TW201803885A - Method for preparing precursor of gene expression probe - Google Patents

Abstract

A method for preparing a precursor of gene expression probe is revealed. First prepare 2-deoxy-2-fluoro-3,5-di-O-benzoyl-[alpha]-D-arabinofuranosyl bromide and 2,4-bis-O-(trimethylsilyl)-5-bromouracil respectively. Then use 2-deoxy-2-fluoro-3,5-di-O-benzoyl-[alpha]-D-arabinofuranosyl bromide and 2,4-bis-O-(trimethylsilyl)-5-bromouracil to perform a coupling reaction and get a 1-(3,5-di-O-benzoyl-2-deoxy-2-fluoro-[beta] -D-arabinofuranosyl)-5-bromouracil. Next use 1-(3,5-di-O-benzoyl-2-deoxy-2-fluoro-[beta]-D-arabinofuranosyl)-5-bromouracil generated and hexabutylditin to perform a substitution reaction in an anhydrous 1,4-dioxane solution and get 1-(3,5-di-O-benzoyl-2-deoxy-2-fluoro-[beta]-D-arabinofuranosyl)-5-tributylstannyl)uracil. At last carry out debenzoylation of 1-(3,5-di-O-benzoyl-2-deoxy-2-fluoro-[beta]-D-arabino-furanosyl)-5-tributylstannyl)uracil to get 5-tributylstannyl-1-(2-deoxy-2-fluoro-[beta]-D-arabinofuranosyl)uracil (FSAU), a precursor of gene expression probe.

Description

Manufacturing method of gene expression contrast agent precursor

The present invention relates to a method for manufacturing a compound, and particularly to a method for manufacturing a precursor of a gene expression contrast agent.

Gene therapy (gene therapy) is a method that uses gene transfer technology to introduce the therapy into a patient's body with the help of a vector, so that the therapeutic gene can be expressed in the organism to produce a corresponding protein product. Reply to make treatment. Among them, the key to affect the effectiveness of gene therapy is: the carrier used must have high infectivity, so that the DNA can be effectively delivered to the target cells, but it cannot cause an immune response or produce side effects; the DNA delivered to the body , Must not cause genetic or other changes in the body; and the DNA sent to the body must be able to successfully produce protein products in the cell to exert its effect. However, how to judge and confirm whether the therapeutic gene is successfully carried into the target cells is also a very important part of the gene therapy process. Get treatment. Among them, the key to affect the effectiveness of gene therapy is: the carrier used must have high infectivity, so that the DNA can be effectively delivered to the target cells, but it cannot cause an immune response or produce side effects; the DNA delivered to the body , Must not cause genetic or other changes in the body; and the DNA sent to the body must be able to successfully produce protein products in the cell to exert its effect. However, how to judge and confirm whether the therapeutic gene is successfully carried into the target cells is also a very important part of the gene therapy process. Get treatment. Among them, the key to affect the effectiveness of gene therapy is: the carrier used must have high infectivity, so that the DNA can be effectively delivered to the target cells, but it cannot cause an immune response or produce side effects; the DNA delivered to the body , Must not cause genetic or other changes in the body; and the DNA sent to the body must be able to successfully produce protein products in the cell to exert its effect. However, how to judge and confirm whether the therapeutic gene is successfully carried into the target cells is also a very important part of the gene therapy process.

Reporter gene is an auxiliary judgment tool developed in response to the characteristic that the expression of the therapeutic gene in the cell is sometimes difficult to detect in a timely manner. It is usually designed behind the therapeutic gene. Transplanted into the target cells together can not only confirm whether they successfully colonized the target cells, but also can be used to indirectly confirm that the performance of the therapeutic gene is normal. Reporter genes are usually very easy to detect and have the advantages of being sensitive, quantifiable, and reanalyzable. At present, most of them are presented in the form of molecular imaging, which means using optical, magnetic vibration, microbubbles, or gamma The radiography method is used to observe the molecular or cellular changes in the organism, and to observe the gene expression of the reporter gene through different application methods such as promoter or enhancer performance, gene expression and transfection system after the target gene is colonized Infer the performance of the therapeutic gene in the target cell.

Herpes Simplex Virus Type 1 Thymine Phosphorylase Reporter Gene (Herpes Simplex Virus 1 thymidine kinase reporter gene (HSV1-tk)) is a relatively commonly used reporter gene that is delivered to the target using a vector at the same time as the therapeutic gene Intracellular, it is transcribed into messenger ribonucleic acid (mRNA) by intracellular continuous or inducible promoter activation, and then translated into herpes simplex virus type 1 thymidine phosphorylase expression protein (HSV1-TK) in the cell Within the role. In order to observe the performance of HSV1-TK expression protein, at this time, nucleotide analogs with radioactive flags, such as non-uridine (Fialuridine, FIAU), can be worn freely according to the characteristics of their nucleotide analogs. Pass the cell membrane and enter the cell membrane. When the radioactively labeled nucleotide analogues enter the cell, the HSV1-TK expression protein will phosphorylate the radioactively labeled nucleotide analogues. ), So that it no longer has the property of freely penetrating the cell membrane and is left in the cell. At this time, the degree of accumulation of radioactive materials in the cell can directly reflect the activity of the HSV1-TK-expressing protein and the HSV1-tk gene, and then indirectly estimate the therapeutic gene that is carried with the cell. Performance.

The nucleotide analogues are mainly obtained by replacing the five-carbon sugars or partial structures on the bases of purine or pyrimidine-type nucleotides of the human body. Commonly, the non-uridine system replaces the methyl group of the fifth carbon of thymine on the base with an iodine atom, and replaces its five-carbon sugar with a fluorine atom based on a hydrogen atom on the second carbon. In this case, the iodine atom can be further replaced by the isotope iodine 124 to achieve the purpose of radioactive tracking. The non-uridine, which is replaced by the isotope 124, not only has a good contrast effect, but also has less side effects on the human body, but its high cost results in high clinical research costs. In view of this, in order to effectively reduce the clinical research cost of related gene therapy, how to develop an easily prepared non-uridine precursor which not only has high yield, low preparation cost, but also can be easily and effectively replaced with 124 with iodine. The non-uridine, and its effective application in related gene therapy has become a very important subject in related medical research.

The main object of the present invention is to provide a method for manufacturing a gene expression contrast agent precursor. The gene expression contrast agent precursor generated by the manufacturing method has a leaving group that can be easily replaced, and can be more easily linked with iodine. The isotope is substituted to generate the gene expression contrast agent with better production efficiency.

Another object of the present invention is to provide a method for manufacturing a gene expression contrast agent precursor. The manufacturing method has the advantages of simple steps and short time consumption, which can effectively reduce production costs and improve production efficiency.

Yet another object of the present invention is to provide a method for manufacturing a precursor for a gene expression contrast agent. Most of the intermediate steps of the manufacturing method can continue the reaction without complicated separation and purification, which not only saves time-consuming production but also It can effectively improve the yield of intermediate products, can obtain more products at a lower cost, and achieve the purpose of saving costs and improving production efficiency.

In order to achieve the above-mentioned object, the present invention discloses a method for manufacturing a precursor of a gene expression contrast agent, the steps of which are first taking a 2-deoxy-2-fluoro-1,3,5-tri-oxo-benzyl- Bromination reaction of α-D-arabinofuranose with monohydrobromide acetic acid solution to produce 2-deoxy-2-fluoro-3,5-di-oxo-benzylidene-α-D-arabinofuron bromide Glycosyl, and additionally take 5-bromouracil and hexamethyldisilazane for silylation protection reaction to generate 2,4-bis-oxy- (trimethylsilyl) -5-bromouracil Then, the 2-deoxy-2-fluoro-3,5-di-oxy-benzylidene-α-D-brominated arabinofuranosyl group and the 2,4-bis-oxy- (trimethyl Silyl) -5-bromouracil undergoes a coupling reaction to form 1- (3,5-di-oxy-benzylidene-2-deoxy-2-fluoro-β-D-arabinofuranosyl)- 5-bromouracil, and the resulting 1- (3,5-di-oxy-benzylidene-2-deoxy-2-fluoro-β-D-arabinofuranosyl) -5- The substitution reaction of bromouracil and hexa-n-butylditin in anhydrous dioxane solution yields 1- (3,5-di-oxy-benzylidene-2-deoxy-2-fluoro- β-D-arabinofuranosyl) -5- (tributane Stannous) uracil, and finally the 1- (3,5-di-oxy-benzylidene-2-deoxy-2-fluoro-β-D-arabinofuranosyl) -5- ( Tributanetinyl) uracil is removed by phenylhydrazone protection reaction to obtain 5- (tributanestannyl) -1- (2-deoxy-2-fluoro-β-D-arabinofuranosyl) Uracil completes the preparation of the gene expression contrast agent precursor of the present invention.

In an embodiment of the present invention, it is also disclosed that the concentration of the hydrogen bromide acetic acid solution used in the bromination reaction is only 20% to 40% by weight.

In one embodiment of the present invention, it is also disclosed that the reaction temperature of the bromination reaction is between -196 ° C and 13 ° C.

In one embodiment of the present invention, it is also disclosed that the reaction time of the bromination reaction is 15 hours to 25 hours.

In one embodiment of the present invention, it is also disclosed that the silylation protection reaction is based on ammonium sulfate and hexamethyldisilazane.

In one embodiment of the present invention, it is also disclosed that the reaction temperature of the silylation protection reaction is between 120 ° C and 160 ° C.

In one embodiment of the present invention, it is also disclosed that the reaction time of the silylation protection reaction is 2 hours to 6 hours.

In one embodiment of the present invention, it is also disclosed that the reaction temperature of the coupling reaction is between 70 ° C and 110 ° C.

In one embodiment of the present invention, it is also disclosed that the reaction time of the coupling reaction is 15 hours to 25 hours.

In one embodiment of the present invention, it is also disclosed that the substitution reaction uses dichlorobis (triphenylphosphine) palladium as a catalyst.

In one embodiment of the present invention, it is also disclosed that the reaction temperature of the substitution reaction is between 90 ° C and 130 ° C.

In one embodiment of the present invention, it is also disclosed that the reaction time of the substitution reaction is from 18 hours to 30 hours.

In one embodiment of the present invention, it is also disclosed that the protection reaction for removing phenylfluorenyl group is performed in an aqueous solution of ammonium hydroxide, wherein the concentration of the ammonium hydroxide solution is only 15% to 25% by weight.

According to an embodiment of the present invention, it is also disclosed that the reaction temperature of the phenylhydrazone removal protection reaction is between 20 ° C and 30 ° C.

According to an embodiment of the present invention, it is also disclosed that the reaction time of the phenylhydrazone protection reaction is 18 hours to 30 hours.

In one embodiment of the present invention, it is also disclosed that after the phenylhydrazone protection reaction is removed, the 5- (tributanestannyl) -1- (2-deoxy-2-fluoro-β-D- Arabinofuranosyl) uracil can further undergo a substitution reaction with a radionuclide selected from the group consisting of iodine (I) elements to form a radioactive iodine (I) non-uridine.

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Figure 1: This is a flow chart of compound preparation according to a preferred embodiment of the present invention.

In order to make the reviewing committee members have a better understanding and understanding of the features of the present invention and the effects achieved, we would like to provide a better embodiment and a detailed description with the following description:

In the present invention, a novel method for preparing a compound is provided for a situation in which the current cost of radioactive iodine (I) non-uridine is too high, resulting in excessively high costs required for clinical research and treatment. It is directed against the radioiodine (I) non-uracil precursor: 5- (tributanestannyl) -1- (2-deoxy-2-fluoro-β-D-arabinofuranosyl) uracil (5-tributylstannyl-1- (2-deoxy-2-fluoro-β-D-arabinofuranosyl) uracil, FSAU) has been improved. It not only provides different reaction materials and steps to simplify the process of FSAU, but also provides The extraction method with higher recovery rate can reduce unnecessary waste in the production process and increase the yield in the production process, so as to effectively reduce the cost in the preparation process.

In the following, the components, properties and preparation methods included in the method for preparing the gene expression contrast agent precursor of the present invention are further explained:

Please refer to FIG. 1, which is a preparation flow chart of the first embodiment of the present invention. As shown in the figure, the preparation method of the gene expression contrast agent precursor of the present invention includes the following steps:
Step S11: Take 2-deoxy-2-fluoro-1,3,5-tri-oxy-benzylidene-α-D-arabinofuranose and monobromine acetic acid solution to perform bromination reaction to form 2 -Deoxy-2-fluoro-3,5-di-oxo-benzylidene-α-D-arabinofuranosyl bromide;
Step S12: Another 5-bromouracil and hexamethyldisilazane are subjected to a silylation protection reaction to generate 2,4-bis-oxy- (trimethylsilyl) -5-bromouracil;
Step S14: Take the 2-deoxy-2-fluoro-3,5-di-oxy-benzylidene-α-D-brominated arabinofuranosyl group and the 2,4-bis-oxy- (trimethyl Silyl) -5-bromouracil undergoes a coupling reaction to form 1- (3,5-di-oxy-benzylidene-2-deoxy-2-fluoro-β-D-arabinofuranosyl) -5-bromouracil;
Step S16: Take the 1- (3,5-di-oxy-benzylidene-2-deoxy-2-fluoro-β-D-arabinofuranosyl) -5-bromouracil and hexa-n-butyl Ditin in an anhydrous dioxane solution to form a 1- (3,5-di-oxy-benzylidene-2-deoxy-2-fluoro-β-D-arabinofuranosyl group ) -5- (tributanestannyl) uracil; and step S18: take the 1- (3,5-di-oxy-benzylidene-2-deoxy-2-fluoro-β-D- Arabinofuranosyl) -5- (tributanestannyl) uracil is removed by a phenylhydrazone protection reaction to obtain 5- (tributanestannyl) -1- (2-deoxy-2-fluoro- β-D-arabinofuranosyl) uracil.

As shown in step S11 in the figure, the method for preparing a gene expression contrast agent precursor provided by the present invention is based on 2-deoxy-2-fluoro-1,3,5-tri-oxy-benzyl-α -D-arabinofuranose (2-deoxy-2-fluoro-1,3,5-tri-O-benzoyl-α-D-arabinofurance) as a starting reactant, and bromination reaction with acetic acid monobromide solution After that, 2-Deoxy-2-fluoro-3,5-di-oxo-benzylidene-α-D-brominated arabinofuranosyl group (2-Deoxy-2-fluoro-3,5-di -O-benzoyl-α-D-arabinofuranosyl bromide), as shown in the following formula 1:


(Formula 1)

Wherein, the hydrogen bromide acetic acid solution used in this step is the 2-deoxy-2-fluoro-1,3,5-tri-oxy- One of the benzamidine groups in benzamyl-α-D-arabinofuranose is brominated. After the reaction is completed, it is washed with an organic solvent and an alkaline solution, and then the organic layer is removed to remove water. The product was obtained by concentration under pressure.

Furthermore, in the bromination reaction provided by the present invention, the weight percentage concentration of the hydrobromic acid solution used is between 20% and 40%, preferably between 25% and 35%, and In the low temperature environment, the temperature is between -196 ° C and 13 ° C, and the above reaction is preferably performed in an ice bath environment, and the reaction time is between 15 hours and 25 hours.

Using this reactant and reaction solution to carry out the reaction, the product can be recovered by using only ordinary solution after the reaction is completed, without the need for complicated separation and purification steps, and the recovery yield is as high as 86%, which is not only more effective than the existing technology Simplifying the steps greatly improves the yield of the reaction and reduces unnecessary waste of reaction materials in the reaction step.

In addition, as shown in step S12, in the method for preparing a gene expression contrast agent precursor provided by the present invention, another initial reaction is also provided to provide a reactant required for generating a gene expression contrast agent precursor, which is a 5-bromourea The silylation reaction of pyrimidine with hexamethyldisilazane generates a 2,4-bis-oxy- (trimethylsilyl) -5-bromouracil, as shown in the following formula 2:


(Formula 2)

Wherein, the silylation protection reaction provided by the present invention is performed in an environment of ammonium sulfate solution, and the ditrimethylsilicon on the hexamethyldisilazane is heated under the condition of passing nitrogen gas. It is respectively connected to the 5-bromouracil oxy group for silylation protection, and then the solvent is removed without performing any separation and purification step, that is, the subsequent reaction is performed with the obtained crude product.

Furthermore, in the silylation protection reaction provided by the present invention, the temperature of the heating environment is between 120 ° C and 160 ° C, and the reaction time is between 2 hours and 6 hours.

Utilizing the product produced by the reaction in step S12, since no isolation and purification step is required, any loss in yield generated during the isolation and purification step can be avoided, thereby increasing the yield of the gene expression contrast agent precursor. And reduce the cost of preparation.

Next, as shown in step S14, in the method for preparing a gene expression contrast agent precursor provided by the present invention, after collecting the products generated in steps S11 and S12, the 2-deoxy-2-fluoro-3 is then taken, 5-Di-oxo-benzylidene-α-D-brominated arabinofuranosyl group and the 2,4-bis-oxo- (trimethylsilyl) -5-bromouracil are coupled to form a 1- (3,5-Di-oxo-benzylidene-2-deoxy-2-fluoro-β-D-arabinofuranosyl) -5-bromouracil, as shown in the following formula III:


(Formula 3)

Wherein, the coupling reaction provided by the present invention is the 2-deoxy-2-fluoro-3,5-di-oxy-benzylidene-α-D-brominated arabinofuranosyl group and the 2,4- Di-oxy- (trimethylsilyl) -5-bromouracil is heated under reflux in a nitrogen atmosphere to carry out the reaction. After the reaction is completed, it is concentrated under reduced pressure and washed with organic solvents to obtain the solid. Product of the reaction.

In the heating and refluxing process of the aforementioned coupling reaction, the reaction temperature is between 70 ° C and 110 ° C, and the reaction time is between 15 hours and 25 hours.

In this reaction, because the reaction time is lengthened and the product is recovered using only a small amount of organic solvent for cleaning, the complicated separation and purification steps in the prior art are also simplified, so the yield can also be increased to reduce For the purpose of manufacturing cost, the yield of 64% is still higher in this step, which is much higher than the preparation method of the gene expression contrast agent precursor in the prior art.

As shown in step S16, in the method for preparing a gene expression contrast agent precursor provided by the present invention, the product 1- (3,5-di-oxy-benzylidene-2-deoxy-2) generated in step S13 -Fluoro-β-D-arabinofuranosyl) -5-bromouracil will be further substituted with hexa-n-butylditin in an anhydrous dioxane solution environment to form a 1- (3,5- Di-oxy-benzylidene-2-deoxy-2-fluoro-β-D-arabinofuranosyl) -5- (tributanestannyl) uracil, as shown in the following formula four:


(Formula 4)

Wherein, the substitution reaction provided by the present invention is bromine on the 2-deoxy-2-fluoro-3,5-di-oxy-benzylidene-α-D-brominated arabinofuranosyl group through a catalyst. After the radical is oxidized, the bromo group is removed, and then the tributanestannyl group on the hexa-n-butylditin is substituted on the six-membered ring. It is based on the 1- (3,5-di-oxy-benzylidene-2-deoxy-2-fluoro-β-D-arabinofuranosyl) -5-bromouracil in anhydrous dioxane In the solution environment, hexa-n-butyl ditin and the catalyst are heated together under reflux, and then the crude product is concentrated under reduced pressure, and after being extracted with an organic solvent, these organic solvents are separated and purified by liquid chromatography to obtain the 1 -(3,5-di-oxo-benzylidene-2-deoxy-2-fluoro-β-D-arabinofuranosyl) -5- (tributanestannyl) uracil.

Among them, the catalyst used in the substitution reaction provided by the present invention may be a catalyst, a nickel catalyst, or a copper catalyst, and dichlorobis (triphenylphosphine) palladium is more preferred. Is better. Is better.

Furthermore, in the heating and refluxing process described in the substitution reaction provided by the present invention, the reaction temperature is between 90 ° C and 130 ° C, and the reaction time is between 18 hours and 30 hours.

Furthermore, in the liquid chromatography process described above, the organic solvent used to extract the crude product may be ether, ethyl acetate or hexane, and the chromatography tube used in the liquid chromatography method is filled with The system is selected from the 1- (3,5-di-oxy-benzylidene-2-deoxy-2-fluoro-β-D-arabinofuranosyl) -5- (tributane Tin-based) uracil products have a high affinity, preferably filled with silicon dioxide, the mobile phase of the liquid chromatography method is preferably selected from a mixed solution of ethyl acetate and hexane . However, the conditions of the liquid chromatography method used in the present invention should not be limited to this.

By lengthening the heating reflux time of the substitution reaction and the purification method using liquid chromatography, the substitution step provided by the present invention can effectively improve the recovery yield to 55%, which is higher than the preparation method of the prior art. 66%.

As shown in step S18, in the method for preparing a gene expression contrast agent precursor provided by the present invention, the 1- (3,5-di-oxy-benzylidene-2-deoxy-deoxy- 2-Fluoro-β-D-arabinofuranosyl) -5- (tributanestannyl) uracil is removed by phenylhydrazone protection reaction to obtain 5- (tributanestannyl) -1- (2 -Deoxy-2-fluoro-β-D-arabinofuranosyl) uracil, which is the target contrast agent precursor FSAU provided by the present invention, as shown in the following formula 5:


(Formula 5)

Wherein, the protection reaction for removing phenylhydrazone provided by the present invention is the 1- (3,5-di-oxy-benzylidene-2-deoxy-2-fluoro-β-D-arabinofuranosyl) After the -5- (tributanestannyl) uracil is dissolved, the ammonium hydroxide solution is added and stirred to perform the reaction. After the reaction is completed, it is concentrated under reduced pressure and separated and purified by liquid chromatography.

Furthermore, the ammonium hydroxide aqueous solution has a concentration of only 15% to 25% by weight, and the reaction temperature during the stirring process is between 20 ° C and 30 ° C, and the reaction time is between 18 hours and 18 hours. Between 30 hours.

Furthermore, in the liquid chromatography process described above, the filling material of the chromatography tube used in the liquid chromatography method is selected from substances having a high affinity with the FSAU product, preferably Filled with silicon dioxide, and the mobile phase of the liquid chromatography is preferably selected from a mixed solution of ethyl acetate and chloroform. However, the conditions of the liquid chromatography method used in the present invention should not be limited to this.

Through the above reaction process, not only are the steps concise, but also the intermediate products in the middle of each reaction can be obtained without complicated separation and purification process, and the final product is extracted by liquid chromatography with better separation and purification effect. Therefore, it can indeed provide a method for preparing a gene expression contrast agent precursor with higher recovery yield and lower preparation cost, which is a major breakthrough in the current related research fields.

In addition, after completing all the above-mentioned preparation methods of gene expression contrast agent precursors, the generated contrast agent precursor FSAU can be further substituted with a radionuclide selected from the element iodine (I) to generate a radioactive iodine (I). Non-uridine is used as a radioactive probe that reacts with gene reported by gene therapy to observe the effect of gene therapy.

In the following, specific implementation examples are used for the description of the organizational technical content, features, and results of this invention, and can be implemented accordingly, but the scope of protection of the present invention is not limited thereto.

[Example 1]

2-Deoxy-2-fluoro-3,5-di-oxy-benzylidene-α-D-brominated arabinofuranosyl (2-Deoxy-2-fluoro-3,5-di-O-benzoyl -α-D-arabinofuranosyl bromide)

Take 0.9 g (g) of the starting material 2-deoxy-2-fluoro-1,3,5-tri-oxo-benzyl-α-D-arabinofuranose (1.94 mmol) (mmol) 2-deoxy-2-fluoro-1,3,5-tri-O-benzoyl-α-D-arabinofurance) was added to 4 milliliters (mL) of anhydrous dichloromethane, and gaseous nitrogen was passed through it in an ice bath. In a state, a mixed solution of 1.25 ml of a hydrogen bromide acetic acid solution with a concentration of 30% by weight and 5 ml of anhydrous dichloromethane was dropped into the aforementioned starting material solution and stirred for 20 hours.

Next, add 20 ml of dichloromethane, and wash it twice with 20 ml of water and 20 ml of sodium bicarbonate solution. Take out the organic layer in the finished product area and remove the water with sodium sulfate solution. 2-Deoxy-2-fluoro-3,5-di-oxy-benzylidene-α-D-brominated arabinofuranosyl (2-Deoxy-2-fluoro-3,5-di-O-benzoyl -α-D-arabinofuranosyl bromide).

Analytical data for 2-deoxy-2-fluoro-3,5-di-oxo-benzylidene-α-D-arabinofuranosyl bromide: IR (neat) ν1726 (CO) cm-1. 1H NMR (CDCl3) δ8.12-7.40 (m, 10 H, ArH), 6.63 (d, 1 H, H1), 5.68 (d, 1 H, H2), 5.58 (d, 1H, H 3), 4.85-4.70 (m, 3 H, H4 and H5). 13C NMR (CDCl3) δ165.99 and 165.51 (CO) .133.89, 133.23, 129.98, 129.78, 129.38, 128.64, 128.51 and 128.38 (Ph), 101.86 and 99.32 (CHF) , 87.76 and 87.34 (CHBr), 84.70 (CHO), 77.43, 77.01, 76.58, 76.40 and 75.98 (CH2CH), 62.48 (CH2CH). MS m / z 343 (M + -Br).

[Example 2]

Synthesis of 2,4-bis-oxy- (trimethylsilyl) -5-bromouracil (2,4-bis-O- (trimethylsilyl) -5-bromouracil)

Take 0.32 g, 1.68 millimolar (mmol) of 5-bromouracil as a starting material, add 0.22 g, 1.68 millimolar (mmol) of ammonium sulfate and 1.5 ml of hexamethyldisilazane (Hexamethyldisilazane, HMDS), and the mixture was stirred under heating at 140 ° C. for 4 hours under a condition of passing nitrogen gas.

Then, the solvent remaining in the above reaction was removed under reduced pressure to obtain a crude product, which was not purified by isolation, even if it was used in the next step of synthesis.

[Example 3]

1- (3,5-di-oxy-benzylidene-2-deoxy-2-fluoro-β-D-arabinofuranosyl) -5-bromouracil (1- (3,5-di- Synthesis of O-benzoyl-2-deoxy-2-fluoro-β-D-arabinofuranosyl) -5-bromouracil)

Take 0.71 grams, 1.68 millimoles (mmol) of 2-deoxy-2-fluoro-3,5-di-oxo-benzylidene-α-D-brominated arabinofuranosyl group, in a nitrogen-filled Under the conditions, 0.71 g, 1.68 millimoles (mmol) of 2,4-bis-oxy- (trimethylsilyl) -5-bromouracil was added, and the mixture was heated under reflux at 90 ° C. and stirred for 20 hours.

Then, the solvent remaining in the reaction was removed by concentration under reduced pressure, then washed with hexane and filtered with suction, and finally washed with a little ether, and the solid was obtained to obtain 1- (3,5-di-oxy-benzyl Fluorenyl-2-deoxy-2-fluoro-β-D-arabinofuranosyl) -5-bromouracil (1- (3,5-di-O-benzoyl-2-deoxy-2-fluoro-β -D-arabinofuranosyl) -5-bromouracil).

Analytical data of 1- (3,5-di-oxy-benzylidene-2-deoxy-2-fluoro-β-D-arabinofuranosyl) -5-bromouracil: IR (KBr) ν1726, 1716 and 1671 (CO) cm-1. 1H NMR (CDCl3) δ8.39 (s, 1 H, NH), 8.11-8.03 (m, 4H, Ph), 7.92 (d, 1 H, H6), 7.67- 7.58 (m, 2 H, Ph), 7.58-7.45 (m, 4H, Ph), 6.31 (dd, 1H, H1), 5.63 (dd, 1H, H3), 5.33 (dd, 1H, H2), 4.84- 4.81 (m, 2H, H5), 4.56-4.52 (m, 1H, H4) .13C NMR (CDCl3) δ166.10 and 165.51 (CO) .158.10, 149.50, 141.30, 135.20, 133.80, 131.10, 129.80, 129.70 and 128.80 (Ph), 94.80 and 92.70 (CHF), 90.10 and 85.60 (CHBr), 82.10 (CHO), 78.10, 77.80, 77.60, 77,20 and 75.90 (CH2CH), 62.50 (CH2CH). MS m / z 534 ( M) +.

[Example 4]

1- (3,5-Di-oxy-benzylidene-2-deoxy-2-fluoro-β-D-arabinofuranosyl) -5- (tributanestannyl) uracil (1- Synthesis of (3,5-di-O-benzoyl-2-deoxy-2-fluoro-β-D-arabinofuranosyl) -5-tributylstannyl) uracil)

Take 0.51 grams, 0.96 millimoles (mmol) of 1- (3,5-di-oxy-benzylidene-2-deoxy-2-fluoro-β-D-arabinofuranosyl) -5-bromo Add uracil 0.84 ml, 1.68 millimoles (mmol) of hexa-n-butylditin, 40 mg of dichlorobis (triphenylphosphine) palladium and 20 ml of anhydrous dioxane solution. Under conditions, the mixture was heated under reflux at 110 ° C for 24 hours.

Next, the concentrated solvent was removed under reduced pressure, and then the precipitate was dissolved and collected with ether, ethyl acetate, and hexane, and the insoluble matter was removed. Finally, the ether, ethyl acetate, and Hexane and its dissolved recovered matter were separated and purified by liquid chromatography (Liquid Chromatography) using silica dioxide as an adsorption column and a separation phase of ethyl acetate: hexane = 1: 2. , 5-di-oxo-benzylidene-2-deoxy-2-fluoro-β-D-arabinofuranosyl) -5- (tributanestannyl) uracil (1- (3,5 -di-O-benzoyl-2-deoxy-2-fluoro-β-D-arabinofuranosyl) -5-tributylstannyl) uracil).

Analytical data of 1- (3,5-di-oxy-benzylidene-2-deoxy-2-fluoro-β-D-arabinofuranosyl) -5- (tributanetinyl) uracil : IR (neat) ν1715 and 1677 (CO) cm-1. 1H NMR (CDCl3) δ8.97 (s, 1 H, NH), 8.10-7.37 (m, 11 H, Ph), 6.36 (dd, 1 H , H1), 5.63 (dd, 1 H, H3), 5.34 (dd, 1 H, H2) 4.75 (m, 2 H, H5), 4.51 (m, 1 H, H4), 1.48-0.82 (m, 27 H, SnBu3). 13C NMR (CDCl3) δ165.98 and 165.15 (CO), 150.89, 143.93, 143.87, 134.07, 133.33, 129.95, 129.74, 129.33, 128.68, 128.45 and 128.16 (Ph), 112.13 (CHSn), 93.95 and 91.41 (CHF), 84.84 and 84.62 (CHCHO), 81.06 (CH2CH), 76.45 (CHN), 63.53 (CH2CH) 28.67, 27.10, 13.65, and 9.76 (SnCH2CH2CH2CH3) 2. MS m / z 742 (M) +.

[Example 5]

5- (tributanestannyl) -1- (2-deoxy-2-fluoro-β-D-arabinofuranosyl) uracil (5-tributylstannyl-1- (2-deoxy-2-fluoro- β-D-arabinofuranosyl) uracil, FSAU)

Take 0.10 grams, 0.13 millimoles (mmol) of 1- (3,5-di-oxy-benzylidene-2-deoxy-2-fluoro-β-D-arabinofuranosyl) -5- ( Add 4.5 ml of methanol to dissolve tributanetinyl uracil. Then add 4.5 ml of 25% ammonium hydroxide aqueous solution (NH ₄ OH) by volume and stir under reflux at 25 ° C for 24 hours. .

Next, the product after stirring was concentrated under reduced pressure. For the recovered product, liquid chromatography using silica dioxide as the adsorption column and trichloromethane: ethyl acetate = 1: 1 as the separation phase (Liquid Chromatography) After separation and purification, 5- (tributanestannyl) -1- (2-deoxy-2-fluoro-β-D-arabinofuranosyl) uracil (5-tributylstannyl-1- (2 -deoxy-2-fluoro-β-D-arabinofuranosyl) uracil).

Analytical data of 5- (tributanestannyl) -1- (2-deoxy-2-fluoro-β-D-arabinofuranosyl) uracil: IR (neat) ν3390 (OH), 2927 (NH ), 1695 and 1647 (CO) cm-1.1H NMR (CDCl3) δ9.62 (br, 1 H, NH), 7.29 (d, 1H, CHSn), 6.25 (dd, 1 H, H1), 5.09 ( dd, 1H, H2), 4.46 (br, 1 H, OH), 4.43 (dd, 1 H, H3), 4.06 (q, 1 H, H4), 3.85 (m, 2H, H5), 3.23 (br, 1H, OH), 1.54-0.85 (m, 27H, SnBu3) .13C NMR (CDCl3) δ166.76 and 151.42 (CO), 144.91 (CHN), 111.99 (CHSn), 96.18 and 93.63 (C2), 84.35 and 84.15 (C3 and C4), 75.15 and 74.81 (C1), 61.92 (C5), 28.87, 27.22, 13.64 and 9.84 (SnCH2CH2CH2CH3) 2. MS m / z 534 (M) +.

To sum up, the method for preparing the gene expression contrast agent precursor disclosed in the present invention uses methods such as selection of cleaning solution, improvement of reaction materials and related steps, and no excessive extraction of intermediate products, and even uses liquid direction The final product is separated by chromatography to reduce the loss of the target product during the manufacturing process, which not only improves the yield of the target product, but also simplifies the existing separation and purification steps and accelerates the production of the target product, which indeed improves the production efficiency and reduces the production requirements. The cost of this method provides a valuable method of preparation in this patent field. At the same time, further, the preparation method provided by the present invention can be further reacted with an iodine radioisotope to prepare a radioactive iodine (I) non-uridine (I ¹²⁴ -Fialuridine), which can be highly free Penetrating the cell membrane and reacting with proteins generated by reporter genes in gene therapy are used as indirect aids to judge the effectiveness of gene therapy and have high commercial value.

However, the above are only preferred embodiments of the present invention, and are not intended to limit the scope of implementation of the present invention. For example, all changes and modifications of the shapes, structures, features, and spirits in accordance with the scope of the patent application for the present invention are made. Shall be included in the scope of patent application of the present invention.

Claims (16)

A method for manufacturing a precursor of a gene expression contrast agent, the steps include:
Take 2-deoxy-2-fluoro-1,3,5-tri-oxo-benzylidene-α-D-arabinofuranose and mono-brominated acetic acid solution to form a 2-deoxy Oxo-2-fluoro-3,5-di-oxo-benzylidene-α-D-arabinofuranosyl bromide;
In addition, a 5-bromouracil and a hexamethyldisilazane are subjected to a silylation protection reaction to form a 2,4-bis-oxy- (trimethylsilyl) -5-bromouracil;
Take the 2-deoxy-2-fluoro-3,5-di-oxy-benzylidene-α-D-brominated arabinofuranosyl group and the 2,4-bis-oxy- (trimethylsilyl group) ) -5-bromouracil undergoes a coupling reaction to form 1- (3,5-di-oxo-benzylidene-2-deoxy-2-fluoro-β-D-arabinofuranosyl) -5 -Bromouracil;
Take this 1- (3,5-di-oxy-benzylidene-2-deoxy-2-fluoro-β-D-arabinofuranosyl) -5-bromouracil and hexa-n-butylditin A mono-substitution reaction is carried out in an anhydrous dioxane solution to form a 1- (3,5-di-oxo-benzylidene-2-deoxy-2-fluoro-β-D-arabinofuranosyl)- 5- (tributanestannyl) uracil; and the 1- (3,5-di-oxy-benzylidene-2-deoxy-2-fluoro-β-D-arabinofuranosyl group) -5- (tributanestannyl) uracil is subjected to a protection reaction to remove phenylhydrazone to obtain 5- (tributanetinyl) -1- (2-deoxy-2-fluoro-β-D -Arabinofuranosyl) uracil. According to the manufacturing method of the gene expression contrast agent precursor described in item 1 of the scope of patent application, wherein the weight percentage concentration of the hydrogen bromide acetic acid solution used in the bromination reaction is 20% to 40%. According to the method for manufacturing a gene expression contrast agent precursor described in item 1 of the scope of the patent application, wherein the reaction temperature of the bromination reaction is between -196 ° C and 13 ° C. According to the method for manufacturing a gene expression contrast agent precursor described in item 1 of the scope of the patent application, wherein the reaction time of the bromination reaction is between 15 hours and 25 hours. According to the method for producing a precursor of a gene expression contrast agent as described in item 1 of the scope of the patent application, wherein the silylation protection reaction uses ammonium monosulfate to react with the hexamethyldisilazane. According to the method for manufacturing a gene expression contrast agent precursor described in item 1 of the scope of the patent application, wherein the reaction temperature of the silylation protection reaction is between 120 ° C and 160 ° C. According to the manufacturing method of the gene expression contrast agent precursor as described in the first item of the patent application scope, the reaction time of the silylation protection reaction is between 2 hours and 6 hours. According to the method for manufacturing a gene expression contrast agent precursor described in item 1 of the scope of the patent application, wherein the reaction temperature of the coupling reaction is between 70 ° C and 110 ° C. According to the method for manufacturing a gene expression contrast agent precursor as described in item 1 of the scope of the patent application, wherein the reaction time of the coupling reaction is between 15 hours and 25 hours. According to the method for manufacturing a gene expression contrast agent precursor described in item 1 of the scope of the patent application, wherein the substitution reaction uses dichlorobis (triphenylphosphine) palladium as a catalyst. According to the method for manufacturing the precursor of the gene expression contrast agent described in item 1 of the scope of the patent application, wherein the reaction temperature of the substitution reaction is between 90 ° C and 130 ° C. According to the method for manufacturing a gene expression contrast agent precursor described in item 1 of the scope of the patent application, wherein the reaction time of the substitution reaction is between 18 hours and 30 hours. The method for manufacturing a precursor of a gene expression contrast agent as described in item 1 of the scope of the patent application, wherein the protection reaction for removing phenylhydrazone is performed in an aqueous solution of ammonium hydroxide, wherein the aqueous solution of ammonium hydroxide is only 100% by weight The concentration is 15% to 25%. According to the method for manufacturing the precursor of the gene expression contrast agent described in item 1 of the scope of the patent application, wherein the reaction temperature for removing the phenylhydrazone protection reaction is between 20 ° C and 30 ° C. According to the method for manufacturing a gene expression contrast agent precursor described in item 1 of the scope of the patent application, wherein the reaction time for removing the phenylhydrazone protection reaction is between 18 hours and 30 hours. The method for manufacturing a gene expression contrast agent precursor according to item 1 of the scope of the patent application, wherein the 5- (tributanestannyl) -1- (2- Deoxy-2-fluoro-β-D-arabinofuranosyl) uracil can further undergo a substitution reaction with a radionuclide selected from the group consisting of iodine (I) element to form a radioactive iodine (I) non-uridine.