg, 0.09 mmol) was dissolved in trifluoroacetic acid (5 mL). The reaction was completed after 10 min at room temperature. After concentration under reduced pressure, the mixture was purified using a Sep-Pak C18 column to obtain a light yellow solid compound Pro-PBA-Sia (94%, 80 mg).

[0045] (8) N-Hydroxysuccinimide (NHS, 2.04 g, 20.20 mmol) was added to a dichloromethane (80 mL) solution containing 4-phenylbenzoic acid (BPC, 2.00 g, 10.10 mmol) and EDC (3.88 g, 20.20 mmol). The reaction was completed after stirring at room temperature for 3 h. The organic phase was washed with 1 M aqueous hydrochloric acid (100 mL) and saturated brine (100 mL) in sequence, and then the organic phase was separated and dehydrated with anhydrous sodium sulfate to obtain a crude product of biphenyl-3-carboxylic acid (2,5-dioxo-1-pyrrolidinyl) ester (BPC-NHS) for later use. BPC-NHS (148 mg, 0.50 mmol) was added to a methanol (5 mL) solution containing S6 (150 mg, 0.25 mmol), and then N,N-diisopropylethylamine (DIPEA, 97 mg, 0.75 mmol) was added. The reaction was completed after overnight reaction at room temperature. The organic phase was concentrated under reduced pressure and purified by silica gel column chromatography (eluent: dichloromethane / methanol, 25:1) to obtain the product S8 (80%, 156 mg).

[0046] (9) S8 (100 mg, 0.13 mmol) was dissolved in pyridine (5 mL), and butyric anhydride (205 mg, 1.3 mmol) and 4-dimethylaminopyridine (DMAP, 8 mg, 0.07 mmol) were added in sequence. The reaction was completed after 24 hours at room temperature. After being pumped dry by an oil pump, it was redissolved in ethyl acetate (30 mL), and the organic phase was washed with saturated sodium bicarbonate solution (20 mL), 1M hydrochloric acid aqueous solution (20 mL), and saturated brine (20 mL) in sequence. The organic phase was then separated and dehydrated with anhydrous sodium sulfate. After the organic phase was concentrated under reduced pressure, it was separated and purified by silica gel column chromatography (eluent: ethyl acetate / methanol, 50:1) to obtain a white solid compound (72%, 98 mg). The obtained solid (70 mg, 0.07 mmol) was dissolved in trifluoroacetic acid (5 mL). The reaction was completed after reaction at room temperature for 10 min. After concentration under reduced pressure, the mixture was purified using a Sep-Pak C18 column to obtain a light yellow solid compound Pro-BPC-Sia (95%, 57 mg).

[0047] Example 3

[0048] B16F10 cells were incubated with DMEM culture medium containing 1 mM Az-Sia-Lys-OH, Az-Sia-Phe-OH and Az-Sia-Glu-OH prepared in Example 1 for 24 h, and then washed three times with PBS; then the cells were incubated with 50 mM DBCO-FITC for 1 h, washed three times with PBS, and photographed using a confocal microscope.

[0049] The results are as follows Figure 2 As shown: Compared with cells incubated with Az-Sia-Phe-OH or Az-Sia-Glu-OH, the cell surface incubated with Az-Sia-Lys-OH treated with DBCO-FITC exhibited obvious fluorescence, indicating that lysine removal initiated by carboxypeptidase B led to Az-Sia entering the cell membrane through the sugar metabolism pathway.

[0050] Its principle is as follows Figure 3 As shown: The C-1 masking group prevents 9-azidosialic acid from entering the cell membrane through the sugar metabolism pathway. Carboxypeptidase catalyzes the removal of the C-1 masking group to generate 9-azidosialic acid, which is introduced into the cell membrane surface through the sugar metabolism pathway. By detecting the content of azide on the cell membrane surface by DBCO-FITC, it can be confirmed that the strategy based on enzyme-catalyzed removal of C-1 masked sialic acid is used for selective cancer cell sugar metabolism modification.

[0051] Example 4

[0052] 6-8 week old C57BL / 6 female mice were subcutaneously inoculated with B16F10 cells (5×10 5cells / mouse, volume 100μL), and randomly divided into groups after inoculation, with 6 mice in each group, for a total of 7 groups. When the mouse tumor grew to 80-100mm 3 The drug was administered through the tail vein at the time of vaccination, and starting from the third day after vaccination, each mouse was injected with the drug by intravenous injection. The 7 groups of mice were injected with drugs, respectively: (1) saline (100 μL, blank control group), (2) low-dose (Pro-PBA-Sia) compound (2.2 mmol / ml, 100 μL, low-dose group), (3) high-dose (Pro-PBA-Sia) compound (6.6 mmol / ml, 100 μL, high-dose group), (4) low-dose (Pro-BPC-Sia) compound (2.2 mmol / ml, 100 μL, low-dose group), (5) high-dose (Pro-BPC-Sia) compound (6.6 mmol / ml, 100 μL, high-dose group), (6) low-dose (Pro-PBA-Sia-OH) compound (2.2 mmol / ml, 100 μL, low-dose group), (7) high-dose (Pro-PBA-Sia-OH) compound (6.6 mmol / ml, 100 μL, high-dose group). The administration frequency was once every 2 days. The survival time of the mice was then recorded in relation to the compound dose, and the tumor size and body weight of the mice were recorded.

[0053] The results are as follows Figure 4 As shown. There was no significant decrease in the body weight of mice in all groups, indicating that each group of drugs had good biocompatibility. In the tumor-bearing mouse groups administered with Pro-PBA-Sia and Pro-BPC-Sia at the same concentration, the tumor mass and tumor volume of the Pro-PBA-Sia-administered mouse group were significantly smaller than those of the Pro-BPC-Sia group, indicating that the released functional molecule PBA-Sia was better than BPC-Sia. This is consistent with the literature report that the binding force of PBA-Sia to CD22 is higher than that of BPC-Sia. Under the same concentration conditions, the tumor diameter and mass of the Pro-PBA-Sia group were significantly smaller than those of the Pro-PBA-Sia-OH group, indicating that butyrylation modification can synergize the effect of PBA-Sia to further inhibit tumor growth. In summary, the two components of Pro-PBA-Sia and butyric acid work together to improve the tumor inhibition effect.

[0054] The above description is only a preferred embodiment of the present invention, and therefore cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A functionalized sialic acid compound, characterized in that: Its structural formula is Among them, R is 2. The method for preparing the functionalized sialic acid compound according to claim 1, characterized in that: Its synthetic route is as follows:

3. Use of the functionalized sialic acid compound according to claim 1 in preparing an anti-tumor composition.

4. The use according to claim 3, characterized in that: The anti-tumor composition has a growth inhibitory effect on melanoma.

5. An antitumor composition, characterized in that: The effective component thereof comprises the functionalized sialic acid compound described in claim 1.

6. An antitumor composition according to claim 5, characterized in that: The effective component is the functionalized sialic acid compound.

7. An antitumor composition according to claim 5 or 6, characterized in that: It has a growth inhibitory effect on melanoma.

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