CN114480401A - Clofarabine modified oligonucleotide - Google Patents

Clofarabine modified oligonucleotide Download PDF

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CN114480401A
CN114480401A CN202011173092.5A CN202011173092A CN114480401A CN 114480401 A CN114480401 A CN 114480401A CN 202011173092 A CN202011173092 A CN 202011173092A CN 114480401 A CN114480401 A CN 114480401A
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clofarabine
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phosphoramidite monomer
gemcitabine
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CN114480401B (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 an oligonucleotide modified by clofarabine. The invention provides a clofarabine modified oligonucleotide, which comprises an aptamer segment, wherein the aptamer segment is modified with a clofarabine phosphoramidite monomer group. The nucleotide drugs clofarabine and/or gemcitabine are designed and synthesized into a phosphoramidite monomer for solid phase synthesis, the fixed-point precise functionalization of clofarabine and/or gemcitabine on oligonucleotide is realized by using a solid phase synthesis technology, and the prepared clofarabine and/or gemcitabine modified oligonucleotide can release clofarabine under the action of nuclease, still has higher cytotoxicity on tumor cells, retains the medicinal activity of clofarabine and/or gemcitabine, and has good industrial prospect.

Description

Clofarabine modified oligonucleotide
Technical Field
The invention relates to the field of pharmaceutical chemistry, in particular to an oligonucleotide modified by clofarabine.
Background
Clofarabine is a purine nucleoside antimetabolite, and differs from other purine nucleoside analogs in the presence of chlorine in the purine ring and fluorine 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 intracellularly by deoxycytidine kinase to the active 5 '-monophosphate metabolite and by mono-and diphosphate kinases to the 5' -triphosphate metabolite. Thereby achieving the inhibition of ribonucleotide reductase, terminating the extension of the DNA chain and inhibiting the repair by competitively inhibiting DNA polymerase, and further inhibiting the synthesis of DNA. In preclinical models, clofarabine has been shown to inhibit the ability of 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 childhood Acute Lymphoblastic Leukemia (ALL) in several countries around the world.
Oligonucleotides are short single-stranded or double-stranded DNA or RNA molecules, playing an increasingly important role in molecular biology, can be used for probes for nucleic acid sequence detection, single base diversity analysis, antisense oligonucleotides, and the like, and are very effective research tools. The oligonucleotide is functionally modified, so that the oligonucleotide can be endowed with new functions, and the application range of the oligonucleotide is widened.
Disclosure of Invention
In view of the above-mentioned disadvantages of the prior art, the present invention provides a clofarabine-modified oligonucleotide, which solves the problems of the prior art.
To achieve the above and other related objects, the present invention provides a clofarabine-modified oligonucleotide comprising an aptamer segment modified with a clofarabine phosphoramidite monomer group.
In some embodiments of the invention, the aptamer segment is modified with one or more clofarabine phosphoramidite monomer groups that are modified at the 5 'end of the aptamer segment and/or modified at the 3' end of the aptamer segment and/or added in the middle of the aptamer segment.
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 chemical structure of the clofarabine phosphoramidite monomer group modified at the 5' end of the aptamer fragment is as follows:
Figure BDA0002747900610000021
the chemical structural formula of the intermediate clofarabine phosphoramidite monomer group added to the aptamer fragment is shown as follows:
Figure BDA0002747900610000022
the chemical structural formula of the clofarabine phosphoramidite monomer group modified at the 3' end of the aptamer fragment is shown as follows:
Figure BDA0002747900610000023
in some embodiments of the invention, the aptamer fragment is further modified with a gemcitabine phosphoramidite monomer group.
In some embodiments of the invention, the aptamer segment is modified with one or more gemcitabine phosphoramidite monomer groups modified at the 5 'end of the aptamer segment and/or modified at the 3' end of the aptamer segment and/or added in the middle of the aptamer segment.
In some embodiments of the invention, the gemcitabine phosphoramidite monomer moiety is linked to the aptamer fragment via a phosphodiester linkage.
In some embodiments of the present invention, the gemcitabine phosphoramidite monomer group modified at the 5' end of the aptamer fragment has the following chemical structure:
Figure BDA0002747900610000024
the chemical structure of the gemcitabine phosphoramidite monomer group added to the middle of the aptamer fragment is shown below:
Figure BDA0002747900610000031
the chemical structure of the gemcitabine phosphoramidite monomer group modified at the 3' end of the aptamer fragment is shown below:
Figure BDA0002747900610000032
in some embodiments of the invention, the polynucleotide sequence of the aptamer fragment comprises a sequence as set forth in one of SEQ ID No. 11-20.
In some embodiments of the invention, the polynucleotide sequence of the clofarabine modified oligonucleotide comprises a sequence shown in one of SEQ ID NO. 1-10.
In another aspect, the present invention provides a method for preparing the clofarabine-modified oligonucleotide, which comprises:
the clofarabine phosphoramidite monomer is connected with the aptamer segment through a solid phase synthesis method.
In some embodiments of the present invention, 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%.
In some embodiments of the invention, the chemical structure of the clofarabine phosphoramidite monomer is as follows:
Figure BDA0002747900610000033
in some embodiments of the present invention, the gemcitabine phosphoramidite monomer has the following chemical structure:
Figure BDA0002747900610000041
the invention also provides application of the clofarabine modified oligonucleotide in preparation of a medicament.
Drawings
FIG. 1 is a schematic diagram showing the mass spectrometric identification result of the clofarabine-modified oligonucleotide in example 3 of the present invention.
FIG. 2 is a schematic diagram showing the mass spectrometric identification results of the clofarabine-modified oligonucleotide in example 3 of the present invention.
FIG. 3 is a schematic diagram showing the mass spectrometric identification results of the clofarabine-modified oligonucleotide in example 3 of the present invention.
FIG. 4 is a schematic diagram showing the results of experiments on the inhibition of cancer cells by gemcitabine and clofarabine modified oligonucleotides according to example 4 of the present invention.
FIG. 5 is a schematic diagram showing the results of the experiment for the specific binding of clofarabine-modified oligonucleotide to cancer cells in example 5 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments, and other advantages and effects of the present invention will be readily apparent to those skilled in the art from the disclosure of the present specification.
The inventor of the invention surprisingly discovers that the oligonucleotide modified with clofarabine can not only keep the original medicinal activity of the clofarabine, but also increase the targeting property of the medicament, reduce toxic and side effects and improve the bioavailability after a large number of practical researches, and the invention is completed on the basis.
The invention provides a clofarabine modified oligonucleotide, which comprises an aptamer segment, wherein the aptamer segment is modified with a clofarabine phosphoramidite monomer group. The clofarabine-modified oligonucleotide can be prepared from a clofarabine phosphoramidite monomer by a solid-phase synthesis method, and the prepared clofarabine-modified oligonucleotide can comprise a clofarabine phosphoramidite monomer group formed by the clofarabine phosphoramidite monomer and a nucleic acid aptamer fragment connected with the clofarabine phosphoramidite monomer group.
The oligonucleotide modified by clofarabine provided by the invention can comprise a clofarabine phosphoramidite monomer group. The specific structure of the clofarabine phosphoramidite monomer group will generally correspond to the clofarabine phosphoramidite monomer used. In one embodiment of the present invention, the chemical structural formula of the clofarabine phosphoramidite monomer used is as follows:
Figure BDA0002747900610000051
in the clofarabine modified oligonucleotide provided by the invention, the clofarabine phosphoramidite monomer group can be modified at each position of the aptamer segment, for example, the clofarabine phosphoramidite monomer group can be modified at the 5 'end of the aptamer segment, also can be modified at the 3' end of the aptamer segment, and also can be added in the middle of the aptamer segment. The aptamer fragment can be modified with one or more clofarabine phosphoramidite monomer groups, for example, an oligonucleotide sequence containing 60 bases can be modified with 1-11, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 clofarabine phosphoramidite monomer groups. When the aptamer segment is modified with multiple clofarabine phosphoramidite monomer groups, the individual clofarabine phosphoramidite monomer groups can be discontinuous or at least a portion of the clofarabine phosphoramidite monomer groups can be continuous.
In the clofarabine-modified oligonucleotide provided by the invention, as described above, the clofarabine-modified oligonucleotide can be prepared from a clofarabine phosphoramidite monomer by 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 by a phosphodiester bond. Specifically, the trivalent phosphorus group in the clofarabine phosphoramidite monomer can couple with the hydroxyl group at the 5 'end of the aptamer fragment, thereby forming a phosphodiester bond, the deprotected (e.g., DMT-protected) hydroxyl group in the clofarabine phosphoramidite monomer can couple with the phosphoramidite group at the 3' end of the aptamer fragment, thereby forming a phosphodiester bond, and the deprotected (e.g., DMT-protected) hydroxyl group in the clofarabine phosphoramidite monomer can react with the phosphoramidite group in the clofarabine phosphoramidite monomer, thereby forming a phosphodiester bond
In one embodiment of the present invention, when the clofarabine phosphoramidite monomer group is modified at the 5' end of the aptamer fragment, the chemical structure of the formed group can be as follows:
Figure BDA0002747900610000052
in one embodiment of the present invention, when the clofarabine phosphoramidite monomer group is added to the middle of the aptamer fragment, the chemical structure of the formed group can be as follows:
Figure BDA0002747900610000061
in one embodiment of the present invention, when the clofarabine phosphoramidite monomer group is modified at the 3' end of the aptamer fragment, the chemical structure of the formed group can be as follows:
Figure BDA0002747900610000062
in the clofarabine modified oligonucleotide provided by the invention, the aptamer segment is further modified with gemcitabine phosphoramidite monomer group. The oligonucleotide modified by clofarabine and gemcitabine can be prepared by a solid phase synthesis method from clofarabine phosphoramidite monomers and gemcitabine phosphoramidite monomers, and the prepared oligonucleotide modified by clofarabine and gemcitabine can comprise clofarabine phosphoramidite monomer groups formed correspondingly by the clofarabine phosphoramidite monomers, gemcitabine phosphoramidite monomer groups formed correspondingly by the gemcitabine phosphoramidite monomers, and nucleic acid aptamer fragments connected with the clofarabine phosphoramidite monomer groups and/or gemcitabine phosphoramidite monomer groups.
The clofarabine modified oligonucleotide provided by the invention can comprise a gemcitabine phosphoramidite monomer group. The specific structure of the gemcitabine phosphoramidite monomer group will generally correspond to the gemcitabine phosphoramidite monomer used. In one embodiment of the present invention, the chemical structure of the gemcitabine phosphoramidite monomer used is as follows:
Figure BDA0002747900610000063
in the clofarabine modified oligonucleotide provided by the invention, gemcitabine phosphoramidite monomer groups can be modified at each position of a nucleic acid aptamer fragment, for example, the gemcitabine phosphoramidite monomer group may be modified at the 5 'end of the aptamer fragment, may be modified at the 3' end of the aptamer fragment, may be added in the middle of the aptamer fragment, further, for example, both ends of the gemcitabine phosphoramidite monomer group may each independently be unmodified, linked to the aptamer fragment, gemcitabine phosphoramidite monomer group, or other clofarabine phosphoramidite monomer groups, further, for example, both ends of the gemcitabine phosphoramidite monomer group may each independently be unmodified, linked to the aptamer fragment, clofarabine phosphoramidite monomer group, or other gemcitabine phosphoramidite monomer groups. The aptamer fragment can be modified with one or more gemcitabine phosphoramidite monomer groups, for example, a 60 base oligonucleotide sequence can be modified with 1-10, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 gemcitabine phosphoramidite monomer groups. When the aptamer segment is modified with a plurality of gemcitabine phosphoramidite monomer groups, there may be no continuity between each gemcitabine phosphoramidite monomer group, or there may be continuity between at least some of the gemcitabine phosphoramidite monomer groups.
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 by 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 by a phosphodiester bond, and a gemcitabine phosphoramidite monomer group formed by the gemcitabine phosphoramidite monomer can be connected with an adjacent group or fragment by a phosphodiester bond. Specifically, the trivalent phosphorus group in the clofarabine phosphoramidite monomer, or gemcitabine phosphoramidite monomer can be coupled to a hydroxyl group of an adjacent group or fragment (e.g., aptamer fragment, clofarabine phosphoramidite monomer group, or gemcitabine phosphoramidite monomer group) to form a phosphodiester bond, while the deprotected (e.g., DMT-protected) hydroxyl group in the clofarabine phosphoramidite monomer, or gemcitabine phosphoramidite monomer group can be coupled to a phosphoramidite group of an adjacent group or fragment (e.g., aptamer fragment, clofarabine phosphoramidite monomer group, or gemcitabine phosphoramidite monomer group) to form a phosphodiester bond.
In one embodiment of the invention, when the gemcitabine phosphoramidite monomer moiety is modified on the aptamer segmentThe chemical structure of the group formed at the 5' end of (a) can be as follows:
Figure BDA0002747900610000071
in one embodiment of the present invention, when gemcitabine phosphoramidite monomer groups are added to the middle of an aptamer fragment, the chemical structure of the resulting groups can be as follows:
Figure BDA0002747900610000072
in one embodiment of the present invention, when the gemcitabine phosphoramidite monomer group is modified at the 3' end of the aptamer fragment, the chemical structure of the resulting group can be as follows:
Figure BDA0002747900610000081
the clofarabine modified oligonucleotide provided by the invention can comprise an aptamer segment. The selection of the specific sequence of the aptamer segment largely determines the targeting property of the oligonucleotide and largely determines the overall stability of the oligonucleotide, and the polynucleotide sequence of the aptamer segment (i.e. the polynucleotide sequence before the unmodified clofarabine phosphoramidite monomer group) can comprise a sequence shown in one of SEQ ID NO. 11-20. Aptamer fragments are typically targeted to their corresponding proteins, e.g., aptamer fragments as shown above can be targeted to protein tyrosine kinase 7(PTK7), all of which enable specific targeting of tumor cells. For another example, the polynucleotide sequence of the clofarabine-modified oligonucleotide can include a sequence shown in one of SEQ ID NO. 1-10.
In a specific embodiment of the present invention, a group formed by a clofarabine phosphoramidite monomer group in the clofarabine modified oligonucleotide can be one of the following groups:
Figure BDA0002747900610000082
wherein n is1Is 0, or a positive integer, for example, can be 0, 1,2, 3, 4, 5, 6, 7, 8, or 9;
n2is a positive integer, for example, can be 1,2, 3, 4, 5, 6, 7, 8, 9, or 10;
n3is 0, or a positive integer, for example, can be 0, 1,2, 3, 4, 5, 6, 7, 8, or 9;
Figure BDA0002747900610000091
represents other fragments of the clofarabine modified oligonucleotide linked to the clofarabine phosphoramidite monomer group.
In another embodiment of the present invention, the group formed by gemcitabine phosphoramidite monomer group in the clofarabine modified oligonucleotide may be one of the following groups:
Figure BDA0002747900610000092
wherein n is4Is 0, or a positive integer, for example, can be 0, 1,2, 3, 4, 5, 6, 7, 8, or 9;
n5is a positive integer, for example, can be 1,2, 3, 4, 5, 6, 7, 8, 9, or 10;
n6is 0, or a positive integer, for example, can be 0, 1,2, 3, 4, 5, 6, 7, 8, or 9;
Figure BDA0002747900610000093
other fragments of clofarabine-modified oligonucleotides linked to gemcitabine phosphoramidite monomer groups are shown.
In a second aspect, the present invention provides a method for preparing a clofarabine-modified oligonucleotide provided in the first aspect, comprising: the clofarabine phosphoramidite monomer and/or gemcitabine phosphoramidite monomer are linked to the aptamer segment by solid phase synthesis.
In the method for preparing the clofarabine modified oligonucleotide provided by the invention, suitable conditions for the solid phase synthesis method should be known to those skilled in the art, and for example, the reaction conditions for the solid phase synthesis method can be A, T, C, G and other natural base coupling reaction conditions. 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 can be 30% -70%, 30% -35%, 35% -40%, 40% -45%, 45% -50%, 50% -55%, 55% -60%, 60% -65%, or 65% -70%; the solvent may be nitrile solvent, ether solvent, halogenated alkane solvent, etc., and specifically may be 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: a compound of formula I is condensed with 2-cyanoethyl N, N-diisopropylphosphoramidite to provide clofarabine phosphoramidite monomer according to the following equation:
Figure BDA0002747900610000101
in the above condensation reaction, the reaction may be carried out in the presence of a base, which may be an organic base, and specifically, for example, DIPEA, triethylamine, DMPA, pyridine, or the like. The base is generally used in excess relative to the compound of formula I, so that the conversion of the reaction is ensured and the reaction proceeds sufficiently in the forward direction. 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: 5-6, 1: 6-7, 1: 7-8, 1: 8-9, 1: 9-10, 1: 10-11, or 1: 11 to 12, preferably 1: 5.5 to 6.5.
In the above condensation reaction, the amount of 2-cyanoethyl N, N-diisopropylphosphoramidite used is usually in excess relative to the compound of formula I, 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 above condensation reaction, the molar ratio of the compound of formula I to 2-cyanoethyl N, N-diisopropylphosphoroamidite may be 1: 1.5-6, 1: 1.5-2, 1: 2-2.5, 1: 2.5-3, 1: 3-3.5, 1: 3.5-4, 1: 4-4.5, 1: 4.5-5, 1: 5-5.5, or 1: 5.5-6, preferably 1: 2.5 to 3.5.
In the above 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 generally an aprotic solvent, and the kind and amount of a suitable reaction solvent should be known to those skilled in the art, and for example, in the condensation reaction, the reaction solvent may be a haloalkane-based solvent, a sulfoxide-based solvent, or the like, and more specifically, dichloromethane, dimethyl sulfoxide, chloroform, 1, 2-dichloroethane, or the like.
In the above condensation reactions, it is generally necessary to avoid carrying out the reaction at too high a 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, etc., and for example, the reaction time of the condensation reaction can be 0.5-3 h, 0.5-1 h, 1-1.5 h, 1.5-2 h, or 2-3 h.
In the above condensation reaction, the reaction is usually carried out under a gas protection. Suitable methods of providing a gas shield will be known to those skilled in the art, for example, conditions under which a gas shield can be provided by nitrogen, inert gases, and the like, and the inert gases can be, in particular, helium, neon, argon, krypton, and the like.
In the above condensation reaction, the product obtained by the reaction may be subjected to a post-treatment by a person skilled in the art by selecting an appropriate method, and for example, may include: desolventizing and purifying. After the reaction is completed, the solvent can be removed from the product, and after further purification, the clofarabine phosphoramidite monomer can be provided. Suitable purification methods will 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 also comprise the following steps: a first hydroxyl protection reaction of the compound of formula II with 4,4' -bis-methoxytrityl chloride (DMTrCl) provides the compound of formula I, the reaction equation is as follows:
Figure BDA0002747900610000111
in the first hydroxyl group-protecting reaction, the reaction may be carried out in the presence of a base which may be an organic base, and specifically, for example, pyridine, DMAP, triethylamine, diethylamine and the like. The base is usually used in a large excess relative to the compound of formula I, and may itself act as a solvent for the reaction system.
In the first hydroxyl protecting reaction, the reaction may be carried out in the presence of a reaction solvent, the reaction solvent used in the first hydroxyl protecting reaction may be generally an aprotic solvent, and the kind and amount of a suitable reaction solvent should be known to those skilled in the art, for example, in the first hydroxyl protecting reaction, the reaction solvent may be a haloalkane-type solvent, a sulfoxide-type solvent, or the like, and more specifically, may be dichloromethane, dimethyl sulfoxide, chloroform, 1, 2-dichloroethane, or the like.
In the first hydroxyl protection reaction, DMTrCl is usually used in an amount substantially equal to or in excess with respect to the compound of formula II, 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 first hydroxyl protection reaction described above, the molar ratio of the compound of formula II to DMTrCl may be 1: 1-1.5, 1: 1-1.1, 1: 1.1-1.2, 1: 1.2-1.3, 1: 1.3 to 1.4, or 1: 1.4 to 1.5, preferably 1: 1.15 to 1.25.
In the first hydroxyl group-protecting reaction, the reaction may be carried out usually at a temperature ranging from 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 those skilled in the art according to the reaction progress, for example, in the first hydroxyl protection reaction, the reaction progress of the first hydroxyl protection reaction can be determined by methods such as TLC and chromatography, and for example, the reaction time of the first hydroxyl protection reaction can be 2-24 h, 2-3 h, 3-4 h, 4-6 h, 6-8 h, 8-12 h, 12-16 h, 16-20 h, or 20-24 h.
In the first hydroxyl protecting reaction, the reaction is usually carried out under a gas atmosphere. Suitable methods of providing a gas shield will be known to those skilled in the art, for example, conditions under which a gas shield can be provided by nitrogen, inert gases, and the like, and the inert gases can be, in particular, helium, neon, argon, krypton, and the like.
In the first hydroxyl protection reaction, the skilled person can select a suitable method to carry out the post-treatment of the product obtained from the reaction, and for example, the method may include: desolventizing and purifying. After the reaction is complete, the product may be freed from the solvent and, after further purification, the compounds of the formula I may be provided. Suitable purification methods will 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 also comprise the following steps: reacting the compound of formula III with tetrabutylammonium fluoride (TBAF) for TBDMS removal to provide a compound of formula II, the reaction equation is as follows:
Figure BDA0002747900610000121
in the TBDMS removal reaction described above, the reaction may be carried out usually in the presence of an acid, which may be usually an organic acid, and specifically, for example, formic acid, acetic acid, propionic acid, and the like. The acid is generally used in excess with respect to the compound of formula III, so as to ensure conversion and to enable the reaction to proceed fully in the forward direction. 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: 5-6, 1: 6-8, 1: 8-10, 1: 10-12, 1: 12-14, 1: 14-16, 1: 16-18, or 1: 18 to 20, preferably 1: 10 to 14.
In the TBDMS removal reaction, tetrabutylammonium fluoride is usually used in an amount substantially equal to or in excess relative to 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 and 1: 2-3, 1: 3-4, 1: 4-6, 1: 6-8, or 1: 8-10, preferably 1: 2 to 4.
In the 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 amount of the 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 haloalkane solvent, a sulfoxide solvent, an ether solvent, an amide solvent, and the like, and more specifically tetrahydrofuran, trichloromethane, N-dimethylformamide, dimethylsulfoxide, 1, 2-dichloroethane, and the like.
In the TBDMS removal reaction described above, it is generally necessary to avoid carrying out the reaction 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 those 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 determined by TLC, chromatography, and the like, 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, the reaction is generally carried out under the protection of gas. Suitable methods of providing a gas shield will be known to those skilled in the art, for example, conditions under which a gas shield can be provided by nitrogen, inert gases, and the like, and the inert gases can be, in particular, helium, neon, argon, krypton, and the like.
In the TBDMS removal reaction, the skilled person can select a suitable method to perform the post-treatment of the reaction product, and for example, the method may include: desolventizing and purifying. After the reaction is finished, the solvent can be removed from the product, and after further purification, the clofarabine phosphoramidite monomer can be provided. Suitable purification methods will 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 also comprise the following steps: subjecting the compound of formula IV to an amino protection reaction with benzoyl chloride (BzCl) to provide a compound of formula III, the reaction equation is as follows:
Figure BDA0002747900610000131
in the above-mentioned amino group protection reaction, the reaction may be carried out usually in the presence of a base, which may be usually an organic base, and specifically, for example, imidazole, triethylamine, or N, N-diisopropylethylamine, and the like may be mentioned. The base is generally used in excess relative to the compound of formula III, so that the conversion of the reaction is ensured and the reaction proceeds sufficiently in the forward direction. For example, in the amino protection reaction described above, the molar ratio of the compound of formula I to the base may be 1: 3-30, 1: 3-4, 1: 4-6, 1: 6-8, 1: 8-10, 1: 10-12, 1: 12-14, 1: 14-16, 1: 16-18, 1: 18-20, 1: 20-25, or 1: 25-30, preferably 1: 16 to 20.
In the above amino protection reaction, the benzoyl chloride is usually used in an amount substantially equal to or in excess relative to the compound of formula IV, 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 amino protection reaction described above, the molar ratio of the compound of formula IV to benzoyl chloride may be 1: 2-20, 1: 2-3, 1: 3-4, 1: 4-6, 1: 6-8, 1: 8-10, 1: 10-12, 1: 12-14, 1: 14-16, 1: 16-18, or 1: 18 to 20, preferably 1: 6-8.
In the above-mentioned amino group protecting reaction, the reaction may be carried out in the presence of a reaction solvent, the reaction solvent used in the amino group protecting reaction may be generally an aprotic solvent, and the kind and amount of a suitable reaction solvent should be known to those skilled in the art, and for example, in the amino group protecting reaction, the reaction solvent may specifically be a haloalkane-based solvent, a sulfoxide-based solvent, an amide-based solvent, and the like, and more specifically, may be dichloromethane, trichloromethane, N-dimethylformamide, dimethylsulfoxide, 1, 2-dichloroethane, and the like.
In the above-mentioned amino protection reaction, the reaction is usually carried out at an excessively high temperature to be avoided. For example, the reaction temperature in the amino 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 methods such as TLC and chromatography, and for example, the reaction time of the amino protection reaction can be 4-12 h, 4-6 h, 6-8 h, 8-10 h, or 10-12 h.
In the above-mentioned amino group protection reaction, the reaction is usually carried out under a gas atmosphere. Suitable methods of providing a gas shield will be known to those skilled in the art, for example, conditions under which a gas shield can be provided by nitrogen, inert gases, and the like, and the inert gases can be, in particular, helium, neon, argon, krypton, and the like.
In the above amino protection reaction, the product obtained by the reaction may be subjected to a post-treatment by a suitable method selected by those skilled in the art, and may include, for example: desolventizing and purifying. After the reaction is finished, the solvent can be removed from the product, and after further purification, the clofarabine phosphoramidite monomer can be provided. Suitable purification methods will 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 also comprise the following steps: subjecting the compound of formula V to a second deprotection reaction with t-butyldimethylsilyl chloride (TBDMSCl) to provide a compound of formula IV, the reaction equation is as follows:
Figure BDA0002747900610000141
in the second deprotection reaction, the reaction may be carried out in the presence of a base, which may be an organic base, and specifically, for example, imidazole, triethylamine, N-diisopropylethylamine, or the like. The base is generally used in a large excess relative to the compound of formula V, for example, in the second deprotection reaction described above, the molar ratio of the compound of formula V to the base may be 1: 4 to 25, preferably 1: 6 to 10
In the second deprotection reaction, the reaction may be carried out in the presence of a reaction solvent, the reaction solvent used in the second deprotection reaction may be an aprotic solvent, and the kind and the amount of a suitable reaction solvent are known to those skilled in the art, and for example, in the second deprotection reaction, the reaction solvent may be a haloalkane-based solvent, a sulfoxide-based solvent, an amide-based solvent, or the like, and more specifically, N-dimethylformamide, dimethylsulfoxide, 1, 2-dichloroethane, or the like.
In the second hydroxyl protection reaction, the amount of tert-butyldimethylsilyl chloride used is usually in excess 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 hydroxyl protection reaction described above, the molar ratio of the compound of formula V to t-butyldimethylsilyl chloride may be 1: 2-10, 1: 2-3, 1: 3-4, 1: 4-6, 1: 6-8, or 1: 8-10, preferably 1: 3 to 4.
In the second deprotection reaction, the reaction may be carried out at a temperature ranging from room temperature to the boiling point of the solvent. For example, the reaction temperature in the second hydroxyl 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 second hydroxyl protection reaction, the reaction progress of the second hydroxyl protection reaction can be judged by TLC, chromatography and the like, and for example, the reaction time of the second hydroxyl protection reaction can be 3 to 24 hours.
In the second deprotection reaction, the reaction is usually carried out under a gas blanket. Suitable methods of providing a gas shield will be known to those skilled in the art, for example, conditions under which a gas shield can be provided by nitrogen, inert gases, and the like, and the inert gases can be, in particular, helium, neon, argon, krypton, and the like.
In the second hydroxyl protection reaction, the product obtained by the reaction may be subjected to post-treatment by a person skilled in the art by selecting an appropriate method, and for example, the method may include: desolventizing and purifying. After the reaction is complete, the product may be freed from the solvent and, after further purification, the compounds of the formula I may be provided. Suitable purification methods will 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 condensation reaction of a compound of formula VI with 2-cyanoethyl N, N-diisopropylphosphoramidite to provide gemcitabine phosphoramidite monomer is as follows:
Figure BDA0002747900610000161
in the above condensation reaction, the reaction may be carried out in the presence of a base, which may be an organic base, and specifically, for example, DIPEA, triethylamine, DMPA, pyridine, or the like. The base is generally used in excess relative to the compound of formula VI to ensure conversion and to allow the reaction to proceed in a substantially forward direction. 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: 5-6, 1: 6-7, 1: 7-8, 1: 8-9, 1: 9-10, 1: 10-11, or 1: 11 to 12, preferably 1: 5.5 to 6.5.
In the above condensation reaction, the amount of 2-cyanoethyl N, N-diisopropylphosphoramidite used is usually in excess relative to the compound of formula VI, so that the conversion rate of the reaction can be ensured and the reaction can be sufficiently proceeded 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-diisopropylphosphorochloridite may be 1: 1.5-6, 1: 1.5-2, 1: 2-2.5, 1: 2.5-3, 1: 3-3.5, 1: 3.5-4, 1: 4-4.5, 1: 4.5-5, 1: 5-5.5, or 1: 5.5-6, preferably 1: 2.5 to 3.5.
In the above 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 amount of a suitable reaction solvent are known to those skilled in the art, and for example, in the condensation reaction, the reaction solvent may be a haloalkane-type solvent, an ether-type solvent, a nitrile-type solvent, and the like, and more specifically, methylene chloride, tetrahydrofuran, acetonitrile, and the like.
In the above condensation reactions, it is generally necessary to avoid carrying out the reaction at too high a 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 determined by TLC, chromatography, and 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 above condensation reaction, the reaction is usually carried out under a gas blanket. Suitable methods of providing a gas shield will be known to those skilled in the art, for example, conditions under which a gas shield can be provided by nitrogen, inert gases, and the like, and the inert gases can be, in particular, helium, neon, argon, krypton, and the like.
In the above condensation reaction, the product obtained by the reaction may be subjected to a post-treatment by a person skilled in the art by selecting an appropriate method, and for example, may include: desolventizing and purifying. After the reaction is complete, the product may be desolventized and, after further purification, gemcitabine phosphoramidite monomer may be provided. Suitable purification methods will 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 also comprise the following steps: the compound of formula VII is subjected to a hydroxyl protection reaction with 4,4' -bis-methoxytrityl chloride (DMTrCl) to provide the compound of formula VI, which is given by the following equation:
Figure BDA0002747900610000171
in the above-mentioned hydroxyl group-protecting reaction, the reaction can be usually carried out in the presence of a base which may be usually an organic base, and specifically, for example, pyridine, DMAP, triethylamine, diethylamine and the like. The base is usually used in a large excess relative to the compound of formula VII, and may itself act as a solvent for the reaction system.
In the above-mentioned hydroxyl protecting reaction, the reaction may be carried out in the presence of a reaction solvent, the reaction solvent used in the hydroxyl protecting reaction may be generally an aprotic solvent, and the kind and amount of a suitable reaction solvent should be known to those skilled in the art, and for example, in the hydroxyl protecting reaction, the reaction solvent may be selected from a haloalkane-based solvent, a sulfoxide-based solvent, and the like, and more specifically, methylene chloride, dimethyl sulfoxide, chloroform, 1, 2-dichloroethane, and the like.
In the above-mentioned hydroxyl protection reaction, DMTrCl is usually used in an amount substantially equal to or in excess with respect to the compound of formula VII, so that the conversion rate of the reaction can be secured and the reaction can be sufficiently carried out in the forward direction. For example, in the above-described hydroxyl protection reaction, the molar ratio of the compound of formula VII and DMTrCl may be 1: 1-1.5, 1: 1-1.1, 1: 1.1-1.2, 1: 1.2-1.3, 1: 1.3-1.4, 1: or 1.4 to 1.5, preferably 1: 1.15 to 1.25.
In the above-mentioned hydroxyl group-protecting reaction, the reaction can be carried out usually under a temperature condition of from 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 protection reaction, the reaction progress of the hydroxyl protection reaction can be judged by methods such as TLC and chromatography, and for example, the reaction time of the hydroxyl protection reaction can be 2-24 h, 2-3 h, 3-4 h, 4-6 h, 6-8 h, 8-12 h, 12-16 h, 16-20 h, or 20-24 h.
In the above-mentioned hydroxyl protecting reaction, the reaction is usually carried out under a gas atmosphere. Suitable methods of providing a gas shield will be known to those skilled in the art, for example, conditions under which a gas shield can be provided by nitrogen, inert gases, and the like, and the inert gases can be, in particular, helium, neon, argon, krypton, and the like.
In the above-mentioned hydroxyl protecting reaction, the person skilled in the art can select a suitable method to post-treat the product obtained from the reaction, and for example, the method may include: desolventizing and purifying. After the reaction is complete, the product may be desolventized and, after further purification, the compound of formula VI may be provided. Suitable purification methods will 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 also comprise the following steps: subjecting a compound of formula VIII to an amino protection reaction with acetic anhydride to provide a compound of formula VII, the reaction equation is as follows:
Figure BDA0002747900610000181
in the above-mentioned amino protection reaction, acetic anhydride is usually used in an amount substantially equal to or in excess with respect to the compound of formula VIII, so that the conversion rate of the reaction can be secured and the reaction can be sufficiently carried out in the forward direction. For example, in the above-described amino protection reaction, the molar ratio of the compound of formula VIII to acetic anhydride may be 1: 1-1.5, 1: 1-1.1, 1: 1.1-1.2, 1: 1.2-1.3, 1: 1.3-1.4, 1: 1.4 to 1.5, preferably 1: 1.05 to 1.15.
In the above-mentioned amino group protection reaction, the reaction may be carried out in the presence of a reaction solvent, the reaction solvent used in the amino group protection reaction may be generally an aprotic solvent, and the kind and amount of a suitable reaction solvent should be known to those skilled in the art, and for example, in the amino group protection reaction, the reaction solvent may be selected from amide solvents, ether solvents, halogenated alkane solvents, and the like, and more specifically, DMF, tetrahydrofuran, dioxane, 1, 2-dichloroethane, and the like may be mentioned.
In the above-mentioned amino group-protecting reaction, the reaction can be usually carried out at a temperature ranging from 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 methods such as TLC and chromatography, and for example, the reaction time of the amino protection reaction can be 5-6 h, 6-8 h, 8-10 h, 10-12 h, 12-16 h, 16-24 h, or 24-36 h.
In the above-mentioned amino group protection reaction, the reaction is usually carried out under a gas atmosphere. Suitable methods of providing a gas shield will be known to those skilled in the art, for example, conditions under which a gas shield can be provided by nitrogen, inert gases, and the like, and the inert gases can be, in particular, helium, neon, argon, krypton, and the like.
In the above amino protection reaction, the skilled person can select a suitable method to carry out the post-treatment of the product obtained from the reaction, and for example, the method may include: desolventizing and purifying. After the reaction is complete, the product may be desolventized and, after further purification, the compound of formula VII may be provided. Suitable purification methods will 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 a use of the clofarabine modified oligonucleotide provided in the first aspect of the invention in preparation of a medicament. The oligonucleotide modified by clofarabine and/or gemcitabine provided by the invention has good specificity and targeting property for target cells (for example, tumor cells, specifically pancreatic ductal adenocarcinoma, acute lymphocytic leukemia, acute T lymphocytic leukemia, triple negative breast cancer, colorectal cancer and the like), can keep the original medicinal activity of clofarabine, can reduce toxic and side effects and improve bioavailability, and can be used as a tumor treatment medicament.
The nucleotide drugs clofarabine and/or gemcitabine are designed and synthesized into a phosphoramidite monomer for solid phase synthesis, the fixed-point precise functionalization of clofarabine and/or gemcitabine on oligonucleotide is realized by using a solid phase synthesis technology, and the prepared clofarabine and/or gemcitabine modified oligonucleotide can release clofarabine under the action of nuclease, still has higher cytotoxicity on tumor cells, retains the medicinal activity of clofarabine and/or gemcitabine, and has good industrial 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) in a single neck flask was added clofarabine (1 eq), 20mL of N, N-dimethylformamide, imidazole (15 eq) and tert-butyldimethylchlorosilane (3.2 eq) was added to the solution at room temperature. The mixture was stirred for 12 h. The solvent was removed in vacuo using a rotary evaporator and purified by silica gel column chromatography to give TBDMS-clofarabine (99%) as a white solid.1H 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.
The reaction formula of reacting clofarabine with tert-butyldimethylsilyl chloride (TBDMSCl) in N, N-Dimethylformamide (DMF) under the action of imidazole (imidazole) is as follows:
Figure BDA0002747900610000191
2) TBDMS-clofarabine (1 eq.) and triethylamine (18 eq.) were added to a single vial, 50mL of dichloromethane were added, and benzoyl chloride (6.8 eq.) was added to the solution at 0 ℃. Reacting for 8 hours, removing the solvent in vacuum by using a rotary evaporator, and separating and purifying by silica gel column chromatography to obtain colorless oily TBDMS-Bz2Clofarabine (80%).1H 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:
Figure BDA0002747900610000201
3) adding TBDMS-Bz into a single-mouth bottle2Clofarabine (1 eq) and acetic acid (12 eq), 100mL tetrahydrofuran was added, and tetrabutylammonium fluoride (3 eq) was added at 0 ℃. The reaction was carried out at room temperature for 6 hours. Removing solvent with rotary evaporator, and separating and purifying by flash column chromatography to obtain Bz as white solid2Clofarabine (71%). 1H NMR (400MHz, Chloroform-d) δ 8.39(s,1H),7.81(d, J ═ 7.7Hz,4H),7.49(t, J ═ 7.4Hz,2H),7.36(t, J ═ 7.6Hz,4H),6.40(dd, J ═ 15.4,3.6Hz,1H),5.02(d, J ═ 51.8Hz,1H),4.52(d, J ═ 17.8Hz,1H),3.96-3.91(m,1H),3.82-3.67(m,2H) ppm.
TBDMS-Bz2Reaction of clofarabine with tetrabutylammonium fluoride (TBAF) in tetrahydrofuran with acetic acid of the formula:
Figure BDA0002747900610000202
4) adding Bz into a single-mouth bottle2Clofarabine (1 eq), dissolved in 40mL pyridine, and DMTrCl (1.2 eq) added to the solution in 3 portions at room temperature. Mixing the mixture in N2Stirring for 8h under protection, removing solvent in vacuum with rotary evaporator, and separating and purifying by flash column chromatography to obtain white foamy DMTr-Bz2Clofarabine (82%). 1H NMR (400MHz, acetonitril-d 3), δ 8.26(d, J ═ 2.0Hz,1H),7.82(d, J ═ 7.4Hz,4H),7.58(t, J ═ 7.5Hz,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(d, J ═ 18, 3.7, 4.3Hz,1H),5.19 (dd, J ═ 51.9,3.9Hz,1H), 4.55.55 (1, 18, 3.9, 3.3, 3H), 3.09, 3.3H, 3, 13.73 ppm (d, 3, H, 3, d, 3, d.
Bz2Reaction of clofarabine with 4,4' -bismethoxytrityl chloride (DMTrCl) in pyridine is as follows:
Figure BDA0002747900610000211
5) adding DMTr-Bz into a single-mouth bottle2Clofarabine (1 eq) and DIPEA (6 eq) dissolved in 100mL dichloromethane. At 0 ℃ and N2To the mixed solution was added 2-cyanoethyl N, N-diisopropylphosphorochloridite (3 equivalents) with protection. After 10min the mixture was brought to room temperature and stirring was continued for 1 h. The solvent was removed in vacuo using a rotary evaporator at room temperature and purified by flash column chromatography to give a white foamy solid as the clofarabine phosphoramidite monomer (73%).1H NMR(400MHz,Acetonitrile-d3)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.31P NMR(162MHz,Acetonitrile-d3)δ150.54ppm.
DMTr-Bz2Reaction of clofarabine with 2-cyanoethyl N, N-diisopropylphosphoramidite in dichloromethane is as follows:
Figure BDA0002747900610000212
example 2
Preparation of gemcitabine phosphoramidite monomer:
1) gemcitabine (710mg, 2.7mmol), 20mL DMF was added to a 100mL single neck round bottom flask and acetic anhydride (280. mu.L, 2.96mmol) was added to the solution at room temperature. N is a radical of2The mixture was stirred overnight with protection. The solvent DMF was removed in vacuo using a rotary evaporator, and the residue was combined with silica gel and purified by silica gel column chromatography to give Ac-gemcitabine (616mg, 75%) as a white solid.1H NMR(400MHz,DMSO-d6)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)2O) in N, N-Dimethylformamide (DMF) the reaction is as follows:
Figure BDA0002747900610000221
2) a100 mL single neck round bottom flask was charged with Ac-gemcitabine (2.12g, 6.95mmol) dissolved in 50mL pyridine and DMTrCl (2.53g, 7.65mmol) was added to the solution in 3 portions at room temperature. Mixing the mixture in N2After stirring for 8h under protection, the solvent pyridine was removed in vacuo using a rotary evaporator, and the residue was combined with silica gel and purified by flash column chromatography to give DMTr-Ac-gemcitabine as a white foamy solid (3.04g, 72%).1H NMR(400MHz,DMSO-d6)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' -bis-methoxytrityl chloride (DMTrCl) in pyridine is shown below:
Figure BDA0002747900610000222
3) a100 mL single neck round bottom flask was charged with DMTr-Ac-gemcitabine (637mg, 1.05mmol) and DIPEA (1.055mL, 6.30mmol), 20mL dichloromethane. At 0 ℃ and N2To the mixed solution was added 2-cyanoethyl N, N-diisopropylphosphoramidite chloride (755. mu.L, 3.148mmol) with protection. After 10min the mixture was brought to room temperature and stirring was continued for 1 h. The solvent was removed in vacuo using a rotary evaporator at room temperature, and the residue was combined with silica gel and purified by flash column chromatography to give gemcitabine phosphoramidite monomer as a white foamy solid (795mg, 95%).1H NMR(400MHz,Acetonitrile-d3)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.31P NMR(162MHz,Acetonitrile-d3)δ151.74ppm.
The reaction of DMTr-Ac-gemcitabine with 2-cyanoethyl N, N-diisopropylphosphoramidite in dichloromethane is shown below:
Figure BDA0002747900610000231
example 3
Preparing clofarabine modified oligonucleotide:
the clofarabine phosphoramidite monomer and the gemcitabine phosphoramidite monomer are respectively dissolved in anhydrous acetonitrile to be prepared into the concentration of 0.1M, and the solution is used for solid phase synthesis of a DNA solid phase synthesizer. Coupling is carried out at 25-35 ℃ for 5 minutes twice, clofarabine and/or gemcitabine modified oligonucleotides with different sequences are synthesized on Universal CPG at the 3' end, and the polynucleotide sequences of the coupled oligonucleotides are shown in Table 1. After coupling, the oligonucleotides were cleaved from the solid phase support using concentrated ammonia and purified using HPLC. After purification, mass spectrometry characterization is performed, the molecular weight of each sequence of substances obtained by mass spectrometry molecular measurement is consistent with the theoretical molecular weight, and the following characterization results are given as an example: sequence 5 mass spectrometry results are shown in fig. 1, MS: calibrated 18471.9(Found:18472.9), sequence 8 mass spectrum characterization results are shown in FIG. 2, MS: summarized: 18106.2(Found:18102.6), sequence 10 mass spectrum characterization results are shown in FIG. 3, MS: calibrated 12670.2(Found: 12667.6).
TABLE 1 Clofarabine and/or Gemcitabine modified oligonucleotide sequences
Figure BDA0002747900610000232
Figure BDA0002747900610000241
Example 4
Gemcitabine and clofarabine modified oligonucleotides (SEQ ID No.5 to SEQ ID No.9) prepared in example 3 were used in cancer cell inhibition experiments, and gemcitabine and clofarabine were used as comparative examples. Dispersing HCT-116 cells of human colon cancer cells in RPMI-1640 culture medium according to the cell density of 5 ten thousand/mL, preparing culture medium solutions of different concentrations of drugs, mixing the drug solutions with the cell solution at a ratio of 1:1, adding the mixture into a 96-well plate, adding 100 mu L of the mixture into each well, and placing the mixture in a constant-temperature incubator for culture. Removing the culture medium after 72h, adding 100 μ L of fresh culture medium solution dissolved with CCK-8, shaking, mixing, incubating at 37 deg.C for 1h, and measuring the absorption at 450nm with Synergy-2 multi-functional enzyme-labeling instrument, with the experimental results shown in FIG. 4. As shown in FIG. 4, the gemcitabine and clofarabine modified oligonucleotide have higher inhibitory activity on cancer cells, and the IC50 value is about 12.82nM to 139.0nM, and still basically maintains the original pharmaceutical activity of the two drugs.
Example 5
The oligonucleotides (SEQ ID NO.1-SEQ ID NO.4) modified by clofarabine prepared in the example 3 are used for the specific recognition of cancer cells to realize the targeted delivery of the clofarabine, and blank cells are used as a comparative example. Taking the flow cytometry experiment of human acute lymphoblastic leukemia CCRF-CEM cells as an example, firstly measuring a proper volume of CCRF-CEM cell suspension according to the amount of 15 ten thousand cells of each sample, centrifuging for 3min at the rotating speed of 800r/min, removing the supernatant, adding 200 mu L of binding buffer solution into each sample, adding a certain volume of aptamer mother liquor, mixing to the final concentration of 200nM, and placing for 1h at 4 ℃ in a dark place. Centrifuging at 800r/min for 3min, removing supernatant, adding cleaning buffer, mixing, centrifuging, and washing for three times. The experiment was performed using a flow cytometer by adding 400. mu.L of washing buffer, 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, and the targeted delivery of clofarabine drugs is realized.
In conclusion, the present invention effectively overcomes various disadvantages of the prior art and has high industrial utilization value.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Sequence listing
<110> university of Hunan
<120> 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 (10)

1. A clofarabine modified oligonucleotide comprising an aptamer segment modified with a clofarabine phosphoramidite monomer group.
2. The clofarabine-modified oligonucleotide of claim 1, wherein the aptamer segment is modified with one or more clofarabine phosphoramidite monomer groups, wherein the clofarabine phosphoramidite monomer groups are modified at the 5 'end of the aptamer segment and/or modified at the 3' end of the aptamer segment and/or added in the middle of the aptamer segment.
3. The clofarabine modified oligonucleotide of claim 2, wherein the clofarabine phosphoramidite monomer group is linked to the aptamer fragment via a phosphodiester linkage;
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:
Figure FDA0002747900600000011
the chemical structural formula of the intermediate clofarabine phosphoramidite monomer group added to the aptamer fragment is shown as follows:
Figure FDA0002747900600000012
the chemical structural formula of the clofarabine phosphoramidite monomer group modified at the 3' end of the aptamer fragment is shown as follows:
Figure FDA0002747900600000013
4. the clofarabine modified oligonucleotide of claim 1, wherein the aptamer fragment is further modified with a gemcitabine phosphoramidite monomer moiety.
5. The gemcitabine-modified oligonucleotide of claim 4, wherein the aptamer segment is modified with one or more gemcitabine phosphoramidite monomer groups, wherein the gemcitabine phosphoramidite monomer groups are modified at the 5 'end of the aptamer segment and/or at the 3' end of the aptamer segment and/or added in the middle of the aptamer segment.
6. The gemcitabine-modified oligonucleotide of claim 5, wherein the gemcitabine phosphoramidite monomer moiety is linked to the aptamer segment by a phosphodiester linkage;
and/or, the chemical structural formula of the gemcitabine phosphoramidite monomer group modified at the 5' end of the aptamer fragment is shown as follows:
Figure FDA0002747900600000021
the chemical structure of the gemcitabine phosphoramidite monomer group added to the middle of the aptamer fragment is shown below:
Figure FDA0002747900600000022
the chemical structure of the gemcitabine phosphoramidite monomer group modified at the 3' end of the aptamer fragment is shown below:
Figure FDA0002747900600000023
7. the clofarabine modified oligonucleotide according to claim 1, wherein the polynucleotide sequence of the aptamer fragment comprises a sequence shown in any one of SEQ ID nos. 11 to 20.
8. The clofarabine modified oligonucleotide of claim 1, wherein the polynucleotide sequence of the clofarabine modified oligonucleotide comprises a sequence shown as one of SEQ ID No. 1-10.
9. The method for preparing the clofarabine modified oligonucleotide according to any one of claims 1 to 8, comprising:
connecting the clofarabine phosphoramidite monomer and/or gemcitabine phosphoramidite monomer with the aptamer segment by a solid phase synthesis method;
preferably, 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:
Figure FDA0002747900600000031
and/or, the chemical structural formula of the gemcitabine phosphoramidite monomer is shown as follows:
Figure FDA0002747900600000032
10. use of the clofarabine-modified oligonucleotide of any one of claims 1-8 in the preparation of a medicament.
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