Functionalized sialic acid compounds, methods of making and using the same

Abstract

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

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicinal compounds, and particularly relates to a functional sialic acid compound and a preparation method and application thereof. BACKGROUND

[0002] The occurrence and development of tumors is a complex multi-factor process, involving tumor cell proliferation, immune escape, and dynamic regulation of the microenvironment. The chemical prodrug strategy based on tumor-specific characteristics provides a new direction for efficient inhibition of tumor growth and reduction of systemic toxicity. Selective release of functional molecules through the catalytic action of tumor cell-specific enzymes can achieve targeted therapy for tumors, which is expected to reduce damage to normal tissues.

[0003] Sialic acid (Sia) is a class of 9-carbon monosaccharide derivatives often located at the end of the cell surface glycocalyx. Various tumor cells overexpress sialic acid on the glycocalyx. This overexpression helps tumor immune escape, and the overexpression of sialic acid on the tumor membrane surface also indicates that tumor cells need a large amount of sialic acid for 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 anabolic metabolism in cells. The C-5 N-acylated sialic acid generated in cells is expressed on the cell surface glycocalyx through a metabolic pathway, thereby introducing non-natural sialic acid or sialic acid derivatives on the cell membrane surface. However, N-acylmannosamine lacks cell and tissue specificity, resulting in the expression of N-acylated sialic acid on the cell surface of various tissues in animals, making it difficult to achieve tumor-specific sialic acid metabolic labeling. In contrast, C-1 or C-6 hydroxyl-modified N-acylmannosamine is activated by specific biological species in cancer cells, releasing N-acylmannose for tumor-specific cell membrane sugar metabolic labeling. These strategies can introduce non-natural C-5 N-acylated C-9 hydroxyl-containing sialic acid on the cell membrane surface. C-6 hydroxylation of N-acylmannose is a necessary intermediate for the biotransformation of N-acylmannose into sialic acid precursors, which limits the feasibility of introducing C-9 functional sialic acid on the cell membrane surface by using N-acylmannose as an exogenous sialic acid precursor.

[0004] James C. Paulson group reported a variety of Siglec agglutinin hemoglobin high binding force sugar ligand, which is characterized by containing different substituents at the C-9 position of sialic acid, such as PBA-Sia recognizing B cell CD22 (Siglec-2), TCC-Sia recognizing macrophage CD169 (Siglec-1). B cells are an important type of immune cells, and their functions include antibody production and secretion, antigen presentation, cytokine secretion, etc. In addition, B cells can activate various immune cells such as DC cells and NK cells. Tumor tissues also contain tumor-associated B cells. However, so far, there are still relatively few small molecule tumor immunomodulation methods based on B cells. SUMMARY

[0005] The present application aims to overcome the defects of the prior art, and provides a functionalized sialic acid compound which can be de-caged in specific cells to release functionalized sialic acid molecules, and is suitable for membrane surface glycosylation modification of specific cells.

[0006] Another object of the present application is to provide a preparation method of the above-mentioned functionalized sialic acid compound.

[0007] Still another object of the present application is to provide an application of the above-mentioned functionalized sialic acid compound.

[0008] The technical solutions of the present application are as follows:

[0009] A functionalized sialic acid compound (Pro-PBA-Sia) has the following structural formula: Among them,

[0010] R is

[0011] The preparation method of the above-mentioned functionalized sialic acid compound is characterized by the following synthesis route:

[0012]

[0013] The above-mentioned functionalized sialic acid compound is used in the preparation of an anti-tumor composition.

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

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

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

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

[0018] The beneficial effects of this invention are:

[0019] 1. This invention involves the uptake of substances by tumor cells, followed by catalysis by highly expressed esterases and carboxypeptidases within the tumor, releasing butyric acid and PBA-Sia (…). Figure 1 Butyric acid, as a known tumor growth inhibitor, can exert its effects by regulating the cell cycle and inducing apoptosis. Butyric acid has an inhibitory effect on a variety of tumors. PBA-Sia, a highly binding ligand of the B cell membrane protein CD22 introduced to the surface of cancer cells through glucose metabolism, can enhance the ability of B immune cells to recognize and attack tumors. Figure 1 PBA-Sia on the surface of cancer cells works synergistically with butyric acid released intracellularly to inhibit tumor cell proliferation.

[0020] 2. The functionalized sialic acid compound of the present invention can be activated by a specific intracellular enzyme to uncage the carboxylic acid at the C-1 position in the cell, thereby releasing the functionalized sialic acid molecule. The carboxylic acid used to encapsulate the C-1 position is L-lysine, and L-lysine can also be replaced with other molecules within an understandable range for the glycosylation modification of the membrane surface of specific cell types. Attached Figure Description

[0021] Figure 1 This diagram illustrates the chemical reaction mechanism by which the functionalized sialic acid compound of the present invention is catalyzed by carboxypeptidase in melanoma to release PBA-Sia and butyric acid. As can be seen, the released PBA-Sia is introduced into the tumor cell membrane via a glucose metabolism pathway, where it recognizes and binds to the CD22 protein of B cells, thereby inhibiting tumor growth.

[0022] Figure 2 This is a tumor cell membrane glucose metabolism marker for the uncoupling of Az-Sia-Lys-OH induced by carboxypeptidase in melanoma cancer cells, as described in Example 3 of this invention. Specifically, compared to cells incubated with Az-Sia-Glu-OH and Az-Sia-Phe-OH after treatment with DBCO-FITC, cells incubated with Az-Sia-K after treatment with DBCO-FITC showed significant fluorescence on their surface, indicating that lysine cleavage induced by carboxypeptidase B leads to the entry of Az-Sia into the cell membrane via the glucose metabolism pathway.

[0023] Figure 3This is a schematic diagram illustrating the chemical reaction used in Example 3 of the present invention to confirm the selective glucose metabolism modification of cancer cells based on masked sialic acid and enzyme uncoating using the C-1 fluorescence imaging method. Specifically: the C-1 masking group prevents 9-azidosialic acid from entering the cell membrane via the glucose metabolism pathway; carboxypeptidase catalyzes the removal of the C-1 masking group to generate 9-azidosialic acid, which is then introduced to the cell membrane surface via the glucose metabolism pathway; the content of azide on the cell membrane surface is detected by DBCO-FITC, confirming that the strategy of enzyme-catalyzed removal of C-1 masked sialic acid is used for selective glucose metabolism modification of cancer cells.

[0024] Figure 4 This demonstrates the inhibitory effect of Pro-PBA-Sia and its structural analogs on B16F10 tumors in Example 4 of the present invention. Detailed Implementation

[0025] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0026] Example 1

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

[0028]

[0029] Specifically, the steps include the following:

[0030] (1) 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 1.05 g, 5.46 mmol) was added to a solution of pyridine (40.00 mL) containing S1 (1.20 g, 2.73 mmol) and N6-(tert-butoxycarbonyl)-L-lysine tert-butyl ester (989 mg, 3.28 mmol). The reaction was stirred overnight at room temperature until the reactants were completely converted, and then concentrated. The crude product was further 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. The mixture was reacted at room temperature for 10 min and then concentrated. The crude product was further 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), and 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 dried by an oil pump to obtain a pale 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 solution of pyridine (20.00 mL) containing S1 (400 mg, 0.91 mmol) and L-phenylalanine tert-butyl ester (241 mg, 1.09 mmol). The reaction was stirred overnight at room temperature until the reactants were completely converted. After concentration under reduced pressure, the organic phase was washed with 1 M hydrochloric acid aqueous solution (40 mL), and then separated and dehydrated with anhydrous sodium sulfate. The crude product was further 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 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 carried out at room temperature for 10 min until the reaction was complete. After concentration under reduced pressure, the mixture was redissolved in water several times and then dried by an oil pump to obtain a pale 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 solution of pyridine (20.00 mL) containing S1 (400 mg, 0.91 mmol) and L-glutamic acid di-tert-butyl hydrochloride (322 mg, 1.09 mmol). The reaction was stirred overnight at room temperature until the reactants were completely converted. After concentration under reduced pressure, the organic phase was washed with 1 M hydrochloric acid aqueous solution (40 mL), and then separated and dehydrated with anhydrous sodium sulfate. The crude product was further 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 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 carried out at room temperature for 10 min until the reaction was complete. After concentration under reduced pressure, the mixture was reconstituted with water several times and then dried by an oil pump to obtain a pale yellow solid compound Az-Sia-Glu-OH (94%, 76 mg).

[0037] Example 2

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

[0039]

[0040] Specifically, the steps include the following:

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

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

[0043] (5) S7 (70 mg, 0.09 mmol) was dissolved in trifluoroacetic acid (5 mL), and 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 pale 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 sequentially. The reaction was allowed to proceed at room temperature for 24 hours until complete. After drying by pumping, the solution was redissolved in ethyl acetate (30 mL), and the organic phase was washed sequentially with saturated sodium bicarbonate solution (30 mL), 1 M hydrochloric acid aqueous solution (30 mL), and saturated brine (30 mL). The organic phase was then separated and dehydrated with anhydrous sodium sulfate. After concentrating the organic phase under reduced pressure, the solution was purified by silica gel column chromatography (eluent: dichloromethane / methanol, 50:1) to obtain a pale yellow solid compound (76%, 144 mg). The obtained solid compound (100 mg, 0.09 mmol) was dissolved in trifluoroacetic acid (5 mL), and the reaction was completed after 10 min at room temperature. After concentration under reduced pressure, it was purified using a Sep-Pak C18 column to obtain the pale yellow solid compound Pro-PBA-Sia (94%, 80 mg).

[0045] (8) N-hydroxysuccinimide (NHS, 2.04 g, 20.20 mmol) was added to a solution of dichloromethane (80 mL) containing 4-phenylbenzoic acid (BPC, 2.00 g, 10.10 mmol) and EDC (3.88 g, 20.20 mmol). After stirring at room temperature for 3 h, the reaction was complete. The organic phase was washed successively with 1 M hydrochloric acid aqueous solution (100 mL) and saturated saline solution (100 mL). The organic phase was then separated and dehydrated with anhydrous sodium sulfate to obtain the crude product biphenyl-3-carboxylic acid (2,5-dioxo-1-pyrrolidine) 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), followed by the addition of N,N-diisopropylethylamine (DIPEA, 97 mg, 0.75 mmol). The reaction was allowed to proceed overnight at room temperature until complete. After concentrating the organic phase under reduced pressure, the product S8 (80%, 156 mg) was purified by silica gel column chromatography (eluent: dichloromethane / methanol, 25:1).

[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 sequentially. The reaction was allowed to proceed at room temperature for 24 hours until complete. After drying by pumping, the solution was redissolved in ethyl acetate (30 mL), and the organic phase was washed sequentially with saturated sodium bicarbonate solution (20 mL), 1 M hydrochloric acid aqueous solution (20 mL), and saturated brine (20 mL). The organic phase was then separated and dehydrated with anhydrous sodium sulfate. After concentrating the organic phase under reduced pressure, it was 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), and the reaction was completed after 10 min at room temperature. After concentration under reduced pressure, the solid was purified using a Sep-Pak C18 column to give a pale yellow solid compound Pro-BPC-Sia (95%, 57 mg).

[0047] Example 3

[0048] B16F10 cells were incubated for 24 h with DMEM culture medium containing 1 mM of Az-Sia-Lys-OH, Az-Sia-Phe-OH and Az-Sia-Glu-OH prepared in Example 1, and then washed three times with PBS. After that, the cells were incubated with 50 mM DBCO-FITC for 1 h, washed three times with PBS, and then 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, cells treated with DBCO-FITC exhibited significant fluorescence on the cell surface after incubation with Az-Sia-Lys-OH. This indicates that lysine cleavage induced by carboxypeptidase B leads to the entry of Az-Sia into the cell membrane via the glucose metabolism pathway.

[0050] Its principle is as follows Figure 3 As shown, the C-1 masking group hinders the entry of 9-azidosialic acid (ASA) into the cell membrane via the glycolytic pathway. Carboxypeptidase catalyzes the removal of the C-1 masking group to generate 9-azidosialic acid, which is then introduced to the cell membrane surface via the glycolytic pathway. DBCO-FITC assays confirm the effectiveness of an enzyme-catalyzed C-1 masking ASA removal strategy for selectively modifying the glycolytic metabolism of cancer cells.

[0051] Example 4

[0052] Female C57BL / 6 mice aged 6-8 weeks were subcutaneously inoculated with B16F10 cells (5×10⁻⁶). 5Cells / mouse (100 μL volume) were inoculated and randomly divided into 7 groups of 6 mice each. The mice were allowed to grow tumors to 80-100 mm in size. 3 The drug was administered via the tail vein. Starting from the third day after inoculation, the drug was injected into each mouse via intravenous injection. Seven groups of mice were injected with the following drugs: (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), and (7) high-dose (Pro-PBA-Sia-OH) compound (6.6 mmol / ml, 100 μL, high-dose group). The injections were administered once every two days. Then, the correlation between mouse survival and compound dosage was recorded, along with mouse tumor size and mouse weight.

[0053] The results are as follows Figure 4 As shown in the figure, there was no significant decrease in body weight in any group of mice, indicating good biocompatibility of the drugs. In the tumor-bearing mouse groups treated with the same concentrations of Pro-PBA-Sia and Pro-BPC-Sia, the tumor mass and volume in the Pro-PBA-Sia group were significantly smaller than those in the Pro-BPC-Sia group, indicating that the released functional molecule PBA-Sia is more effective than BPC-Sia. This is consistent with the literature report that PBA-Sia has a higher binding affinity to CD22 than BPC-Sia. Under the same concentration conditions, the tumor diameter and mass in the Pro-PBA-Sia group were significantly smaller than those in the Pro-PBA-Sia-OH group, indicating that butyrylation modification can synergistically inhibit tumor growth in conjunction with the effect of PBA-Sia. In conclusion, the combined effect of Pro-PBA-Sia and butyric acid enhances the tumor-suppressive effect.

[0054] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A functionalized sialic acid compound, characterized by: The structural formula is wherein, R is .

2. The method of producing a functionalized sialic acid compound according to claim 1, characterized by: The synthetic route is as follows: 。 3. Use of the functionalized sialic acid compound according to claim 1 for the preparation of an antitumor composition, characterized in that: The antitumor composition has a growth inhibitory effect on melanoma.

4. An antitumor composition, characterized by: The effective component includes the functionalized sialic acid compound of claim 1.

5. An antitumor composition according to claim 4, wherein the compound is: ###0001### The effective component is the functionalized sialic acid compound.

6. An antitumor composition according to claim 4 or 5, characterized in that: The effective component is the functionalized sialic acid compound. The effective component is the functionalized sialic acid compound.

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