A folic acid phospholipid derivative, its preparation method and application

Abstract

The present invention belongs to the technical field of pharmaceutical technology and relates to a folic acid phosphatide derivative, whose molecular structure is shown in formula (I). The present invention also provides a preparation method of the above-mentioned folic acid phosphatide derivative, and the method comprises the following steps: (1) Glycerophosphatidylcholine and triphosgene react under the condition of the presence of a basic reagent to prepare a phospholipid intermediate; (2) Decanediol and folic acid react to prepare a folic acid decanediol monoester intermediate; (3) Under the condition of the presence of a basic reagent, the phospholipid intermediate and the folic acid decanediol monoester intermediate react to obtain the folic acid phosphatide derivative. The present invention also provides its application in the field of cancer targeting drug carriers. The folic acid phosphatide derivative provided by the present invention is a polymer molecule with a novel chemical structure having a targeting effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical technology and relates to a folic acid phosphatide derivative, a preparation method thereof, and an application thereof. Background Art

[0002] Folic acid is a water-soluble vitamin, which was first extracted and purified from spinach leaves in 1941, so it is named folic acid. Folic acid plays an important role in the synthesis of proteins and nucleic acids and the metabolism of various amino acids. If humans lack folic acid, it will cause megaloblastic anemia and leukopenia, which is especially important for pregnant women. Therefore, pregnant women often need to take folic acid supplements. In feed applications, folic acid is widely used as a feed additive for poultry and livestock, including two contents of 98% and 80%, and its market sales volume increases year by year. In addition, it is worth noting that due to the discovery that folic acid and its metabolites have excellent properties in aspects such as human tumor targeting and transmembrane transport, its application prospects in the fields of new drug / drug excipient research and development have also attracted more and more attention.

[0003] For example, Chinese Patent CN106750252A discloses a distearoyl phosphatidylethanolamine-polyethylene glycol 2000-bifolic acid and a preparation method and application thereof. In this invention, 1,2-distearoyl-SN-glycero-3-phosphoethanolamine-polyethylene glycol-N-hydroxysuccinimide is first synthesized, and it is linked to the activated terminal amino folic acid through aminoadipic acid, thus synthesizing the lipid of the bifolic acid molecule. This invention also provides the application of distearoyl phosphatidylethanolamine-polyethylene glycol 2000-bifolic acid in the preparation of a bifolic acid-targeted ultrasound contrast agent. Distearoyl phosphatidylethanolamine-polyethylene glycol 2000-bifolic acid can significantly improve the targeted binding ability of the ultrasound contrast agent to breast cancer MCF-7.

[0004] In order to further obtain the best folic acid-based tumor-targeted drug carrier material, it is very necessary to synthesize new folic acid derivatives. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides a new folic acid phosphatide derivative, which is a polymer molecule with a novel chemical structure having a targeting effect.

[0006] To solve the above technical problems, the object of the present invention is achieved through the following technical solutions:

[0007] A folic acid phosphatide derivative, whose molecular structure is shown in formula (I):

[0008]

[0009] The present invention also provides a preparation method of the above folic acid phosphatide derivative, and the method includes the following steps:

[0010] (1) Glycerophosphatidylcholine and triphosgene react under the condition of the presence of a basic reagent to prepare a phospholipid intermediate of the compound shown in formula (II).

[0011]

[0012] (2) Decanediol and folic acid react as follows to prepare a folic acid decanediol monoester intermediate of the compound shown in formula (III).

[0013]

[0014] (3) Under the condition of the presence of a basic reagent, the phospholipid intermediate of the compound shown in formula (II) and the folic acid decanediol monoester intermediate of the compound shown in formula (III) react as follows to obtain a folic acid phospholipidated derivative of the compound shown in formula (I).

[0015]

[0016] In the above method for preparing a folic acid phospholipidated derivative, in the step (1), the reaction solvent is anhydrous N,N-dimethylformamide or dichloromethane; in the step (2), the reaction solvent is N,N-dimethylformamide.

[0017] In the above method for preparing a folic acid phospholipidated derivative, in the step (1), the basic reagent is 4-dimethylaminopyridine; in the step (3), the basic reagent is 4-dimethylaminopyridine.

[0018] In the above method for preparing a folic acid phospholipidated derivative, in the step (1), the molar ratio of glycerophosphatidylcholine to triphosgene is 1:(3 - 3.5); in the step (2), the molar ratio of decanediol to folic acid is 1:(1 - 1.5); in the step (3), the molar ratio of the phospholipid intermediate of the compound shown in formula (II) to the folic acid decanediol monoester intermediate of the compound shown in formula (III) is 1:(2.1 - 2.5).

[0019] In the above method for preparing a folic acid phospholipidated derivative, the step (1) includes: dissolving glycerophosphatidylcholine in anhydrous N,N-dimethylformamide, adding triphosgene and 4-dimethylaminopyridine after stirring for a period of time, continuing to stir overnight, cooling to 0 °C with an ice bath after the reaction ends, a large amount of white flocculent precipitate is formed, filtering, washing and purifying to obtain a phospholipid intermediate of the compound shown in formula (II). Preferably, the washing solvent is anhydrous ether, and the purification method is column chromatography, and the elution solvent is a 1:1 dichloromethane and methanol solution.

[0020] In the above-mentioned method for preparing a phospholipid derivative of folic acid, the step (2) comprises: adding folic acid and decanediol to N,N-dimethylformamide, heating and stirring to react, cooling to 0°C after the reaction, filtering and purifying to obtain a compound folic acid decanediol monoester intermediate as shown in formula (III). Preferably, the heating temperature is 80°C, the purification method is column chromatography, and the elution solvent is a 1:1 solution of dichloromethane and methanol.

[0021] In the above-mentioned method for preparing a phospholipid derivative of folic acid, the step (3) comprises: adding the compound phospholipid intermediate as shown in formula (II) to dichloromethane, stirring to dissolve, then adding 4-dimethylaminopyridine and the compound folate decanediol monoester intermediate as shown in formula (III), heating to reflux reaction, dialyzing the resulting solution with a 1000Da dialysis bag after the reaction, and freeze-drying to obtain the compound folate derivative as shown in formula (I). Preferably, the reflux temperature is 40°C.

[0022] Another object of the present invention is to provide a novel application of phospholipidated folic acid derivatives in the field of cancer targeted drug carriers.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The present invention utilizes the high affinity, low immunogenicity and easy modification of folic acid to folic acid receptors, couples it with phosphatidylcholine, and provides a novel phospholipidated folic acid derivative with folic acid receptor targeting effect, which can be used as a tumor-targeted drug carrier material.

[0025] 2. As a class of amphiphilic compounds, phospholipid derivatives can be used as raw materials to better prepare various new preparations such as microcapsules and micelles. The preparations prepared using the phospholipid derivatives of folic acid provided by the present invention as excipients have the advantages of simple structure, simple and diverse preparation process, stable quality, etc., and are widely used in pharmaceutical excipients and various additives. The phospholipid derivatives of folic acid provided by the present invention are widely used in the field, and have great overlap with folic acid monomers, which can maximize the use of existing customers and markets, and have broad prospects.

[0026] 3. The phospholipid derivatives of folic acid provided by the present invention have a short synthetic route, mature preparation technology, easy-to-control process route, high environmental friendliness, considerable yield, and low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is the H NMR spectrum of the phospholipid derivative of folic acid of the compound represented by formula (I).

[0028] Figure 2 It is the H NMR spectrum of the phospholipid intermediate of the compound represented by formula (II).

[0029] Figure 3 It is the 1H NMR spectrum of the folic acid decanediol monoester intermediate, a compound shown in formula (III).

[0030] Figure 4 It is the result of the MTT assay (KB cells).

[0031] Figure 5 It is the result of the MTT assay (PC9 cells). Specific Embodiments

[0032] The present invention will be further described below through the description of specific embodiments. However, this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of the present invention. As long as they do not depart from the basic idea of the present invention, they are within the scope of the present invention.

[0033] All raw materials and reagents used in the embodiments of the present invention are commercially available unless otherwise specified.

[0034] Example 1: Preparation of Phospholipid Intermediate

[0035] Dissolve glycerophosphatidylcholine (1 mmol) in anhydrous DMF and stir at 35 °C for 4 h. Add triphosgene (3 mmol) and 4-dimethylaminopyridine (1 mmol), stir at room temperature overnight. The reaction solution becomes slightly turbid, and a small amount of DMAP hydrochloride precipitates. After the reaction is completed, cool to 0 °C in an ice bath, a large amount of white flocculent precipitate forms, filter, and wash the filter cake twice with ice-cold anhydrous ether. The obtained solid is dissolved in dichloromethane and purified by column chromatography (dichloromethane: methanol = 1:1) to obtain the phospholipid intermediate with a yield of 75%. 1 H NMR(600MHz,CDCl 3 )δ5.76(m,1H),4.35-4.33(s,6H),3.38-3.66(m,2H),3.34(s,9H).

[0036] Example 2: Preparation of Folic Acid Decanediol Monoester Intermediate

[0037] Add 1 mmol of folic acid and 1,10-decanediol (1 mmol) to DMF, stir and react at 80 °C for 5 h. After the reaction is completed, cool to 0 °C, filter, and the obtained solid is purified by column chromatography (dichloromethane: methanol = 1:1) to obtain phospholipid intermediate 2 with a yield of 70%. 1 H NMR(600MHz,CDCl 3)δ8.14(s,1H),7.69(s,1H),7.43(s,1H),7.10(d,3H),4.41(t,2H),4.14 - 4.12(m,4H),3.66 - 3.62(m,7H),1.81(t,2H),1.67 - 1.65(m,4H),1.42(t,2H),1.34 - 1.28(m,12H).

[0038] Example 3: Preparation of folic acid phospholipid derivatives

[0039] Intermediate 1 (1 mmol) was added to dichloromethane and stirred until dissolved. Then DMAP (1 mmol) and Intermediate 2 (2.5 mmol) were added, and the mixture was refluxed at 40 °C for 12 h. Then the resulting solution was dialyzed through a 1000 Da dialysis bag for 48 h and freeze-dried to obtain the target product with a yield of 65%. 1 H NMR(600MHz,CDCl 3 )δ14.65(br,4H),10.78(s,1H),9.78 - 9.75(m,3H),8.18 - 8.09(m,9H),6.59(br,2H),6.12 - 6.10(br,4H),5.35 - 5.34(m,1H),3.61 - 3.46(m,9H),3.02 - 2.97(m,9H),2.71(s,9H),2.09 - 1.26(m,32H).

[0040] Example 4: Cytotoxicity assay

[0041] Using the MTT method, 1.0×104 - 1.5×104 cells / well were seeded in 96-well plates. At the time of the experiment, KB and PC9 cells were cultured in 1640 folic acid-free medium. Folic acid (FA), folic acid phospholipid derivative (FA-GC), glycerophosphatidylcholine (GC), glycerol (GL), and folic acid + glycerophosphatidylcholine (FA+GC) were prepared at different concentrations of 20, 40, 60, 80, 100 μg / mL, and their effects on cell proliferation rate were measured. The results are as Figure 4-5 shown.

[0042] As can be seen above, folic acid (FA), folic acid phospholipid derivative (FA-GC), glycerophosphatidylcholine (GC), and glycerol (GL) did not show obvious cytotoxicity to KB human oral epidermoid carcinoma cells and PC9 human lung cancer cells. In the experiment, only the control group of folic acid + glycerophosphatidylcholine (FA+GC) inhibited cell proliferation to a certain extent at high concentrations, indicating that the folic acid phospholipid derivative disclosed in the present invention has good safety.

[0043] Example 5: Cell uptake experiment

[0044] Flow cytometry was used to detect the uptake effects of folate receptor-positive cells and folate receptor-negative cells on folate phospholipid derivatives and folic acid, so as to determine the targeting of the compounds. The results showed that on folate receptor-positive cell lines KB and PC9, the uptake of folate phospholipid derivatives by cells was 9 - 13 times higher than that of folic acid; on cell lines HELA and SKOV-3, the uptake of folate phospholipid derivatives by cells was 7 - 9 times higher than that of folic acid; on cell lines U87 and HEK293, the uptake of folate phospholipid derivatives by cells was 3 - 7 times higher than that of folic acid. While on folate receptor-negative cell line A549, there was no significant difference in the uptake of folate phospholipid derivatives by cells compared with folic acid. Thus, it can be seen that folate receptor-positive cells can selectively recognize and uptake folate phospholipid derivatives, thus showing good targeting.

[0045] Example 6: Cell Uptake Experiment - Observation by Confocal Microscope

[0046] Furthermore, a confocal microscope was used to observe the uptake effects of folate receptor-positive cells and folate receptor-negative cells on the compounds. The experiments showed that folate phospholipid derivatives had higher brightness on folate receptor-positive cell lines KB and PC9; had certain brightness on cell lines HELA, SKOV-3, U87 and HEK293; and had basically no brightness on folate receptor-negative cell line A549. Thus, it was further proved that folate phospholipid derivatives can be targeted and enriched in folate receptor-positive cells, and have good prospects for developing targeted drugs / auxiliaries.

Claims

1. A folic acid phosphatidyl derivative, characterized in that, the molecular structure of the folic acid phosphatidyl derivative is shown in formula (I):

2. A preparation method of the folic acid phosphatidyl derivative according to claim 1, characterized in that, the method comprises the following steps: (1) Glycerophosphatidylcholine and triphosgene react under the condition of an alkaline reagent to prepare a compound phospholipid intermediate shown in formula (II), (2) Decanediol and folic acid react to prepare a compound folic acid decanediol monoester intermediate shown in formula (III), (3) Under the condition of an alkaline reagent, the compound phospholipid intermediate shown in formula (II) and the compound folic acid decanediol monoester intermediate shown in formula (III) react to obtain a compound folic acid phosphatidyl derivative shown in formula (I), 3. According to the preparation method of a folic acid phosphatidyl derivative described in claim 2, characterized in that, in the step (1), the reaction solvent is anhydrous N, N-dimethylformamide or dichloromethane; in the step (2), the reaction solvent is N, N-dimethylformamide.

4. According to the preparation method of a folic acid phosphatidyl derivative described in claim 2, characterized in that, in the step (1), the alkaline reagent is 4-dimethylaminopyridine; in the step (3), the alkaline reagent is 4-dimethylaminopyridine.

5. According to the preparation method of a folic acid phosphatidyl derivative described in claim 2, characterized in that, in the step (1), the molar ratio of glycerophosphatidylcholine to triphosgene is 1:(3 - 3.5); in the step (2), the molar ratio of decanediol to folic acid is 1:(1 - 1.5); in the step (3), the molar ratio of the compound phospholipid intermediate shown in formula (II) to the compound folic acid decanediol monoester intermediate shown in formula (III) is 1:(2.1 - 2.5).

6. According to the preparation method of a folic acid phosphatidyl derivative described in claim 2, characterized in that, the step (1) includes: dissolving glycerophosphatidylcholine in anhydrous N, N-dimethylformamide, adding triphosgene and 4-dimethylaminopyridine after stirring for a period of time, continuing to stir overnight, cooling to 0 °C with an ice bath after the reaction ends, a large amount of white flocculent precipitate is formed, filtering, washing and purifying to obtain a compound phospholipid intermediate shown in formula (II).

7. According to the preparation method of a folic acid phosphatidyl derivative described in claim 2, characterized in that, the step (2) includes: adding folic acid and decanediol to N, N-dimethylformamide, heating and stirring for reaction, cooling to 0 °C after the reaction ends, filtering and purifying to obtain a compound folic acid decanediol monoester intermediate shown in formula (III).

8. According to the preparation method of a folic acid phosphatidyl derivative described in claim 2, characterized in that, Step (3) includes: adding the compound phospholipid intermediate shown in formula (II) into dichloromethane, stirring to dissolve, then adding 4-dimethylaminopyridine and the compound folic acid decanediol monoester intermediate shown in formula (III), heating under reflux for reaction, after the reaction is completed, dialyzing the obtained solution with a 1000 Da dialysis bag, and freeze-drying to obtain the folic acid phospholipid derivative shown in formula (I).

9. Use of a folic acid phospholipid derivative as described in claim 1, characterized in that the folic acid phospholipid derivative shown in formula (I) is used in the field of cancer-targeted drug carriers.

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