CN114480401B - Clofarabine modified oligonucleotide - Google Patents

Clofarabine modified oligonucleotide Download PDF

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CN114480401B
CN114480401B CN202011173092.5A CN202011173092A CN114480401B CN 114480401 B CN114480401 B CN 114480401B CN 202011173092 A CN202011173092 A CN 202011173092A CN 114480401 B CN114480401 B CN 114480401B
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clofarabine
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phosphoramidite monomer
gemcitabine
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CN114480401A (en
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谭蔚泓
王雪强
何嘉轩
彭天欢
司马颖钰
符婷
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Hunan University
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Abstract

The invention relates to the field of pharmaceutical chemistry, in particular to a clofarabine modified oligonucleotide. The invention provides a clofarabine modified oligonucleotide, which comprises a nucleic acid aptamer segment, wherein the nucleic acid aptamer segment is modified with a clofarabine phosphoramidite monomer group. The invention designs and synthesizes nucleoside drug clofarabine and/or gemcitabine into phosphoramidite monomer which can be used for solid phase synthesis, uses solid phase synthesis technology to realize accurate functionalization of clofarabine and/or gemcitabine on the oligonucleotide, and the prepared clofarabine and/or gemcitabine modified oligonucleotide can release clofarabine under the action of nuclease, has higher cytotoxicity on tumor cells, retains the pharmaceutical activity of clofarabine and/or gemcitabine, and has good industrialization prospect.

Description

Clofarabine modified oligonucleotide
Technical Field
The invention relates to the field of pharmaceutical chemistry, in particular to a clofarabine modified oligonucleotide.
Background
Clofarabine is a purine nucleoside antimetabolite, unlike other purine nucleoside analogues in which chlorine is present in the purine ring and fluorine is present in the ribose moiety of clofarabine. Clofarabine prevents the growth of cancer cells by interfering with the synthesis of nucleic acids to prevent the production of DNA and RNA by the cells. Clofarabine is metabolized by deoxycytidine kinase in the cell to an active 5 '-monophosphate metabolite and by mono-and diphosphate kinases to a 5' -triphosphate metabolite. Thereby achieving the inhibition effect on ribonucleotide reductase, and stopping the extension of DNA chain and inhibiting repair by competitively inhibiting DNA polymerase, thereby inhibiting the synthesis of DNA. In preclinical models, clofarabine has been shown to inhibit DNA repair by incorporating DNA strands during repair. Clofarabine 5' -triphosphate also disrupts the integrity of the mitochondrial membrane, resulting in the release of pro-apoptotic mitochondrial proteins, cytochrome C and apoptosis-inducing factors, leading to programmed cell death. Currently, clofarabine has been approved for clinical treatment of pediatric Acute Lymphoblastic Leukemia (ALL) in several countries worldwide.
Oligonucleotides are short single-or double-stranded DNA or RNA molecules that are playing an increasing role in molecular biology, and can be used as probes for nucleic acid sequence detection, single-base diversity analysis, antisense oligonucleotides, etc., as a very effective research tool. By functionally modifying the oligonucleotide, a new function can be given to the oligonucleotide, and the application range of the oligonucleotide is widened.
Disclosure of Invention
In view of the above-described drawbacks of the prior art, an object of the present invention is to provide a clofarabine-modified oligonucleotide for solving the problems of the prior art.
To achieve the above and other related objects, the present invention provides a clofarabine modified oligonucleotide comprising a nucleic acid aptamer segment modified with a clofarabine phosphoramidite monomer group.
In some embodiments of the invention, the aptamer fragment is modified with one or more clofarabine phosphoramidite monomer groups, which are modified at the 5 'end of the aptamer fragment and/or modified at the 3' end of the aptamer fragment and/or added in the middle of the aptamer fragment.
In some embodiments of the invention, the clofarabine phosphoramidite monomer group is linked to the aptamer fragment via a phosphodiester linkage.
In some embodiments of the invention, the clofarabine phosphoramidite monomer group modified at the 5' end of the aptamer fragment has the chemical structural formula:
the chemical structural formula of the clofarabine phosphoramidite monomer group added in the middle of the aptamer fragment is shown as follows:
the chemical structural formula of the clofarabine phosphoramidite monomer group modified at the 3' -end of the aptamer fragment is shown as follows:
in some embodiments of the invention, the nucleic acid aptamer fragment is further modified with a gemcitabine phosphoramidite monomer group.
In some embodiments of the invention, the aptamer fragment is modified with one or more gemcitabine phosphoramidite monomer groups, which are modified at the 5 'end of the aptamer fragment and/or modified at the 3' end of the aptamer fragment and/or added in the middle of the aptamer fragment.
In some embodiments of the invention, the gemcitabine phosphoramidite monomer group is linked to the aptamer fragment via a phosphodiester linkage.
In some embodiments of the invention, the gemcitabine phosphoramidite monomer group modified at the 5' end of the aptamer fragment has the chemical structural formula:
the chemical structural formula of the gemcitabine phosphoramidite monomer group added to the middle of the aptamer fragment is shown as follows:
the chemical structural formula of the gemcitabine phosphoramidite monomer group modified at the 3' end of the aptamer fragment is shown as follows:
in some embodiments of the invention, the polynucleotide sequence of the nucleic acid aptamer fragment comprises a sequence as shown in one of SEQ ID NOS.11-20.
In some embodiments of the invention, the polynucleotide sequence of the clofarabine modified oligonucleotide comprises a sequence as set forth in one of SEQ ID NO. 1-10.
In another aspect, the present invention provides a method for preparing the clofarabine-modified oligonucleotide described above, comprising:
the clofarabine phosphoramidite monomer is linked to the aptamer fragment by solid phase synthesis.
In some embodiments of the invention, the reaction temperature of the solid phase synthesis is 15-35 ℃, the reaction time is 1-20 minutes, and the air humidity is 30% -70%.
In some embodiments of the invention, the clofarabine phosphoramidite monomer has the chemical structural formula:
In some embodiments of the invention, the gemcitabine phosphoramidite monomer has the chemical structural formula:
in another aspect, the invention provides the use of the clofarabine modified oligonucleotide in the preparation of a medicament.
Drawings
FIG. 1 is a schematic diagram showing the mass spectrum identification results of clofarabine modified oligonucleotides in example 3 of the present invention.
FIG. 2 is a schematic diagram showing the mass spectrum identification results of clofarabine modified oligonucleotides in example 3 of the present invention.
FIG. 3 is a schematic diagram showing the mass spectrum identification results of clofarabine modified oligonucleotides in example 3 of the present invention.
FIG. 4 is a schematic representation of the results of experiments on the inhibition of cancer cells by gemcitabine and clofarabine modified oligonucleotides of example 4 of the present invention.
FIG. 5 is a schematic diagram showing the results of experiments for specific binding of clofarabine modified oligonucleotides to cancer cells in example 5 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantageous technical effects of the present invention more apparent, the present invention will be further described in detail with reference to the following examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the disclosure of the present specification.
The inventor of the present invention has found that, after a lot of practical researches, the oligonucleotide modified with clofarabine not only can maintain the original pharmaceutical activity of clofarabine, but also can increase the targeting of the drug, reduce the toxic and side effects and improve the bioavailability, and the present invention is completed on the basis.
In a first aspect, the invention provides a clofarabine-modified oligonucleotide comprising a nucleic acid aptamer segment modified with a clofarabine phosphoramidite monomer group. The clofarabine modified oligonucleotide can be prepared from clofarabine phosphoramidite monomer through solid phase synthesis, and the prepared clofarabine modified oligonucleotide can comprise clofarabine phosphoramidite monomer groups correspondingly formed from the clofarabine phosphoramidite monomer and aptamer fragments connected with the clofarabine phosphoramidite monomer groups.
The clofarabine modified oligonucleotide provided by the invention can comprise clofarabine phosphoramidite monomer groups. The specific structure of the clofarabine phosphoramidite monomer group generally corresponds to the clofarabine phosphoramidite monomer used. In one embodiment of the present invention, the clofarabine phosphoramidite monomer used has the following chemical structural formula:
In the clofarabine modified oligonucleotide provided by the invention, clofarabine phosphoramidite monomer groups can be modified at various positions of the nucleic acid aptamer segment, for example, clofarabine phosphoramidite monomer groups can be modified at the 5 'end of the nucleic acid aptamer segment, can be modified at the 3' end of the nucleic acid aptamer segment, and can be added in the middle of the nucleic acid aptamer segment. The aptamer fragment may be modified with one or more clofarabine phosphoramidite monomer groups, for example, an oligonucleotide sequence containing 60 bases may be modified with 1-11, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 clofarabine phosphoramidite monomer groups. When the aptamer fragment is modified with multiple clofarabine phosphoramidite monomer groups, each clofarabine phosphoramidite monomer group may be discontinuous or at least part of the clofarabine phosphoramidite monomer groups may be continuous.
In the clofarabine modified oligonucleotide provided by the invention, as described above, the clofarabine modified oligonucleotide can be generally prepared from a clofarabine phosphoramidite monomer through a solid phase synthesis method, so that in the formed clofarabine modified oligonucleotide, a clofarabine phosphoramidite monomer group formed by the clofarabine phosphoramidite monomer and a (modified) aptamer fragment can be connected through a phosphodiester bond. Specifically, the trivalent phosphorus group in the clofarabine phosphoramidite monomer can be coupled to the 5 '-terminal hydroxyl group of the aptamer fragment to form a phosphodiester bond, while the deprotected (e.g., DMT-protected) hydroxyl group in the clofarabine phosphoramidite monomer can be coupled to the 3' -terminal phosphoramidite group of the aptamer fragment to form a phosphodiester bond, while the deprotected (e.g., DMT-protected) hydroxyl group in the clofarabine phosphoramidite monomer can be reacted with the phosphoramidite group in the clofarabine phosphoramidite monomer to form a phosphodiester bond
In one embodiment of the invention, when the clofarabine phosphoramidite monomer group is modified at the 5' end of the aptamer fragment, the chemical structural formula of the formed group can be as follows:
in one embodiment of the invention, when clofarabine phosphoramidite monomer groups are added in the middle of a nucleic acid aptamer fragment, the chemical structure of the groups formed can be as follows:
in one embodiment of the invention, when the clofarabine phosphoramidite monomer group is modified at the 3' end of the aptamer fragment, the chemical structural formula of the formed group can be as follows:
in the clofarabine modified oligonucleotide provided by the invention, the aptamer fragment is further modified with a gemcitabine phosphoramidite monomer group. Oligonucleotides modified with both clofarabine and gemcitabine can be prepared from clofarabine phosphoramidite monomers and gemcitabine phosphoramidite monomers by solid phase synthesis, and the prepared oligonucleotides comprising both clofarabine and gemcitabine can comprise clofarabine phosphoramidite monomer groups formed from clofarabine phosphoramidite monomers, gemcitabine phosphoramidite monomer groups formed from gemcitabine phosphoramidite monomers, and aptamer fragments linked to clofarabine phosphoramidite monomer groups and/or gemcitabine phosphoramidite monomer groups.
The clofarabine modified oligonucleotide provided by the invention can comprise gemcitabine phosphoramidite monomer groups. The specific structure of the gemcitabine phosphoramidite monomer group generally corresponds to the gemcitabine phosphoramidite monomer used. In one embodiment of the present invention, the gemcitabine phosphoramidite monomer used has the following chemical structural formula:
in the clofarabine-modified oligonucleotide provided by the invention, the gemcitabine phosphoramidite monomer group can be modified at each position of the aptamer fragment, for example, the gemcitabine phosphoramidite monomer group can be modified at the 5 'end of the aptamer fragment, can be modified at the 3' end of the aptamer fragment, can be added in the middle of the aptamer fragment, and for example, two ends of the clofarabine phosphoramidite monomer group can be respectively and independently unmodified and connected with the aptamer fragment, the gemcitabine phosphoramidite monomer group or other clofarabine phosphoramidite monomer groups, and for example, two ends of the gemcitabine phosphoramidite monomer group can be respectively and independently connected with the aptamer fragment, the clofarabine phosphoramidite monomer group or other gemcitabine phosphoramidite monomer groups. The aptamer fragment may be modified with one or more gemcitabine phosphoramidite monomer groups, for example, an oligonucleotide sequence containing 60 bases may be modified with 1-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 gemcitabine phosphoramidite monomer groups. When the aptamer fragment is modified with multiple gemcitabine phosphoramidite monomer groups, each gemcitabine phosphoramidite monomer group may be discontinuous or at least some gemcitabine phosphoramidite monomer groups may be continuous.
In the clofarabine modified oligonucleotide provided by the invention, as described above, the clofarabine and gemcitabine modified oligonucleotide can be generally prepared from a clofarabine phosphoramidite monomer and a gemcitabine phosphoramidite monomer through a solid phase synthesis method, so that in the formed clofarabine and gemcitabine modified oligonucleotide, a clofarabine phosphoramidite monomer group formed by the clofarabine phosphoramidite monomer can be connected with an adjacent group or fragment through a phosphodiester bond, and a gemcitabine phosphoramidite monomer group formed by the gemcitabine phosphoramidite monomer can be connected with an adjacent group or fragment through a phosphodiester bond. In particular, a trivalent phosphorus group in a clofarabine phosphoramidite monomer, or a gemcitabine phosphoramidite monomer, can be coupled to a hydroxyl group of an adjacent group or fragment (e.g., a aptamer fragment, a clofarabine phosphoramidite monomer group, or a gemcitabine phosphoramidite monomer group) such that a phosphodiester bond can be formed, and a deprotected (e.g., DMT protected) hydroxyl group in a clofarabine phosphoramidite monomer, or a gemcitabine phosphoramidite monomer can be coupled to a phosphoramidite group of an adjacent group or fragment (e.g., a aptamer fragment, a clofarabine phosphoramidite monomer group, or a gemcitabine phosphoramidite monomer group) such that a phosphodiester bond can be formed.
In one embodiment of the invention, when gemcitabine phosphoramidite monomer groups are modified at the 5' end of a nucleic acid aptamer fragment, the chemical structural formula of the resulting group may be as follows:
in one embodiment of the invention, when a gemcitabine phosphoramidite monomer group is added in the middle of a nucleic acid aptamer fragment, the chemical structural formula of the formed group may be as follows:
in one embodiment of the invention, when the gemcitabine phosphoramidite monomer group is modified at the 3' end of the aptamer fragment, the chemical structural formula of the formed group may be as follows:
the clofarabine modified oligonucleotide provided by the invention can comprise a nucleic acid aptamer fragment. The choice of the specific sequence of the aptamer fragment largely determines the targeting of the oligonucleotide and also largely determines the stability of the oligonucleotide as a whole, and the polynucleotide sequence of the aptamer fragment (i.e. the polynucleotide sequence before the unmodified clofarabine phosphoramidite monomer group) may comprise a sequence as shown in one of SEQ ID NO. 11-20. The nucleic acid aptamer fragments are generally targeted to the proteins to which they correspond, e.g., the nucleic acid aptamer fragments as shown above can target protein tyrosine kinase 7 (PTK 7), all of which can achieve specific targeting of tumor cells. For another example, the polynucleotide sequence of the clofarabine modified oligonucleotide may comprise a sequence as set forth in one of SEQ ID NOS.1-10.
In one embodiment of the invention, the groups formed by the monomer groups of clofarabine phosphoramidite in the clofarabine modified oligonucleotide may be one of the following:
wherein n is 1 Is 0, or a positive integer, for example, may be 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9;
n 2 is a positive integer, and may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
n 3 is 0, or a positive integer, for example, may be 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9;
represents the other fragments in the clofarabine modified oligonucleotide linked to the clofarabine phosphoramidite monomer group.
In another embodiment of the invention, the gemcitabine phosphoramidite monomer groups in the clofarabine modified oligonucleotide may be one of the following:
wherein n is 4 Is 0, or a positive integer, for example, may be 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9;
n 5 is a positive integer, and may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
n 6 is 0, or a positive integer, for example, may be 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9;
represents the other fragments in the clofarabine modified oligonucleotide linked to the gemcitabine phosphoramidite monomer group.
In a second aspect, the invention provides a method for preparing a clofarabine modified oligonucleotide according to the first aspect, comprising: the clofarabine phosphoramidite monomer and/or the gemcitabine phosphoramidite monomer are linked to the aptamer fragment by solid phase synthesis.
In the preparation method of the clofarabine modified oligonucleotide provided by the invention, the conditions of a suitable solid phase synthesis method should be known to those skilled in the art, for example, the reaction conditions of the solid phase synthesis may be coupling reaction conditions of natural bases such as A, T, C, G. For another example, the reaction temperature of the solid phase synthesis method may be 15 to 35 ℃, 15 to 20 ℃, 20 to 25 ℃, 25 to 30 ℃, or 30 to 35 ℃; the reaction time may be 1 to 20 minutes, 1 to 2 minutes, 2 to 4 minutes, 4 to 6 minutes, 6 to 8 minutes, 8 to 10 minutes, 10 to 15 minutes, or 15 to 20 minutes; the air humidity may be 30% to 70%, 30% to 35%, 35% to 40%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, or 65% to 70%; the solvent may be nitrile solvent, ether solvent, halogenated hydrocarbon solvent, etc., specifically acetonitrile, tetrahydrofuran, chloroform, 1, 2-dichloroethane, etc.
In the preparation method of the clofarabine modified oligonucleotide provided by the invention, the preparation method of the clofarabine phosphoramidite monomer can comprise the following steps: the compound of formula I is condensed with 2-cyanoethyl N, N-diisopropylchlorophosphamide to provide clofarabine phosphoramidite monomer as follows:
in the above condensation reaction, the reaction may be generally carried out in the presence of a base, which may be generally an organic base, specifically, DIPEA, triethylamine, DMPA, pyridine, etc. The amount of base is generally in excess relative to the compound of formula I, so that the conversion of the reaction is ensured and the reaction is allowed to proceed sufficiently forward. For example, in the above condensation reaction, the molar ratio of the compound of formula I to the base may be 1: 3-12, 1: 3-4, 1: 4-5, 1:5 to 6, 1:6 to 7, 1: 7-8, 1: 8-9, 1: 9-10, 1:10 to 11, or 1:11 to 12, preferably may be 1:5.5 to 6.5.
In the above condensation reaction, the amount of 2-cyanoethyl N, N-diisopropylchlorophosphamide is usually excessive relative to the compound of formula I, so that the conversion of the reaction can be ensured and the reaction can be sufficiently carried out in the forward direction. For example, in the above condensation reaction, the molar ratio of the compound of formula I to 2-cyanoethyl N, N-diisopropylchlorophosphamide may be 1:1.5 to 6, 1: 1.5-2, 1: 2-2.5, 1:2.5 to 3, 1:3 to 3.5, 1:3.5 to 4, 1:4 to 4.5, 1:4.5 to 5, 1:5 to 5.5, or 1:5.5 to 6, preferably may be 1:2.5 to 3.5.
In the above-mentioned condensation reaction, the reaction may be carried out in the presence of a reaction solvent, the reaction solvent used in the condensation reaction may be generally an aprotic solvent, and the kind and the amount of a suitable reaction solvent should be known to those skilled in the art, for example, in the condensation reaction, the reaction solvent may be a halogenated hydrocarbon solvent, a sulfoxide solvent or the like, more specifically dichloromethane, dimethyl sulfoxide, chloroform, 1, 2-dichloroethane or the like.
In the above condensation reaction, the reaction is usually required to be avoided from being carried out at an excessively high temperature. For example, the reaction temperature in the condensation reaction may be 15 to 30 ℃, 15 to 20 ℃, 20 to 25 ℃, or 25 to 30 ℃. The reaction time can be adjusted by those skilled in the art according to the reaction progress, for example, in the condensation reaction, the reaction progress of the condensation reaction can be judged by TLC, chromatography, or the like, and for example, the reaction time of the condensation reaction can be 0.5 to 3 hours, 0.5 to 1 hour, 1 to 1.5 hours, 1.5 to 2 hours, or 2 to 3 hours.
In the condensation reaction, the reaction is usually carried out under a gas-shielded condition. Suitable methods of providing gas protection should be known to those skilled in the art, for example, the conditions under which gas protection may be provided by nitrogen, inert gases, etc., which may be, in particular, helium, neon, argon, krypton, etc.
In the above condensation reaction, the person skilled in the art may select an appropriate method to post-treat the product obtained by the reaction, and may include, for example: desolventizing and purifying. After the reaction is completed, the solvent may be removed from the product, and after further purification, clofarabine phosphoramidite monomer may be provided. Suitable purification methods should be known to those skilled in the art and may be, for example, column chromatography or the like.
The preparation method of the clofarabine modified oligonucleotide provided by the invention can further comprise the following steps: the compound of formula II was subjected to a first hydroxy protection reaction with 4,4' -dimethoxytrityl chloride (DMTrCl) to provide the compound of formula I as follows:
in the first hydroxyl group protecting reaction described above, the reaction may be usually carried out in the presence of a base, which may be usually an organic base, specifically, pyridine, DMAP, triethylamine, diethylamine, etc. The amount of base used is generally in large excess relative to the compound of formula I and can itself also act as solvent for the reaction system.
In the above-mentioned first hydroxyl group-protecting reaction, the reaction may be carried out in the presence of a reaction solvent, the reaction solvent used in the first hydroxyl group-protecting reaction may be usually an aprotic solvent, and the kind and the amount of a suitable reaction solvent should be known to those skilled in the art, for example, in the first hydroxyl group-protecting reaction, the reaction solvent may be a halogenated hydrocarbon solvent, a sulfoxide solvent or the like, more specifically dichloromethane, dimethyl sulfoxide, chloroform, 1, 2-dichloroethane or the like.
In the first hydroxy-protecting reaction, DMTrCl is generally used in an amount substantially equal to or in excess of the compound of formula II, so that the conversion of the reaction can be ensured and the reaction can be sufficiently carried out in the forward direction. For example, in the first hydroxy protection reaction described above, the molar ratio of the compound of formula II to DMTrCl may be 1:1 to 1.5, 1:1 to 1.1, 1:1.1 to 1.2, 1:1.2 to 1.3, 1:1.3 to 1.4, or 1:1.4 to 1.5, preferably may be 1:1.15 to 1.25.
In the first hydroxyl group protection reaction described above, the reaction may be generally carried out under a temperature condition of room temperature to the boiling point of the solvent. For example, the reaction temperature in the first hydroxyl group protection reaction may be 15 to 45 ℃, 15 to 20 ℃, 20 to 25 ℃, 25 to 30 ℃, 30 to 35 ℃, 35 to 40 ℃, or 40 to 45 ℃. The reaction time can be adjusted by a person skilled in the art according to the reaction progress, for example, in the first hydroxyl group protection reaction, the reaction progress of the first hydroxyl group protection reaction can be judged by TLC, chromatography, or the like, and for example, the reaction time of the first hydroxyl group protection reaction can be 2 to 24 hours, 2 to 3 hours, 3 to 4 hours, 4 to 6 hours, 6 to 8 hours, 8 to 12 hours, 12 to 16 hours, 16 to 20 hours, or 20 to 24 hours.
In the first hydroxyl group-protecting reaction described above, the reaction is usually carried out under gas-protected conditions. Suitable methods of providing gas protection should be known to those skilled in the art, for example, the conditions under which gas protection may be provided by nitrogen, inert gases, etc., which may be, in particular, helium, neon, argon, krypton, etc.
In the first hydroxyl protecting reaction, the person skilled in the art may select an appropriate method to post-treat the product obtained by the reaction, and for example, may include: desolventizing and purifying. After the reaction is completed, the product may be freed from the solvent and further purified to provide the compound of formula I. Suitable purification methods should be known to those skilled in the art and may be, for example, column chromatography or the like.
The preparation method of the clofarabine modified oligonucleotide provided by the invention can further comprise the following steps: the compound of formula III is subjected to TBDMS removal reaction with tetrabutylammonium fluoride (TBAF) to provide the compound of formula II, the reaction equation is as follows:
in the above-described TBDMS removal reaction, the reaction may be generally carried out in the presence of an acid, which may be generally an organic acid, specifically, formic acid, acetic acid, propionic acid, or the like, for example. The amount of acid is generally in excess relative to the compound of formula III, so that the conversion of the reaction is ensured and the reaction is allowed to proceed sufficiently forward. For example, in the TBDMS removal reaction described above, the molar ratio of the compound of formula I to the acid may be 1: 5-20, 1:5 to 6, 1: 6-8, 1: 8-10, 1:10 to 12, 1: 12-14, 1:14 to 16, 1:16 to 18, or 1:18 to 20, preferably may be 1:10 to 14.
In the TBDMS removal reaction, tetrabutylammonium fluoride is used in an amount substantially equal to or in excess of the compound of formula III, so that the conversion rate of the reaction can be ensured and the reaction can be sufficiently carried out in the forward direction. For example, in the TBDMS removal reaction described above, the molar ratio of the compound of formula III to tetrabutylammonium fluoride may be 1: 1-10, 1: 1-2, 1: 2-3, 1: 3-4, 1:4 to 6, 1:6 to 8, or 1:8 to 10, preferably may be 1:2 to 4.
In the above-mentioned TBDMS removal reaction, the reaction may be carried out in the presence of a reaction solvent, the reaction solvent used in the TBDMS removal reaction may be generally an aprotic solvent, and the kind and the amount of a suitable reaction solvent should be known to those skilled in the art, for example, in the TBDMS removal reaction, the reaction solvent may be specifically a halogenated hydrocarbon solvent, a sulfoxide solvent, an ether solvent, an amide solvent or the like, and more specifically tetrahydrofuran, chloroform, N-dimethylformamide, dimethyl sulfoxide, 1, 2-dichloroethane or the like.
In the TBDMS removal reactions described above, the reaction is generally required to avoid being carried out at too high a temperature. For example, the reaction temperature in the TBDMS removal reaction may be 15 to 30 ℃, 15 to 20 ℃, 20 to 25 ℃, or 25 to 30 ℃. The reaction time can be adjusted by a person skilled in the art according to the reaction progress, for example, in the TBDMS removal reaction, the reaction progress of the condensation reaction can be judged by a method such as TLC, chromatography, etc., and for example, the reaction time of the TBDMS removal reaction can be 3 to 12 hours, 3 to 4 hours, 4 to 6 hours, 6 to 8 hours, or 8 to 12 hours.
In the TBDMS removal reaction described above, the reaction is usually carried out under gas-shielded conditions. Suitable methods of providing gas protection should be known to those skilled in the art, for example, the conditions under which gas protection may be provided by nitrogen, inert gases, etc., which may be, in particular, helium, neon, argon, krypton, etc.
In the TBDMS removal reaction, a person skilled in the art may select an appropriate method to post-treat the product obtained by the reaction, and may include, for example: desolventizing and purifying. After the reaction is completed, the solvent may be removed from the product, and after further purification, clofarabine phosphoramidite monomer may be provided. Suitable purification methods should be known to those skilled in the art and may be, for example, column chromatography or the like.
The preparation method of the clofarabine modified oligonucleotide provided by the invention can further comprise the following steps: the compound of formula IV is subjected to an amino protection reaction with benzoyl chloride (BzCl) to provide a compound of formula III, the reaction equation is as follows:
in the above amino-protecting reaction, the reaction may be usually carried out in the presence of a base, which may be usually an organic base, specifically, imidazole, triethylamine, N-diisopropylethylamine or the like, for example. The amount of base is generally in excess relative to the compound of formula III, so that the conversion of the reaction is ensured and the reaction is allowed to proceed sufficiently forward. For example, in the above amino protection reaction, the molar ratio of the compound of formula I to the base may be 1: 3-30, 1: 3-4, 1:4 to 6, 1: 6-8, 1: 8-10, 1:10 to 12, 1: 12-14, 1:14 to 16, 1: 16-18, 1: 18-20, 1:20 to 25, or 1:25 to 30, preferably may be 1: 16-20.
In the above amino-protecting reaction, the benzoyl chloride is generally used in an amount substantially equal to or in excess of the compound of formula IV, so that the conversion of the reaction can be ensured and the reaction can be sufficiently carried out in the forward direction. For example, in the above amino protection reaction, the molar ratio of the compound of formula IV to benzoyl chloride may be 1: 2-20, 1: 2-3, 1: 3-4, 1:4 to 6, 1: 6-8, 1: 8-10, 1:10 to 12, 1: 12-14, 1:14 to 16, 1:16 to 18, or 1:18 to 20, preferably may be 1:6 to 8.
In the above-mentioned amino-protecting reaction, the reaction may be carried out in the presence of a reaction solvent, the reaction solvent used in the amino-protecting reaction may be usually an aprotic solvent, and the kind and the amount of a suitable reaction solvent should be known to those skilled in the art, for example, in the amino-protecting reaction, the reaction solvent may be specifically a halogenated hydrocarbon solvent, a sulfoxide solvent, an amide solvent or the like, more specifically methylene chloride, chloroform, N-dimethylformamide, dimethyl sulfoxide, 1, 2-dichloroethane or the like.
In the above amino protection reaction, the reaction is usually required to be carried out at an excessively high temperature. For example, the reaction temperature in the amino group protection reaction may be room temperature, 15 to 30 ℃, 15 to 20 ℃, 20 to 25 ℃, or 25 to 30 ℃. The reaction time can be adjusted by those skilled in the art according to the reaction progress, for example, in the amino protection reaction, the reaction progress of the condensation reaction can be judged by TLC, chromatography, or the like, and for example, the reaction time of the amino protection reaction can be 4 to 12 hours, 4 to 6 hours, 6 to 8 hours, 8 to 10 hours, or 10 to 12 hours.
In the above amino group-protecting reaction, the reaction is usually carried out under gas-protecting conditions. Suitable methods of providing gas protection should be known to those skilled in the art, for example, the conditions under which gas protection may be provided by nitrogen, inert gases, etc., which may be, in particular, helium, neon, argon, krypton, etc.
In the above amino protection reaction, the person skilled in the art may select an appropriate method to post-treat the product obtained by the reaction, and for example, may include: desolventizing and purifying. After the reaction is completed, the solvent may be removed from the product, and after further purification, clofarabine phosphoramidite monomer may be provided. Suitable purification methods should be known to those skilled in the art and may be, for example, column chromatography or the like.
The preparation method of the clofarabine modified oligonucleotide provided by the invention can further comprise the following steps: the compound of formula V is subjected to a second hydroxy protection reaction with t-butyldimethylchlorosilane (TBDMSCl) to provide a compound of formula IV, the reaction equation is as follows:
in the above second hydroxyl group protecting reaction, the reaction may be usually carried out in the presence of a base, which may be usually an organic base, specifically, imidazole, triethylamine, N-diisopropylethylamine or the like, for example. The amount of base used is generally in large excess relative to the compound of formula V, for example, in the second hydroxy-protecting reaction described above, the molar ratio of compound of formula V to base may be 1:4 to 25, preferably may be 1:6 to 10
In the above-mentioned second hydroxyl group-protecting reaction, the reaction may be carried out in the presence of a reaction solvent, the reaction solvent used in the second hydroxyl group-protecting reaction may be generally an aprotic solvent, and the kind and the amount of a suitable reaction solvent should be known to those skilled in the art, for example, in the second hydroxyl group-protecting reaction, the reaction solvent may be a halogenated hydrocarbon solvent, a sulfoxide solvent, an amide solvent or the like, and more specifically may be N, N-dimethylformamide, dimethylsulfoxide, 1, 2-dichloroethane or the like.
In the second hydroxyl group-protecting reaction, the t-butyldimethylchlorosilane is used in an excessive amount relative to the compound of formula V, so that the conversion rate of the reaction can be ensured and the reaction can be sufficiently carried out in the forward direction. For example, in the second hydroxy protection reaction described above, the molar ratio of the compound of formula V to t-butyldimethylchlorosilane may be 1: 2-10, 1: 2-3, 1: 3-4, 1:4 to 6, 1:6 to 8, or 1:8 to 10, preferably may be 1:3 to 4.
In the above second hydroxyl group protection reaction, the reaction may be generally carried out under a temperature condition of room temperature to the boiling point of the solvent. For example, the reaction temperature in the second hydroxyl group protection reaction may be 15 to 45 ℃, 15 to 20 ℃, 20 to 25 ℃, 25 to 30 ℃, 30 to 35 ℃, 35 to 40 ℃, or 40 to 45 ℃. The reaction time can be adjusted by a person skilled in the art according to the reaction progress, for example, in the second hydroxyl group protection reaction, the reaction progress of the second hydroxyl group protection reaction can be judged by TLC, chromatography, or the like, and for example, the reaction time of the second hydroxyl group protection reaction can be 3 to 24 hours.
In the second hydroxyl group-protecting reaction, the reaction is usually carried out under gas-protecting conditions. Suitable methods of providing gas protection should be known to those skilled in the art, for example, the conditions under which gas protection may be provided by nitrogen, inert gases, etc., which may be, in particular, helium, neon, argon, krypton, etc.
In the second hydroxyl protecting reaction, the person skilled in the art may select an appropriate method to post-treat the product obtained by the reaction, and may include, for example: desolventizing and purifying. After the reaction is completed, the product may be freed from the solvent and further purified to provide the compound of formula I. Suitable purification methods should be known to those skilled in the art and may be, for example, column chromatography or the like.
In the preparation method of the clofarabine modified oligonucleotide provided by the invention, the preparation method of the gemcitabine phosphoramidite monomer can comprise the following steps: the compound of formula VI is condensed with 2-cyanoethyl N, N-diisopropylchlorophosphamide to provide gemcitabine phosphoramidite monomer as follows:
in the above condensation reaction, the reaction may be generally carried out in the presence of a base, which may be generally an organic base, specifically, DIPEA, triethylamine, DMPA, pyridine, etc. The amount of base is generally in excess relative to the compound of formula VI, so that the conversion of the reaction is ensured and the reaction is allowed to proceed sufficiently forward. For example, in the above condensation reaction, the molar ratio of the compound of formula VI to the base may be 1: 3-12, 1: 3-4, 1: 4-5, 1:5 to 6, 1:6 to 7, 1: 7-8, 1: 8-9, 1: 9-10, 1:10 to 11, or 1:11 to 12, preferably may be 1:5.5 to 6.5.
In the above condensation reaction, the amount of 2-cyanoethyl N, N-diisopropylchlorophosphamide is usually excessive relative to the compound of formula VI, so that the conversion of the reaction can be ensured and the reaction can be sufficiently carried out in the forward direction. For example, in the above condensation reaction, the molar ratio of the compound of formula VI to 2-cyanoethyl N, N-diisopropylchlorophosphamide may be 1:1.5 to 6, 1: 1.5-2, 1: 2-2.5, 1:2.5 to 3, 1:3 to 3.5, 1:3.5 to 4, 1:4 to 4.5, 1:4.5 to 5, 1:5 to 5.5, or 1:5.5 to 6, preferably may be 1:2.5 to 3.5.
In the above-mentioned condensation reaction, the reaction may be carried out in the presence of a reaction solvent, and the reaction solvent used in the condensation reaction may be usually an aprotic solvent, and the kind and the amount of a suitable reaction solvent should be known to those skilled in the art, for example, in the condensation reaction, the reaction solvent may be a halogenated hydrocarbon solvent, an ether solvent, a nitrile solvent or the like, more specifically dichloromethane, tetrahydrofuran, acetonitrile or the like.
In the above condensation reaction, the reaction is usually required to be avoided from being carried out at an excessively high temperature. For example, the reaction temperature in the condensation reaction may be 15 to 30 ℃, 15 to 20 ℃, 20 to 25 ℃, or 25 to 30 ℃. The reaction time can be adjusted by those skilled in the art according to the reaction progress, for example, in the condensation reaction, the reaction progress of the condensation reaction can be judged by TLC, chromatography, or the like, and for example, the reaction time of the condensation reaction can be 0.5 to 3 hours, 0.5 to 1 hour, 1 to 1.5 hours, 1.5 to 2 hours, or 2 to 3 hours.
In the condensation reaction, the reaction is usually carried out under a gas-shielded condition. Suitable methods of providing gas protection should be known to those skilled in the art, for example, the conditions under which gas protection may be provided by nitrogen, inert gases, etc., which may be, in particular, helium, neon, argon, krypton, etc.
In the above condensation reaction, the person skilled in the art may select an appropriate method to post-treat the product obtained by the reaction, and may include, for example: desolventizing and purifying. After the reaction is completed, the product may be freed from solvent and further purified to provide gemcitabine phosphoramidite monomer. Suitable purification methods should be known to those skilled in the art and may be, for example, column chromatography or the like.
The preparation method of the clofarabine modified oligonucleotide provided by the invention can further comprise the following steps: hydroxy protection of a compound of formula VII with 4,4' -dimethoxytrityl chloride (DMTrCl) to provide a compound of formula VI, the reaction equation is as follows:
in the above-mentioned hydroxyl group protection reaction, the reaction may be usually carried out in the presence of a base, which may be usually an organic base, specifically, pyridine, DMAP, triethylamine, diethylamine, etc. The amount of base used is generally in large excess relative to the compound of formula VII, and can itself be used as a solvent for the reaction system.
In the above-mentioned hydroxyl group-protecting reaction, the reaction may be carried out in the presence of a reaction solvent, the reaction solvent used in the hydroxyl group-protecting reaction may be generally an aprotic solvent, the kind and the amount of the suitable reaction solvent should be known to those skilled in the art, and for example, in the hydroxyl group-protecting reaction, the reaction solvent may be selected from halogenated hydrocarbon solvents, sulfoxide solvents and the like, more specifically dichloromethane, dimethyl sulfoxide, chloroform, 1, 2-dichloroethane and the like.
In the above hydroxy-protecting reaction, DMTrCl is generally used in an amount substantially equal to or in excess of the compound of formula VII, so that the conversion of the reaction can be ensured and the reaction can be sufficiently carried out in the forward direction. For example, in the above hydroxy protection reaction, the molar ratio of the compound of formula VII to DMTrCl may be 1:1 to 1.5, 1:1 to 1.1, 1:1.1 to 1.2, 1:1.2 to 1.3, 1:1.3 to 1.4, 1: or 1.4 to 1.5, preferably may be 1:1.15 to 1.25.
In the above-mentioned hydroxyl group protection reaction, the reaction may be generally carried out under a temperature condition of room temperature to the boiling point of the solvent. For example, the reaction temperature in the hydroxyl group protection reaction may be 15 to 45 ℃, 15 to 20 ℃, 20 to 25 ℃, 25 to 30 ℃, 30 to 35 ℃, 35 to 40 ℃, or 40 to 45 ℃. The reaction time can be adjusted by those skilled in the art according to the reaction progress, for example, in the hydroxyl-protecting reaction, the reaction progress of the hydroxyl-protecting reaction can be judged by TLC, chromatography, or the like, and for example, the reaction time of the hydroxyl-protecting reaction can be 2 to 24 hours, 2 to 3 hours, 3 to 4 hours, 4 to 6 hours, 6 to 8 hours, 8 to 12 hours, 12 to 16 hours, 16 to 20 hours, or 20 to 24 hours.
In the above-mentioned hydroxyl group protection reaction, the reaction is usually carried out under gas-shielded conditions. Suitable methods of providing gas protection should be known to those skilled in the art, for example, the conditions under which gas protection may be provided by nitrogen, inert gases, etc., which may be, in particular, helium, neon, argon, krypton, etc.
In the above hydroxy-protecting reaction, the person skilled in the art may select an appropriate method to post-treat the product obtained by the reaction, and may include, for example: desolventizing and purifying. After the reaction is completed, the product may be freed of solvent and further purified to provide the compound of formula VI. Suitable purification methods should be known to those skilled in the art and may be, for example, column chromatography or the like.
The preparation method of the clofarabine modified oligonucleotide provided by the invention can further comprise the following steps: the compound of formula VIII is subjected to an amino-protecting reaction with acetic anhydride to provide a compound of formula VII, the reaction equation is as follows:
in the above amino-protecting reaction, the amount of acetic anhydride to be used is usually substantially equal or excessive relative to the compound of formula VIII, so that the conversion of the reaction can be ensured and the reaction can be sufficiently carried out in the forward direction. For example, in the above amino protection reaction, the molar ratio of the compound of formula VIII to acetic anhydride may be 1:1 to 1.5, 1:1 to 1.1, 1:1.1 to 1.2, 1:1.2 to 1.3, 1:1.3 to 1.4, 1:1.4 to 1.5, preferably may be 1:1.05 to 1.15.
In the above amino-protecting reaction, the reaction may be carried out in the presence of a reaction solvent, the reaction solvent used in the amino-protecting reaction may be generally an aprotic solvent, and the kind and the amount of a suitable reaction solvent should be known to those skilled in the art, for example, in the amino-protecting reaction, the reaction solvent may be selected from amide-based solvents, ether-based solvents, haloalkane-based solvents, etc., more specifically DMF, tetrahydrofuran, dioxane, 1, 2-dichloroethane, etc.
In the above amino group protecting reaction, the reaction may be generally carried out under a temperature condition of room temperature to the boiling point of the solvent. For example, the reaction temperature in the amino group protection reaction may be 15 to 35 ℃, 15 to 20 ℃, 20 to 25 ℃, 25 to 30 ℃, or 30 to 35 ℃. The reaction time can be adjusted by those skilled in the art according to the reaction progress, for example, in the amino protection reaction, the reaction progress of the condensation reaction can be judged by TLC, chromatography, or the like, and for example, the reaction time of the amino protection reaction can be 5 to 6 hours, 6 to 8 hours, 8 to 10 hours, 10 to 12 hours, 12 to 16 hours, 16 to 24 hours, or 24 to 36 hours.
In the above amino group-protecting reaction, the reaction is usually carried out under gas-protecting conditions. Suitable methods of providing gas protection should be known to those skilled in the art, for example, the conditions under which gas protection may be provided by nitrogen, inert gases, etc., which may be, in particular, helium, neon, argon, krypton, etc.
In the above amino protection reaction, the person skilled in the art may select an appropriate method to post-treat the product obtained by the reaction, and for example, may include: desolventizing and purifying. After the reaction is completed, the product may be freed of solvent and further purified to provide the compound of formula VII. Suitable purification methods should be known to those skilled in the art and may be, for example, column chromatography or the like.
In a third aspect, the invention provides the use of a clofarabine modified oligonucleotide as provided in the first aspect of the invention in the manufacture of a medicament. The clofarabine and/or gemcitabine modified oligonucleotide provided by the invention has good specificity and targeting to target cells (for example, tumor cells, specifically pancreatic duct adenocarcinoma, acute lymphoblastic leukemia, acute T lymphoblastic leukemia, triple negative breast cancer, colorectal cancer and the like), can keep the original pharmaceutical activity of clofarabine, can reduce toxic and side effects and improve bioavailability, and can be used as a tumor therapeutic drug.
The invention designs and synthesizes nucleoside drug clofarabine and/or gemcitabine into phosphoramidite monomer which can be used for solid phase synthesis, uses solid phase synthesis technology to realize accurate functionalization of clofarabine and/or gemcitabine on the oligonucleotide, and the prepared clofarabine and/or gemcitabine modified oligonucleotide can release clofarabine under the action of nuclease, has higher cytotoxicity on tumor cells, retains the pharmaceutical activity of clofarabine and/or gemcitabine, and has good industrialization prospect.
The invention of the present application is further illustrated by the following examples, which are not intended to limit the scope of the present application.
Example 1
Preparation of clofarabine phosphoramidite monomer:
1) To the solution was added 3.2 equivalents of t-butyldimethylsilyl chloride (3.2 equivalents) at room temperature, clofarabine (1 equivalent) was added, 20mL of N, N-dimethylformamide, and imidazole (15 equivalents) was added to the single-necked flask. The mixture was stirred for 12h. The solvent was removed in vacuo using a rotary evaporator, and the mixture was separated and purified by silica gel column chromatography to give TBDMS-clofarabine (99%) as a white solid. 1 H NMR(400MHz,Chloroform-d)δ8.05(d,J＝2.6Hz,1H),6.51(s,2H),6.43(dd,J＝17.5,3.9Hz,1H),4.99(dt,J＝52.1,2.9Hz,1H),4.61(dt,J＝18.0,2.9Hz,1H),3.94(q,J＝4.5Hz,1H),3.84(d,J＝4.6Hz,2H),0.92(d,J＝3.8Hz,18H),0.14(d,J＝1.9Hz,6H),0.10(s,6H)ppm.
Clofarabine is reacted with t-butyldimethylchlorosilane (TBDMSCl) in N, N-Dimethylformamide (DMF) under the action of imidazole (imidozole) as follows:
2) TBDMS-clofarabine (1 eq) and triethylamine (18 eq) were added in a single-necked flask, 50mL of dichloromethane was added, and benzoyl chloride (6.8 eq) was added to the solution at 0deg.C. The reaction was carried out for 8 hours, the solvent was removed in vacuo using a rotary evaporator, and the reaction mixture was separated and purified by silica gel column chromatography to give TBDMS-Bz as a colorless oil 2 Clofarabine (80%). 1 H NMR(400MHz,Chloroform-d)δ8.27(d,J＝2.5Hz,1H),7.86(s,2H),7.84-7.83(m,2H),7.49(q,J＝7.5Hz,2H),7.37(t,J＝7.7Hz,4H),6.48(dd,J＝18.9,3.4Hz,1H),5.07-4.89(m,1H),4.66-4.57(m,1H),3.97(q,J＝4.4Hz,1H),3.83(d,J＝4.6Hz,2H),0.92(d,J＝6.9Hz,18H),0.15(d,J＝2.4Hz,6H),0.09-0.08(m,6H)ppm.
The reaction formula of TBDMS-clofarabine and benzoyl chloride (BzCl) in dichloromethane under the action of triethylamine is as follows:
3) Adding TBDMS-Bz in a Single Vial 2 Clofarabine (1 eq.) and acetic acid (12 eq.) are added to 100mL of tetrahydrofuran and tetrabutylammonium fluoride (3 eq.) is added at 0 ℃. The reaction was carried out at room temperature for 6 hours. Solvent was removed using a rotary evaporator and purified by flash column chromatography to give Bz as a white solid 2 Clofarabine (71%). 1H NMR (400 MHz, chloroform-d) δ8.39 (s, 1H), 7.81 (d, J=7.7 Hz, 4H), 7.49 (t, J=7.4 Hz, 2H), 7.36 (t, J=7.6 Hz, 4H), 6.40 (dd, J=15.4, 3.6Hz, 1H), 5.02 (d, J=51.8 Hz, 1H), 4.52 (d, J=17.8 Hz, 1H), 3.96-3.91 (m, 1H), 3.82-3.67 (m, 2H) ppm.
TBDMS-Bz 2 The reaction of clofarabine with tetrabutylammonium fluoride (TBAF) in tetrahydrofuran under the action of acetic acid is given by:
4) Bz addition in a Single Vial 2 Clofarabine (1 eq.) was dissolved in 40mL pyridine and DMTrCl (1.2 eq.) was added to the solution in 3 portions at room temperature. The mixture is put under N 2 Stirring for 8h under protection, removing solvent in vacuum by rotary evaporator, and separating and purifying by flash column chromatography to obtain white foaming DMTR-Bz 2 Clofarabine (82%). 1H NMR (400 MHz, acetonitrile-d 3) δ8.26 (d, J=2.0 Hz, 1H), 7.82 (d, J=7.4 Hz, 4H), 7.58 (t, J=7.5 Hz, 2H)),7.42(t,J＝7.8Hz,6H),7.30(d,J＝8.6Hz,4H),7.25(t,J＝7.3Hz,2H),7.19(t,J＝7.1Hz,1H),6.83(d,J＝8.7Hz,4H),6.44(dd,J＝14.7,4.3Hz,1H),5.19(dt,J＝51.9,3.9Hz,1H),4.55(dt,J＝18.1,3.9Hz,1H),4.13-4.09(m,1H),3.73(s,6H),3.38(ddd,J＝34.7,10.5,4.9Hz,2H)ppm.
Bz 2 The reaction of clofarabine with 4,4' -dimethoxytrityl chloride (DMTrCl) in pyridine is as follows:
5) DMTr-Bz addition to a single vial 2 Clofarabine (1 eq.) and DIPEA (6 eq.) in 100mL dichloromethane. At 0 ℃ and N 2 To the mixed solution was added 2-cyanoethyl N, N-diisopropylchlorophosphamide (3 eq.) under protection. After 10min the mixture was returned to room temperature and stirring was continued for 1h. The solvent was removed in vacuo at room temperature using a rotary evaporator and purified by flash column chromatography to give a white foamy solid as clofarabine phosphoramidite monomer (73%). 1 H NMR(400MHz,Acetonitrile-d 3 )δ8.30(s,1H),7.82(d,J＝7.8Hz,4H),7.59(t,J＝7.4Hz,2H),7.43(t,J＝7.6Hz,6H),7.31(d,J＝8.7Hz,4H),7.25(t,J＝7.5Hz,2H),7.22-7.17(m,1H),6.83(d,J＝8.0Hz,4H),6.45(dd,J＝14.0,4.5Hz,1H),5.35(dt,J＝51.9,4.1Hz,1H),4.84-4.73(m,1H),4.23(q,J＝5.0Hz,1H),3.74(s,6H),3.69-3.63(m,2H),3.63-3.55(m,2H),3.44(t,J＝5.2Hz,2H),2.52(t,J＝6.0Hz,2H),1.16(dd,J＝12.1,6.8Hz,12H)ppm. 31 P NMR(162MHz,Acetonitrile-d 3 )δ150.54ppm.
DMTr-Bz 2 The reaction of clofarabine with 2-cyanoethyl N, N-diisopropylchlorophosphamide in dichloromethane is given by:
example 2
Preparation of gemcitabine phosphoramidite monomer:
1) Gemcitabine (710 mg,2.7 mmol) was added to a 100mL single neck round bottom flask, 20mL DMF, and acetic anhydride (280. Mu.L, 2.96 mmol) was added to the solution at room temperature. N (N) 2 The mixture was stirred overnight under protection. The solvent DMF was removed in vacuo using a rotary evaporator, the residue was mixed with silica gel and purified by column chromatography on silica gel to give Ac-gemcitabine (616 mg, 75%) as a white solid. 1 H NMR(400MHz,DMSO-d 6 )δ11.02(s,1H),8.24(d,J＝7.6Hz,1H),7.25(d,J＝7.6Hz,1H),6.33(d,J＝6.6Hz,1H),6.17(t,J＝7.4Hz,1H),5.31(t,J＝5.4Hz,1H),4.24-4.14(m,1H),4.11(q,J＝5.3Hz,1H),3.89(dt,J＝8.5,3.0Hz,1H),3.80(ddd,J＝12.7,5.2,2.4Hz,1H),3.65(ddd,J＝12.7,5.9,3.6Hz,1H),3.16(d,J＝5.3Hz,2H),2.11(s,3H)ppm.
Gemcitabine and acetic anhydride (Ac) 2 O) in N, N-Dimethylformamide (DMF) as follows:
2) Ac-gemcitabine (2.12 g,6.95 mmol) was added to a 100mL single neck round bottom flask and dissolved in 50mL pyridine, DMTrCl (2.53 g,7.65 mmol) was added to the solution in 3 portions at room temperature. The mixture is put under N 2 After stirring for 8h under protection, the solvent pyridine was removed in vacuo using a rotary evaporator, the residue was mixed with silica gel and purified by flash column chromatography to give DMTR-Ac-gemcitabine (3.04 g, 72%) as a white foam. 1 H NMR(400MHz,DMSO-d 6 )δ11.04(s,1H),8.15(d,J＝7.6Hz,1H),7.39(d,J＝7.7Hz,2H),7.34(t,J＝7.6Hz,2H),7.27(d,J＝8.2Hz,5H),7.11(d,J＝7.6Hz,1H),6.92(d,J＝8.3Hz,4H),6.42(d,J＝6.7Hz,1H),6.23(t,J＝7.2Hz,1H),4.44-4.30(m,1H),4.07(d,J＝7.3Hz,2H),3.75(s,6H),3.43(dd,J＝11.4,4.5Hz,1H),3.32(s,1H),2.11(s,3H)ppm.
The reaction of Ac-gemcitabine with 4,4' -dimethoxytrityl chloride (DMTrCl) in pyridine is as follows:
3) DMTr-Ac-gemcitabine (637 mg,1.05 mmol) and DIPEA (1.055 mL,6.30 mmol) were added to a 100mL single neck round bottom flask, 20mL dichloromethane. At 0 ℃ and N 2 To the mixture was added 2-cyanoethyl N, N-diisopropylchlorophosphamide (755. Mu.L, 3.148 mmol) under protection. After 10min the mixture was returned to room temperature and stirring was continued for 1h. The solvent was removed in vacuo using a rotary evaporator at room temperature, and the residue was mixed with silica gel and purified by flash column chromatography to give a white foamy solid as gemcitabine phosphoramidite monomer (795 mg, 95%). 1 H NMR(400MHz,Acetonitrile-d 3 )δ8.95(d,J＝10.8Hz,1H),8.10(d,J＝7.6Hz,1H),7.46(d,J＝7.5Hz,2H),7.34(t,J＝9.8Hz,5H),7.28(d,J＝7.4Hz,1H),7.11(d,J＝7.9Hz,1H),6.90(d,J＝7.7Hz,4H),6.30(t,J＝7.7Hz,1H),4.60(p,J＝10.6Hz,1H),4.20(d,J＝8.3Hz,1H),3.80(s,6H),3.57(q,J＝13.1,10.0Hz,3H),3.50-3.44(m,1H),3.15(p,J＝6.7,6.3Hz,1H),2.65(t,J＝5.8Hz,2H),2.18(s,3H),1.84(s,2H),1.16(d,J＝6.5Hz,6H),1.01(d,J＝6.6Hz,6H)ppm. 31 P NMR(162MHz,Acetonitrile-d 3 )δ151.74ppm.
DMTr-Ac-gemcitabine and 2-cyanoethyl N, N-diisopropylchlorophosphamide in dichloromethane are reacted as follows:
Example 3
Preparation of clofarabine modified oligonucleotides:
the clofarabine phosphoramidite monomer and the gemcitabine phosphoramidite monomer are respectively dissolved in anhydrous acetonitrile to prepare the concentration of 0.1M, and the solution is used for solid phase synthesis by a DNA solid phase synthesizer. Coupling is carried out at 25-35 ℃ for 5 minutes each time, two times of coupling are carried out, clofarabine and/or gemcitabine modified oligonucleotides with different sequences are synthesized on a Universal-CPG at the 3' end, and the polynucleotide sequences of the coupled oligonucleotides are shown in table 1. After the coupling, the oligonucleotide was cleaved from the solid phase support using concentrated ammonia and purified using HPLC. After purification, mass spectrum characterization is carried out, the molecular weight of the substances of each sequence obtained by mass spectrum molecular measurement is consistent with the theoretical molecular weight, and the following characterization results are given in an exemplary way: the results of the mass spectrum characterization of sequence 5 are shown in fig. 1, MS: calculated 18471.9 (Found: 18472.9), sequence 8 mass spectrum characterization results are shown in FIG. 2, MS: calculated 18106.2 (Found: 18102.6), sequence 10 mass spectrum characterization results are shown in FIG. 3, MS: calculated 12670.2 (Found: 12667.6).
TABLE 1 clofarabine and/or gemcitabine modified oligonucleotide sequences
Example 4
The gemcitabine and clofarabine modified oligonucleotide (SEQ ID NO.5-SEQ ID NO. 9) prepared in example 3 was used for cancer cell inhibition experiments, and gemcitabine and clofarabine were used as comparative examples. Human colon cancer cells HCT-116 are dispersed in RPMI-1640 culture medium according to the cell density of 5 ten thousand/mL, culture medium solutions of medicines with different concentrations are prepared, the medicine solutions are mixed with the cell solutions in a ratio of 1:1, the mixture is added into a 96-well plate, 100 mu L of each well is added, and the mixture is placed in a constant temperature incubator for culture. After 72h, the medium was removed, 100. Mu.L of fresh medium solution in which CCK-8 was dissolved was added, and after shaking and mixing, the mixture was incubated at 37℃for 1h, and the absorption at 450nm was measured using a Synergy-2 multifunctional microplate reader, and the experimental results were shown in FIG. 4. As can be seen from FIG. 4, the gemcitabine and clofarabine modified oligonucleotides have high inhibitory activity on cancer cells, and the IC50 value is about 12.82nM-139.0nM, and the original pharmaceutical activities of the two drugs are still basically maintained.
Example 5
The clofarabine modified oligonucleotides (SEQ ID NO.1-SEQ ID NO. 4) prepared in example 3 were used for specific recognition of cancer cells to achieve targeted delivery of clofarabine, with blank cells as a comparative example. Taking CCRF-CEM cells of human acute lymphoblastic leukemia cells as an example for flow cytometry experiments, firstly taking a proper volume of CCRF-CEM cell suspension according to 15 ten thousand cells per sample, centrifuging at 800r/min for 3min, removing supernatant, adding 200 mu L of binding buffer solution into each sample, adding a certain volume of aptamer mother solution, mixing to a final concentration of 200nM, and placing at 4 ℃ in a dark place for 1h. Centrifuging at 800r/min for 3min, removing supernatant, adding cleaning buffer solution, mixing, centrifuging, and washing three times. The experiment was performed by adding 400. Mu.L of the washing buffer using a flow cytometer, and the experimental results are shown in FIG. 5. As can be seen from fig. 5, the clofarabine modified oligonucleotide can still specifically bind to cancer cells, thus realizing targeted delivery of clofarabine drug.
In summary, the present invention effectively overcomes the disadvantages of the prior art and has high industrial utility value.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
Sequence listing
<110> university of Hunan
<120> a clofarabine modified oligonucleotide
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 41
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
atctmactgc tgcgccgccg ggaaaatact gtacggttag a 41
<210> 2
<211> 41
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
atctamctgc tgcgccgccg ggaaaatact gtacggttag a 41
<210> 3
<211> 41
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
atctaactgc tgcgccgccg ggmaaatact gtacggttag a 41
<210> 4
<211> 41
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
atctaactgc tgcgccgccg ggamaatact gtacggttag a 41
<210> 5
<211> 59
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
kmmactcata gggttagggg ctgctggcca gatactcaga tggtagggtt actatgagc 59
<210> 6
<211> 60
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
kkkmactcat agggttaggg gctgctggcc agatactcag atggtagggt tactatgagc 60
<210> 7
<211> 59
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
kkmactcata gggttagggg ctgctggcca gatactcaga tggtagggtt actatgagc 59
<210> 8
<211> 58
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
kmactcatag ggttaggggc tgctggccag atactcagat ggtagggtta ctatgagc 58
<210> 9
<211> 60
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
kmmmactcat agggttaggg gctgctggcc agatactcag atggtagggt tactatgagc 60
<210> 10
<211> 57
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
mactcatagg gttaggggct gctggccaga tactcagatg gtagggttac tatgagc 57
<210> 11
<211> 41
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
atctaactgc tgcgccgccg ggaaaatact gtacggttag a 41
<210> 12
<211> 41
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
atctaactgc tgcgccgccg ggaaaatact gtacggttag a 41
<210> 13
<211> 41
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
atctaactgc tgcgccgccg ggaaaatact gtacggttag a 41
<210> 14
<211> 41
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
atctaactgc tgcgccgccg ggaaaatact gtacggttag a 41
<210> 15
<211> 56
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
actcataggg ttaggggctg ctggccagat actcagatgg tagggttact atgagc 56
<210> 16
<211> 56
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
actcataggg ttaggggctg ctggccagat actcagatgg tagggttact atgagc 56
<210> 17
<211> 56
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
actcataggg ttaggggctg ctggccagat actcagatgg tagggttact atgagc 56
<210> 18
<211> 56
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 18
actcataggg ttaggggctg ctggccagat actcagatgg tagggttact atgagc 56
<210> 19
<211> 56
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
actcataggg ttaggggctg ctggccagat actcagatgg tagggttact atgagc 56
<210> 20
<211> 56
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 20
actcataggg ttaggggctg ctggccagat actcagatgg tagggttact atgagc 56

Claims (3)

1. A clofarabine-modified oligonucleotide comprising a nucleic acid aptamer fragment modified with a clofarabine phosphoramidite monomer group;
the nucleic acid aptamer segment is modified with one or more clofarabine phosphoramidite monomer groups, and the clofarabine phosphoramidite monomer groups are modified at the 5 'end of the nucleic acid aptamer segment and/or modified at the 3' end of the nucleic acid aptamer segment and/or added in the middle of the nucleic acid aptamer segment;
the clofarabine phosphoramidite monomer group is connected with the nucleic acid aptamer fragment through a phosphodiester bond;
and/or, the chemical structural formula of the clofarabine phosphoramidite monomer group modified at the 5' end of the aptamer fragment is shown as follows:
the chemical structural formula of the clofarabine phosphoramidite monomer group added in the middle of the aptamer fragment is shown as follows:
The chemical structural formula of the clofarabine phosphoramidite monomer group modified at the 3' -end of the aptamer fragment is shown as follows:
the nucleic acid aptamer segment is further modified with gemcitabine phosphoramidite monomer groups;
the nucleic acid aptamer segment is modified with one or more gemcitabine phosphoramidite monomer groups, and the gemcitabine phosphoramidite monomer groups are modified at the 5 'end of the nucleic acid aptamer segment and/or modified at the 3' end of the nucleic acid aptamer segment and/or added in the middle of the nucleic acid aptamer segment;
the gemcitabine phosphoramidite monomer group is connected with the nucleic acid aptamer fragment through a phosphodiester bond;
and/or, the chemical structural formula of the gemcitabine phosphoramidite monomer group modified at the 5' end of the aptamer fragment is as follows:
the chemical structural formula of the gemcitabine phosphoramidite monomer group added to the middle of the aptamer fragment is shown as follows:
the chemical structural formula of the gemcitabine phosphoramidite monomer group modified at the 3' end of the aptamer fragment is shown as follows:
the polynucleotide sequence of the clofarabine modified oligonucleotide is shown as one of SEQ ID NO. 5-9, M in SEQ ID NO. 5-9 is clofarabine, and K is gemcitabine.
2. A method of preparing a clofarabine-modified oligonucleotide according to claim 1 comprising:
connecting a clofarabine phosphoramidite monomer and/or a gemcitabine phosphoramidite monomer with a nucleic acid aptamer segment through a solid phase synthesis method, wherein the reaction temperature of the solid phase synthesis method is 15-35 ℃, the reaction time is 1-20 minutes, and the air humidity is 30-70%;
and/or, the chemical structural formula of the clofarabine phosphoramidite monomer is shown as follows:
and/or, the chemical structural formula of the gemcitabine phosphoramidite monomer is shown as follows:
3. use of a clofarabine-modified oligonucleotide according to claim 1 for the preparation of a medicament for the treatment of human colon cancer diseases.
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CN110179991A (en) * 2019-06-13 2019-08-30 湖南大学 A kind of pro-drug with anti-tumor activity and preparation and application
CN110354268A (en) * 2018-12-29 2019-10-22 湖南大学 Coupling system and the application of a kind of aptamer and its annular divalent aptamer-drug
CN110643609A (en) * 2019-09-20 2020-01-03 上海交通大学 Medicine aptamer constructed by nucleoside analogue medicine molecules and preparation method and application thereof
CN110760516A (en) * 2019-11-12 2020-02-07 赣南医学院 Aptamer derivatives and aminated aptamer derivatives, their use and pharmaceutical conjugates

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104593372A (en) * 2015-02-09 2015-05-06 湖南大学 Aptamer, kit and method for detecting pancreatic duct adenocarcinoma
CN108671235A (en) * 2018-05-21 2018-10-19 上海交通大学 The functional nucleic acid and its derivative of backbone integration nucleoside analogue drugs and application
CN109224080A (en) * 2018-09-26 2019-01-18 河北大学 A kind of targeted nano carrier of support nucleosides series antineoplastic medicament and its preparation method and application
CN110354268A (en) * 2018-12-29 2019-10-22 湖南大学 Coupling system and the application of a kind of aptamer and its annular divalent aptamer-drug
CN110179991A (en) * 2019-06-13 2019-08-30 湖南大学 A kind of pro-drug with anti-tumor activity and preparation and application
CN110643609A (en) * 2019-09-20 2020-01-03 上海交通大学 Medicine aptamer constructed by nucleoside analogue medicine molecules and preparation method and application thereof
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