CN114907274A - 5-fluorouracil-1-alkyl acid derivative, preparation method and application thereof - Google Patents

Abstract

The invention provides a 5-fluorouracil-1-alkyl acid derivative and a preparation method and application thereof, belonging to the technical field of organic synthesis, wherein 5-fluorouracil, 4-bromoalkyl acid ethyl ester and sodium hydroxide are used as raw materials to prepare the 5-fluorouracil-1-alkyl acid derivative, the reaction process has simple steps and operation, and the reaction condition is mild; the synthesized 5-fluorouracil-1-alkyl acid derivative is used as a prodrug of 5-fluorouracil, and can improve the in vivo kinetic process of a medicament and increase the stability of the medicament; and after the colon cancer patient takes the 5-fluorouracil-1-alkyl acid derivative, the 5-fluorouracil-1-alkyl acid derivative can inhibit the growth of tumor cells and weaken the toxic and side effects on the human body.

Description

5-Fluorouracil-1-alkyl acid derivatives, preparation method and application thereof

technical field

The invention belongs to the technical field of organic synthesis, and in particular relates to a 5-fluorouracil-1-alkyl acid derivative, a preparation method and application thereof.

Background technique

5-Fluorouracil (5-Fluorouracil, 5-FU), chemical formula C ₄ H ₃ FN ₂ O ₂ , also known as fluorouracil, is a pyrimidine analog, which is white or off-white crystalline or crystalline powder, in water Slightly soluble in ethanol, almost insoluble in chloroform; soluble in dilute hydrochloric acid or sodium hydroxide solution. As a pyrimidine fluoride, 5-fluorouracil is an antimetabolite and antineoplastic drug, which can inhibit thymidine nucleotide synthase, block the conversion of deoxypyrimidine nucleotides into thymidine nucleoside, and interfere with DNA synthesis. It also has a certain inhibitory effect on RNA synthesis. The mechanism of action of 5-FU is: through its conversion into 5-fluorouracil deoxynucleotide (5F-dUMP) in vivo, it inhibits deoxythymidylate synthase and prevents the conversion of deoxyuridylic acid (dUMP) methyl group to deoxythymidine Acid (dTMP), which affects DNA synthesis; on the other hand, due to the stable CF bond structure and the enhancement of acidity, 5-FU can bind more firmly to enzymes, and after being taken up into cells, replace the tumor nucleic acid at the molecular level. The important precursor uracil, deceptively incorporated into biological macromolecules, forms abnormal RNA and affects the function of nucleic acid, resulting in gene mutation. That is to say, when 5-FU enters the body, it will inhibit the synthesis of DNA and RNA in tumor cells, resulting in the death of tumor cells in the proliferative phase.

In the prior art, 5-FU is clinically used for digestive tract tumors, breast cancer, ovarian cancer, chorioepithelial cancer, cervical cancer, liver cancer, bladder cancer, skin cancer (topical application), leukoplakia (topical application), etc. All have certain curative effects. However, long-term use of 5-FU will cause severe side effects to patients, such as short half-life in vivo, nausea, vomiting and other adverse gastrointestinal reactions.

SUMMARY OF THE INVENTION

In view of this, the present invention provides a 5-fluorouracil-1-alkanoic acid derivative to solve the technical problem in the prior art that long-term use of 5-FU will cause severe side effects to patients.

It is also necessary to provide a preparation method of 5-fluorouracil-1-alkyl acid derivatives.

It is also necessary to provide the use of a 5-fluorouracil-1-alkyl acid derivative.

The technical scheme adopted by the present invention to solve its technical problems is:

A 5-fluorouracil-1-alkyl acid derivative, its chemical structural formula is:

n is a natural number greater than 2.

A preparation method of 5-fluorouracil-1-alkanoic acid derivatives, using 5-fluorouracil, 4-bromoalkanoic acid ethyl ester and sodium hydroxide as raw materials to prepare 5-fluorouracil-1-alkanoic acid derivatives, Include the following steps:

S1: adding organic solvent A and catalyst B to 5-fluorouracil to dissolve, then adding 4-bromo ethyl alkanoate to the reaction solution, reacting for a first predetermined time to obtain a first mixed solution;

S2: adding distilled water to the first mixed solution to quench the reaction, then extracting and washing with extractant C, and then chromatographically purifying to obtain ethyl 5-fluorouracil-1-alkyl acid;

S3: add solution D to 5-fluorouracil-1-alkyl acid ethyl ester, then add NaOH to react for a second predetermined time, adjust the pH of the reaction solution, and obtain a second mixed solution;

S4: rotary-evaporating the second mixed solution, then extracting with extractant E, and then rotary-evaporating to obtain the target product 5-fluorouracil-1-alkyl acid derivative.

Preferably, the step S1 is an anhydrous environment.

Preferably, in the step S1, the first predetermined time is 6-8h.

Preferably, in the step S1, the organic solvent A is N,N-dimethylformamide (DMF), the catalyst B is triethylamine (Et ₃ N), the 5-fluorouracil and 4-bromoalkane are The molar ratio of ethyl ester is 1:(1-1.5).

Preferably, in the step S2, the extractant C is dichloromethane.

Preferably, in the step S3, the second predetermined time is 1.8-2.3h.

Preferably, in the step S3, the solution D is a mixed solution of water and methanol, and the volume ratio of the water and methanol is (1:2).

Preferably, in the step S4, the extractant E is ethyl acetate.

The use of the above-mentioned 5-fluorouracil-1-alkyl acid derivative in a medicament for treating colon cancer.

Compared with the prior art, the beneficial effects of the present invention are:

The invention uses 5-fluorouracil, 4-bromoalkyl acid ethyl ester and sodium hydroxide as raw materials to prepare 5-fluorouracil-1-alkyl acid derivatives, the reaction process steps are simple and the reaction conditions are mild; the synthesized 5-fluorouracil- 1-Alkyl acid derivatives are used as prodrugs of 5-fluorouracil, which can improve the in vivo kinetics of the drug and increase drug stability; and after colon cancer patients take 5-fluorouracil-1-alkyl acid derivatives, 5-fluorouracil -1-Alkyl acid derivatives can inhibit the growth of tumor cells, and the toxic and side effects to the human body are weakened.

Description of drawings

Fig. 1 is the hydrogen nuclear magnetic resonance spectrum of 5-fluorouracil-1-butyric acid ethyl ester in Example 1.

Fig. 2 is the carbon nuclear magnetic resonance spectrum of 5-fluorouracil-1-butyric acid ethyl ester in Example 1.

Fig. 3 is the mass spectrum of 5-fluorouracil-1-butyric acid ethyl ester in Example 1.

Fig. 4 is the hydrogen nuclear magnetic resonance spectrum of 5-fluorouracil-1-butyric acid in Example 1.

Fig. 5 is the carbon nuclear magnetic resonance spectrum of 5-fluorouracil-1-butyric acid in Example 1.

Fig. 6 is the mass spectrum of 5-fluorouracil-1-butyric acid in Example 1

7 is a data analysis diagram of the inhibition rate of 5-fluorouracil-1-butyric acid on the proliferation of HT-29 colon cancer cells in Example 1.

Figure 8 is a data analysis diagram of the inhibition rate of 5-fluorouracil-1-butyric acid on the proliferation of NCM-460 normal colon epithelial cells in Example 1.

Detailed ways

The technical solutions and technical effects of the embodiments of the present invention will be further elaborated below with reference to the accompanying drawings of the present invention.

A 5-fluorouracil-1-alkyl acid derivative, its chemical structural formula is:

n is a natural number greater than 2;

Specifically n=3, 4, 5 . . .

Compared with the prior art, the beneficial effects of the present invention are:

The invention uses 5-fluorouracil, 4-bromoalkyl acid ethyl ester and sodium hydroxide as raw materials to prepare 5-fluorouracil-1-alkyl acid derivatives, the reaction process steps are simple and the reaction conditions are mild; the synthesized 5-fluorouracil- 1-Alkyl acid derivatives are used as prodrugs of 5-fluorouracil, which can improve the in vivo kinetics of the drug and increase drug stability; and after colon cancer patients take 5-fluorouracil-1-alkyl acid derivatives, 5-fluorouracil -1-Alkyl acid derivatives can inhibit the growth of tumor cells, and the toxic and side effects to the human body are weakened. The longer the chain length of 5-fluorouracil-1-alkyl acid derivatives, the better the pharmacokinetic properties, which can prolong the effect of the drug time to enhance the therapeutic effect.

A preparation method of 5-fluorouracil-1-alkanoic acid derivatives, using 5-fluorouracil, 4-bromoalkanoic acid ethyl ester and sodium hydroxide as raw materials to prepare 5-fluorouracil-1-alkanoic acid derivatives, Include the following steps:

S1: adding organic solvent A and catalyst B to 5-fluorouracil to dissolve, then adding 4-bromo ethyl alkanoate to the reaction solution, reacting for a first predetermined time to obtain a first mixed solution;

S2: adding distilled water to the first mixed solution to quench the reaction, then extracting and washing with extractant C, and then chromatographically purifying to obtain ethyl 5-fluorouracil-1-alkyl acid;

S3: add solution D to 5-fluorouracil-1-alkyl acid ethyl ester, then add NaOH to react for a second predetermined time, adjust the pH of the reaction solution, and obtain a second mixed solution;

S4: rotary-evaporating the second mixed solution, then extracting with extractant E, and then rotary-evaporating to obtain the target product 5-fluorouracil-1-alkyl acid derivative.

Further, the step S1 is an anhydrous environment.

Further, in the step S1, the first predetermined time is 6-8h.

Further, in the step S1, the organic solvent A is N,N-dimethylformamide (DMF), the catalyst B is triethylamine (Et ₃ N), the 5-fluorouracil and 4-bromoalkane are The molar ratio of ethyl ester is 1:(1-1.5).

Specifically, the DMF is anhydrous ultra-dry DMF, DMF is added to 5-fluorouracil, Et ₃ N is measured and added to the reaction flask, and after magnetic stirring for 30 minutes, ethyl 4-bromoalkanoate is slowly added to the reaction In the liquid, react for 6-8h to obtain the first mixed solution.

Further, in the step S2, the extractant C is dichloromethane.

Specifically, adding an appropriate amount of distilled water to the first mixed solution to quench the reaction, extracting with dichloromethane three times, washing the extract three times with distilled water, and finally purifying it by silica gel column chromatography to obtain 5-fluorouracil-1-alkyl ethyl acetate.

Further, in the step S3, the second predetermined time is 1.8-2.3h.

Further, in the step S3, the solution D is a mixed solution of water and methanol, and the volume ratio of the water and methanol is (1:2).

Further, in the step S3, the PH=2.

Specifically, water and methanol solution were added to 5-fluorouracil-1-alkyl acid ethyl ester, and then NaOH was added to react for 2 hours, and the pH of the reaction solution was adjusted to 2. It was found that no solid was precipitated, and a second mixed solution was obtained.

Further, in the step S4, the extractant E is ethyl acetate.

Specifically, after the second mixed solution is rotary evaporated to remove methanol, the remaining reaction solution is extracted with ethyl acetate for 4-5 times, and the ethyl acetate layer is rotary dried to obtain the target product 5-fluorouracil-1-alkanoic acid derivative.

The use of the above-mentioned 5-fluorouracil-1-alkyl acid derivative in a medicament for treating colon cancer.

For ease of understanding, the present invention is further illustrated by the following examples:

Example 1:

In a 250mL single-necked flask, add 6.5g (50mmol) of 5-fluorouracil, add 100ml of anhydrous ultra-dry DMF to dissolve it, add 4.17ml of triethylamine as a catalyst, and stir with a magnetic stirrer to fully mix the reaction solution for 30 minutes. minutes, 8.59ml (60mmol) of ethyl 4-bromobutyrate was slowly added to the reaction flask, after the dropwise addition, the reaction was continued at room temperature for 6-8h, after the reaction was completed, an appropriate amount of distilled water was added to the reaction solution to quench the reaction, and dichloride Methane was extracted three times, and the extract was washed three times with water, and then purified by silica gel column chromatography. 5-fluorouracil-1-butyric acid ethyl ester was obtained; then 5-fluorouracil-1-butyric acid ethyl ester was dissolved in a mixed solution of 14 ml of water and 28 ml of methanol, then 0.983 g (24.57 mmol) of NaOH was added and stirring was started for 2 h Then, the pH of the reaction solution was adjusted to 2, and no solid was found. After the methanol was removed by rotary evaporation, the remaining reaction solution was extracted with ethyl acetate for 4-5 times, and the ethyl acetate layer was rotated to dryness to obtain the product 5-fluorouracil-1-butane acid.

Referring to Figures 1, 2, and 3, ⁵ -fluorouracil- ¹ -butyric acid ethyl ester prepared in the above-mentioned embodiment was analyzed by nuclear magnetic resonance and mass spectrometry respectively; Figure 2), mass spectrum (Figure 3):

Referring to Figure 1 and Figure 2, it can be seen that: ¹ H NMR (400MHz, DMSO-d6) δ11.75 (d, 1H, J=5.2Hz, N ₃ -H), 8.06 (d, 1H, J=6.9 Hz), 4.02 (q, 2H, J=7.1 Hz, -OCH ₂ CH ₃ ), 3.64 (t, 2H, J=6.8 Hz, N-CH ₂ -CH ₂ -CH ₂ ), 2.33 (t, 2H, J=14.7 Hz, _-CH2CH2COO ), 1.84 (m, 2H, N- _CH2 - _{CH2 - CH2} ), 1.16 (t, 3H, J=7.1 Hz, _{-COOCH2CH3 )} .

¹³ C NMR (100 MHz, DMSO-d6) δ 172.74, 158.05, 157.79, 150.11, 141.21, 138.94, 130.67, 130.34, 60.37, 47.51, 30.85, 23.96, 14.51.

Mass spectral data: ESI-MS (m/z): 243.2 [MH ⁺ ].

It can be seen from the hydrogen NMR data in Figure 1 that the chemical shift value of 11.75 is the hydrogen atom on the N3 of the pyrimidine ring, and the chemical shift value of 8.06 is the hydrogen atom on the 6th position of the pyrimidine ring. The chemical shift value of the hydrogen atom at the N1 position of the pyrimidine ring of 5-fluorouracil is 10.73, but no hydrogen atom with a chemical shift value of about 10.73 is found in the H NMR spectrum of ethyl 5-fluorouracil-1-butyrate. It shows that the hydrogen atom at the N1 position is completely replaced by ethyl butyrate. In addition, it was detected by electrospray mass spectrometry, and the ion peak at 243.2 was found to be the signal peak of ethyl 5-fluorouracil-1-butyrate in the negative ion mode. All the above results confirmed that 4-bromobutyric acid ethyl ester was linked to the N1 position of 5-fluorouracil to form the intermediate product 5-fluorouracil-1-butyric acid ethyl ester.

The 5-fluorouracil-1-butyric acid prepared in the above example was detected by nuclear magnetic resonance; wherein ¹ H NMR spectrum (Fig. 4), ¹³ C NMR spectrum (Fig. 5), mass spectrum (Fig. 6).

Please refer to Figure 5 and Figure 6, it can be seen that: ¹ H NMR (400MHz, DMSO-d ₆ )δ11.73 (d, 1H, J=5.2Hz, N ₃ -H), 10.51 (s, 1H, -CH) ₂ - _CH2 - _CH2 -COOH), 8.06 (d, 1H, J=6.8Hz), 3.64 (t, 2H, J=6.9Hz, N- _CH2 - _CH2 - _CH2 ), 2.25 (t, 2H, J=7.4Hz, _-CH2CH2COO ), 1.81 (q, 2H, J=7.2Hz, N- _CH2 - _{CH2 - CH2} ).

¹³ C NMR (101 MHz, DMSO-d ₆ ) δ 174.28, 158.07, 157.81, 150.11, 141.18, 138.91, 130.74, 130.41, 47.59, 31.01, 24.08.

Mass spectral data: ESI-MS (m/z): 215.1 [MH ⁺ ].

It can be seen from the H NMR spectrum data in Fig. 4 that the hydrogen signals with chemical shift values of 4.02 and 1.16 disappear. In addition, it was detected by electrospray mass, and the ion peak at 215.1 was found to be the signal peak of 5-fluorouracil-1-butyric acid in negative ion mode. All the above results confirmed that 5-fluorouracil-1-butyric acid ethyl ester was hydrolyzed to 5- Fluorouracil-1-butyric acid.

Therefore, it can be confirmed that the prepared final product solid 5-fluorouracil-1-butyric acid is consistent with the final product in the preset synthetic route.

The prepared 5-fluorouracil-1-butyric acid was subjected to the following experiments.

1. Antitumor activity test

The inhibition of 5-fluorouracil-1-butyric acid on colon cancer cell proliferation was detected by CCK-8 method. Take the logarithmic growth cycle, the human colon cancer HT-29 cells in good growth state are digested and pipetted into a single cell suspension, seeded in 96-well plates with 5×103 cells per well, and placed at 37°C, 5% _CO2 Cultivated for 24h. The cells were synchronized by the serum starvation method, and the blank group (without cells and drugs), the control group (without drugs) and the drug-added experimental group were set up, with 4 replicate wells in each group. 5, 10, 20, 40, 80, and 160 μM concentration were sequentially added with 200 μL of drug-containing medium, and then the 96-well plate was placed in a cell incubator for 24, 48, and 72 hours, respectively. Finally, follow the instructions of the Cell Proliferation and Toxicity Detection Kit (CCK-8), aspirate and discard the old medium, add 100 μl of serum-free medium containing 10% CCK-8 solution to each well, incubate at 37°C for 0.5 h, and use a microplate reader. The absorbance value (450 nm) of each well was measured and repeated three times. The cell viability of each group was calculated according to the following formula: cell viability (%)=(OD value of drug-added experimental group-OD value of blank group)/(OD value of control group-OD value of blank group)×100%. , the experimental results are shown in Figure 7.

As can be seen from Figure 7 (the inhibition rate of 5-fluorouracil-1-butyric acid on the proliferation of HT-29 colon cancer cells), we used the CCK-8 method to carry out in vitro anti-inhibition of the synthesized 5-fluorouracil-1-butyric acid. Tumor activity assessment. Using 5Fu as a positive control group, the cell viability at different concentrations and different time points was studied. The compounds inhibited the proliferation of colon cancer HT-29 cells in a concentration-dependent and time-dependent manner. We found that 5-fluorouracil-1-butyric acid did not show very good antitumor activity, which may be due to the lack of cell culture environment in vitro. Facilitates its release and lacks enzymes required for its metabolism in vitro.

2. Cytotoxicity Experiment

The toxicity of 5-fluorouracil-1-butyric acid to human normal colon epithelial cells was detected by CCK-8 method. Taking the logarithmic growth cycle, the normal human colon epithelial NCM-460 cells in good growth state were digested and pipetted into a single cell suspension, and 1×104 cells per well were inoculated in a 96-well plate and placed at 37°C, 5% CO2 Cultivated for 24h. The cells were synchronized by the serum starvation method, and the blank group (without cells and drugs), the control group (without drugs) and the drug-added experimental group were set up, with 4 replicate wells in each group. 5, 10, 20, 40, 80, and 160 μM concentration were sequentially added with 200 μL of drug-containing medium, and then the 96-well plate was placed in a cell incubator for 24, 48, and 72 hours, respectively. Finally, follow the instructions of the Cell Proliferation and Toxicity Detection Kit (CCK-8), aspirate and discard the old medium, add 100 μl of serum-free medium containing 10% CCK-8 solution to each well, incubate at 37°C for 1.5 hours, and use a microplate reader. The absorbance value (450 nm) of each well was measured and repeated three times. The calculation of cell proliferation activity is based on the above formula, and the experimental results are shown in FIG. 8 .

As can be seen from Figure 8 (the inhibition rate of 5-fluorouracil-1-butyric acid on the proliferation of NCM-460 normal colon epithelial cells), we tested 5-fluorouracil-1- Toxicity of butyric acid to human normal colonic epithelial NCM-460 cells. Compared with the positive control 5Fu, the inhibition of 5-fluorouracil-1-butyric acid on cells did not increase significantly with the increase of the concentration and the prolongation of the action time, which could indicate that 5-fluorouracil-1-butyric acid had a negative effect on the cells. Normal colon cells are less toxic and, therefore, still have some specificity and selectivity for tumor cells. And compared with 5Fu, 5-fluorouracil-1-butyric acid can improve the pharmacokinetic properties, prolong the drug action time, and enhance the therapeutic effect. Therefore, the longer the chain length of 5-fluorouracil is, the more effective the drug is. the better.

The chemical equation of 5-fluorouracil-1-butyric acid prepared by the present invention is:

What is disclosed above is only the preferred embodiment of the present invention, of course, it cannot limit the scope of the right of the present invention. Those of ordinary skill in the art can understand that all or part of the process of realizing the above-mentioned embodiment can be made according to the claims of the present invention. The equivalent changes of the invention still belong to the scope covered by the invention.

Claims (10)

1. A5-fluorouracil-1-alkyl acid derivative characterized in that: the chemical structural formula is as follows:

n is a natural number greater than 2.

2. A preparation method of 5-fluorouracil-1-alkyl acid derivatives is characterized in that: the preparation method of the 5-fluorouracil-1-alkyl acid by using 5-fluorouracil, 4-bromoalkyl ethyl ester and sodium hydroxide as raw materials comprises the following steps:

s1: adding an organic solvent A and a catalyst B into 5-fluorouracil for dissolution, adding 4-ethyl bromoalkyl acid into a reaction solution, and reacting for a first preset time to obtain a first mixed solution;

s2: adding distilled water into the first mixed solution for quenching reaction, extracting and washing by using an extractant C, and then carrying out chromatography purification to obtain 5-fluorouracil-1-alkyl ethyl ester;

s3: adding the solution D into the 5-fluorouracil-1-alkyl ethyl ester, adding NaOH to react for a second preset time, and adjusting the PH of the reaction solution to obtain a second mixed solution;

s4: and (4) carrying out rotary evaporation on the second mixed solution, extracting by using an extracting agent E, and carrying out rotary evaporation again to obtain the target product 5-fluorouracil-1-alkyl acid derivative.

3. The method for producing 5-fluorouracil-1-alkyl acid derivatives according to claim 2, characterized in that: in step S1, the environment is anhydrous.

4. The method for preparing a 5-fluorouracil-1-alkyl acid derivative as claimed in claim 2, wherein the first predetermined time is 6 to 8 hours in step S1.

5. The method for producing 5-fluorouracil-1-alkyl acid derivatives as claimed in claim 2, wherein in the step S1, the organic solvent A is N, N-Dimethylformamide (DMF), and the catalyst B is triethylamine (Et) ₃ N), the molar ratio of the 5-fluorouracil to the ethyl 4-bromoalkyl acid is 1: (1-1.5).

6. The method for preparing 5-fluorouracil-1-alkyl acid derivatives as claimed in claim 2, wherein the extractant C is dichloromethane in step S2.

7. The method for preparing a 5-fluorouracil-1-alkyl acid derivative as claimed in claim 2, wherein the second predetermined time is 1.8 to 2.3 hours in step S3.

8. The method for preparing 5-fluorouracil-1-alkyl acid derivatives according to claim 2, wherein in the step S3, the solution D is a mixed solution of water and methanol, and the volume ratio of the water to the methanol is (1: 2).

9. The method for preparing 5-fluorouracil-1-alkyl acid derivatives as claimed in claim 3, wherein in the step S4, the extractant E is ethyl acetate.

10. The use of 5-fluorouracil-1-alkyl acid derivatives as claimed in claim 1 or 2, characterized in that: the 5-fluorouracil-1-alkyl acid derivative is used in a medicament for treating colon cancer.

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