CN117700468B - Preparation method of nucleotide analogue - Google Patents
Preparation method of nucleotide analogue Download PDFInfo
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- C07F9/02—Phosphorus compounds
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- C07F9/08—Esters of oxyacids of phosphorus
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- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/16—Esters of thiophosphoric acids or thiophosphorous acids
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- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6564—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms
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Abstract
本发明提供了一种核苷酸类似物的制备方法，所述核苷酸类似物如式（I）所示。该方法采用不易吸潮、性质稳定的磷酸盐供体，适用于放大反应；同时采用的磷转移试剂具有对位结构的苯环，可作为较好的离去基团，实现磷转移。通过该方法可实现核苷的二磷酸化或三磷酸化，具有产率高、产物易纯化、副产物易去除等优点。式（I）。The present invention provides a method for preparing a nucleotide analogue, wherein the nucleotide analogue is shown in formula (I). The method uses a phosphate donor that is not easy to absorb moisture and has stable properties, and is suitable for amplification reactions; at the same time, the phosphorus transfer reagent used has a benzene ring with a para-position structure, which can be used as a better leaving group to achieve phosphorus transfer. The method can achieve diphosphorylation or triphosphorylation of nucleosides, and has the advantages of high yield, easy purification of products, and easy removal of by-products. Formula (I).
Description
Technical Field
The invention relates to the field of organic chemistry, in particular to a preparation method of a nucleotide analogue.
Background
Among many nucleoside polyphosphoric acid biomolecules, nucleoside triphosphates (nucleoside triphosphate, NTP) are important substances for maintaining the operation of a living system, nucleoside triphosphates are the basis of substances for gene replication, transcription and translation, and are used as carriers of genetic information, and RNA and DNA are formed by connecting nucleoside triphosphates and deoxynucleoside triphosphates serving as substrates and energy sources by corresponding polymerase. The nucleoside triphosphates include natural nucleotides and modified nucleoside triphosphates, and are mainly used in PCR (polymerase chain reaction) experiment kits. In addition to natural nucleoside triphosphates, many base-modified or sugar ring-modified nucleoside analogs and triphosphates of antiviral nucleoside drugs have important applications in biology and medicine, such as biotin and fluorophore-labeled dUTP are widely used in apoptosis and in protein-NTP interaction studies, and the four-color DNA polymer substrate biochip of the invention taught by Columbia university Jingyue Ju has been widely used in high-throughput DNA sequencing.
Nucleoside triphosphates have wide application and huge market prospects due to important biological functions, however, only a small amount of nucleoside triphosphates can be industrially produced by a bioengineering method at present, which is a direct cause of extremely high price of most of nucleoside triphosphates. The research difficulty of the chemical synthesis of nucleoside triphosphates is that the nucleoside triphosphates themselves and the synthetic raw materials have special physicochemical properties, and specifically comprise the following aspects: (1) the reactants and products have a plurality of reaction sites. The polar nucleophile used in the synthesis reaction of nucleoside triphosphate usually has a plurality of negative charges and reaction sites, and the nucleoside polyphosphate intermediate generated in the reaction also has a certain reaction due to similar reasons, so that the side reaction of the whole synthesis reaction is more, and various polyphosphoric acid byproducts are easy to generate; (2) the difference in solubility of the reactants is large. In the synthesis reaction of nucleoside triphosphate, the inorganic phosphate nucleophile has poor fat solubility, while the nucleoside part is relatively lipophilic, so that it is difficult to find a proper reaction solvent to realize reactants with larger difference of dissolution polarities, and the like.
The existing synthesis methods of nucleoside triphosphates mainly comprise a DCC condensation method, a one-pot three-step method, an enzymatic synthesis method and the like, wherein the DCC condensation method can generate a large amount of byproducts such as nucleoside triphosphates and nucleoside polyphosphates, and the like, and the method has little use value in the synthesis of nucleoside triphosphates; the one-pot three-step method for synthesizing nucleoside triphosphates through nucleoside phosphoryl dichloro intermediates has the advantages that nucleoside does not need to be protected, but has uncertain factors on the regioselectivity of phosphoryl and specificity on nucleoside bases, and the use of tri-n-butylamine containing extremely toxic ammonium pyrophosphate is difficult to purchase and obtain, and the yield is relatively low, which is about 30%; in addition, the nucleoside triphosphate can be synthesized by the nucleoside phosphite ester intermediate, and the method has the problems that the salicylic acid phosphite chloride has active property, is easy to hydrolyze during taking, and impurities formed in the reaction are difficult to remove, and the yield is low (30% -50%); finally, with respect to enzymatic synthesis methods, while biocatalysis can be used for the preparation of non-natural nucleoside triphosphates, a great deal of time and effort must be expended to find the various enzymes that can act on the non-natural nucleoside substrate by screening, while it is highly likely that no enzyme can be found at all that can act on some specific non-natural substrates.
Therefore, there is a need to provide a method for preparing nucleotide analogs with high yields, easy removal of byproducts, and easy purification of the products.
Disclosure of Invention
The present invention aims to solve one of the technical problems in the related art at least to some extent. Therefore, the invention provides a preparation method of nucleotide analogues, by which the di-phosphorylation or tri-phosphorylation of nucleosides can be realized, and the method has the advantages of high yield, easy purification of products, easy removal of byproducts and the like.
To this end, the present invention provides a process for the preparation of a compound of formula (I), comprising:
contacting a compound shown in a formula (II) with a compound shown in a formula (III), and contacting the obtained product with a compound shown in a formula (IV) to obtain a compound shown in a formula (I);
Wherein B is a base or a base derivative;
R 1 is a first phosphate group;
r 2 is H or a first hydroxy protecting group;
R 3 is H, a second hydroxy protecting group or Wherein R 5 is H or a third hydroxy protecting group;
R 4 is a fourth hydroxy protecting group;
R 6 is selected from halogen, NO 2, CN, the following groups which are unsubstituted or substituted by at least one R a: c 1-C10 alkyl, C 1-C10 alkoxy;
r a is independently selected from halogen;
R 7 is selected from H, OH, F, OMe, OMOE or O-Proparyl;
Formula (I)
Formula (II)
Formula (III)
Formula (IV).
In order to solve the defects in the prior art, the invention contacts a compound shown in a formula (II) with a compound shown in a formula (III), and the obtained product contacts a compound shown in a formula (IV), thereby preparing the nucleotide analogue shown in the formula (I). Wherein, compared with other phosphate donors, the compound shown in the formula (II) is not easy to absorb moisture, has stable property and is suitable for amplification reaction; the compound shown in the formula (III) contains benzene rings with para structures, and can be used as a better leaving group to realize phosphorus transfer. The raw materials selected by the invention have better stability, so that complex post-treatment of products is avoided, the generation of impurities is reduced, and the yield is further improved.
According to an embodiment of the invention, B is selected from cytosine or a derivative thereof, thymine or a derivative thereof, adenine or a derivative thereof, guanine or a derivative thereof, uracil or a derivative thereof, or pseudouridine or a derivative thereof.
According to an embodiment of the present invention, the first phosphate group is a biphosphoric acid group or a triphosphoric acid group.
According to an embodiment of the present invention, the first hydroxyl protecting group and the fourth hydroxyl protecting group independently include at least one selected from an alkyl protecting group, a silane protecting group, an acetal protecting group, and an acyl protecting group.
According to an embodiment of the present invention, the alkyl-based protecting group includes at least one selected from the group consisting of methyl, t-butyl, benzyl, p-methoxybenzyl, trityl, azidomethyl.
According to an embodiment of the present invention, the silane-based protecting group includes at least one selected from the group consisting of trimethylsilyl group, t-butyldimethylsilyl group, and t-butyldiphenylsilyl group.
According to an embodiment of the present invention, the acetal-based protecting group includes at least one selected from the group consisting of methoxymethyl, methoxyethoxymethyl, benzyloxymethyl, trimethylsilylethoxymethyl, and tetrahydrofuranyl.
According to an embodiment of the present invention, the acyl-type protecting group includes at least one selected from pivaloyl, benzoyl, acetyl, haloacetyl.
According to an embodiment of the invention, the first hydroxyl protecting group is an azidomethyl group.
According to an embodiment of the invention, the fourth hydroxyl protecting group is acetyl or benzoyl.
According to an embodiment of the invention, the second hydroxyl protecting group comprises a protecting group selected from Fm, ac, bz, piv.
According to an embodiment of the invention, the third hydroxyl protecting group comprises a protecting group selected from Fm, benzyl, p-methoxybenzyl, ms, ts, ns, tf.
According to an embodiment of the invention, R 3 is H, fm or。
According to an embodiment of the invention, R 6 is selected from F, cl, br, NO 2、CN、OCH3、CH3、CF3.
According to an embodiment of the invention, R 6 is selected from Br.
According to the embodiment of the invention, the molar ratio of the compound shown in the formula (II), the compound shown in the formula (III) and the compound shown in the formula (IV) is (1-4): (1-4): (1-4).
According to an embodiment of the present invention, when R 3 is a second hydroxyl-protecting group or R 5 is a third hydroxyl-protecting group, the preparation method further comprises: contacting a compound represented by formula (II), a first deprotection reagent, and a compound represented by formula (III).
According to an embodiment of the invention, the first deprotection reagent comprises at least one selected from DBU, et 3 N, DABCO, TMG, KOtBu, bartons, hunigs, phosphazene.
According to an embodiment of the present invention, when R 2 is a first hydroxyl protecting group, the preparation method further includes: contacting the compound shown in the formula (II) with the compound shown in the formula (III), and contacting the obtained product with the compound shown in the formula (IV) and a second deprotection reagent.
According to an embodiment of the invention, the second deprotection reagent comprises at least one selected from DBU, et 3 N, DABCO, TMG, KOtBu, bartons, hunigs, phosphazene.
According to an embodiment of the invention, the preparation process is carried out in an organic solvent at a temperature of-10 to 60 ℃ for 2 to 48 hours.
According to an embodiment of the present invention, the organic solvent includes at least one selected from the group consisting of anhydrous acetonitrile, DMF, THF.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Detailed Description
Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention.
It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless clearly defined otherwise herein in this document, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
In this document, the terms "comprise" or "include" are used in an open-ended fashion, i.e., to include what is indicated by the present invention, but not to exclude other aspects.
The term "nucleotide" encompasses bases, pentoses, and one or more phosphate groups. Which is a monomeric unit of a nucleic acid sequence. In RNA, the five-carbon sugar is ribose, and in DNA, the five-carbon sugar is deoxyribose. The base may be a purine or pyrimidine base. Purine bases include, but are not limited to, adenine (a) and guanine (G), and modified derivatives or analogs thereof. Pyrimidine bases include, but are not limited to, cytosine (C), thymine (T), uracil (U), and pseudouracil, and modified derivatives or analogs thereof.
The term "base derivative" is understood to mean a modified base.
The term "protecting group" is understood to mean any atom or group of atoms in a molecule that prevents an existing group in the molecule from undergoing an undesired chemical reaction. "hydroxy protecting group" is understood to mean any atom or group of atoms in a molecule that prevents unwanted chemical reactions of the hydroxy group in the molecule.
The following are abbreviations used in the description schemes, preparations and examples:
NO 2 is nitro;
CN is cyano;
OMe is methoxy;
OMOE is 2, 3-dihydro-1, 4-benzodioxane-6-carboxylic acid 4- [ 2-dimethylamino-1- (1-hydroxycyclohexyl) -ethyl ] phenyl ester;
O-Propargyl is oxo-Propargyl;
Fm is S-9-fluorenylmethyl;
ac is acetyl;
bz is tert-butyl;
Piv is pivaloyl;
Ms is methylsulfonyl;
Ts is sulfonyl;
ns is nitrobenzenesulfonyl;
tf is trifluoromethanesulfonyl;
DBU is 1, 8-diazabicyclo [5.4.0] undec-7-ene;
Et 3 N is triethylamine;
DABCO is triethylene diamine;
TMG is tetramethyl guanidine;
KOTBu is potassium tert-butoxide;
Barton s is 2-tert-butyl-1, 3-tetramethylguanidine;
Hunigs is diisopropylethylamine;
phosphazene is hexachlorocyclotriphosphazene;
DMF is N, N-dimethylformamide;
THF is tetrahydrofuran.
According to an embodiment of the present invention, there is provided a method for preparing a compound represented by formula (I), comprising:
contacting a compound shown in a formula (II) with a compound shown in a formula (III), and contacting the obtained product with a compound shown in a formula (IV) to obtain a compound shown in a formula (I);
Wherein B is a base or a base derivative;
R 1 is a first phosphate group;
r 2 is H or a first hydroxy protecting group;
R 3 is H, a second hydroxy protecting group or Wherein R 5 is H or a third hydroxy protecting group;
R 4 is a fourth hydroxy protecting group;
R 6 is selected from halogen, NO 2, CN, the following groups which are unsubstituted or substituted by at least one R a: c 1-C10 alkyl, C 1-C10 alkoxy;
r a is independently selected from halogen;
R 7 is selected from H or OH, F, OMe, OMOE or O-Proparyl;
Formula (I)
Formula (II)
Formula (III)
Formula (IV).
According to a specific embodiment of the invention, B is selected from cytosine or a derivative thereof, thymine or a derivative thereof, adenine or a derivative thereof, guanine or a derivative thereof, or pseudouracil or a derivative thereof.
According to a specific embodiment of the present invention, the first phosphate group is a biphosphoric acid group or a triphosphoric acid group.
According to a specific embodiment of the present invention, the first hydroxyl protecting group and the fourth hydroxyl protecting group independently include at least one selected from an alkyl protecting group, a silane protecting group, an acetal protecting group, and an acyl protecting group.
According to a specific embodiment of the present invention, the alkyl-based protecting group includes at least one selected from the group consisting of methyl, t-butyl, benzyl, p-methoxybenzyl, trityl, azidomethyl.
According to a specific embodiment of the present invention, the silane-based protecting group includes at least one selected from the group consisting of trimethylsilyl, t-butyldimethylsilyl, and t-butyldiphenylsilyl.
According to a specific embodiment of the present invention, the acetal protecting group includes at least one selected from methoxymethyl, methoxyethoxymethyl, benzyloxymethyl, trimethylsilylethoxymethyl, and tetrahydrofuranyl.
According to a specific embodiment of the present invention, the acyl-type protecting group includes at least one selected from pivaloyl, benzoyl, acetyl, haloacetyl.
According to a specific embodiment of the invention, the first hydroxyl protecting group is azidomethyl.
According to a specific embodiment of the present invention, the fourth hydroxyl protecting group is acetyl or benzoyl.
According to a specific embodiment of the invention, the second hydroxyl protecting group comprises a protecting group selected from Fm, ac, bz, piv.
According to a specific embodiment of the invention, the third hydroxyl protecting group comprises a protecting group selected from Fm, benzyl, p-methoxybenzyl, ms, ts, ns, tf.
According to a particular embodiment of the invention, R 3 is H, fm or。
According to a specific embodiment of the invention, R 6 is selected from F, cl, br, NO 2、CN、OCH3、CH3、CF3.
According to a specific embodiment of the invention, R 6 is selected from Br.
According to a specific embodiment of the present invention, the molar ratio of the compound represented by formula (II), the compound represented by formula (III) to the compound represented by formula (IV) is (1-4): (1-4): (1-4).
According to a specific embodiment of the present invention, when R 3 is a second hydroxyl protecting group or R 5 is a third hydroxyl protecting group, the preparation method further comprises: contacting a compound represented by formula (II), a first deprotection reagent, and a compound represented by formula (III).
According to a specific embodiment of the invention, the first deprotection reagent comprises at least one selected from DBU, et 3 N, DABCO, TMG, KOtBu, bartons, hunigs, phosphazene.
According to a specific embodiment of the present invention, when R 2 is a first hydroxyl protecting group, the preparation method further comprises: contacting the compound shown in the formula (II) with the compound shown in the formula (III), and contacting the obtained product with the compound shown in the formula (IV) and a second deprotection reagent.
According to a specific embodiment of the invention, the second deprotection reagent comprises at least one selected from DBU, et 3 N, DABCO, TMG, KOtBu, bartons, hunigs, phosphazene.
According to a specific embodiment of the invention, the preparation process is carried out in an organic solvent at a temperature of from-10 to 60 ℃ for a time period of from 2 to 48 hours.
According to a specific embodiment of the present invention, the organic solvent includes at least one selected from anhydrous acetonitrile, DMF, THF.
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
EXAMPLE 1 preparation of Compound 1-a
(1) Preparation of Compound S2
The method comprises the following specific steps:
(1-1) A round bottom flask equipped with a stirrer was charged with 4-hydroxybenzaldehyde (S1, 12.2g,100 mol), anhydrous THF (100 mL), et 3 N (17.75 mL,120 mmol). The round bottom flask was placed in an ice/water bath, benzoyl chloride (11.6 mL,100 mol) was added and the reaction was allowed to warm to room temperature overnight. The mixture was diluted with ethyl acetate (100 mL) and filtered. The filtrate was washed with saturated NH 4 Cl solution, the organic phase was dried over sodium sulfate, filtered and the solvent was evaporated to give 4-formylbenzoate (23 g);
(1-2) the 4-formylbenzoate (23 g) obtained in the step (1-1) was dissolved in anhydrous THF (100 mL), and the temperature was adjusted to 0 ℃. NaBH 4 (5.65 g,150 mmol) was added in 3 portions and allowed to react for 2h at room temperature, then quenched with saturated NH 4 Cl solution and diluted with ethyl acetate (100 mL). The organic phase was separated and the aqueous phase extracted with ethyl acetate (100 mL). The organic layers were combined, dried over sodium sulfate, filtered, and concentrated under reduced pressure to give phenyl 4- (hydroxymethyl) benzoate (24 g);
(1-3) phenyl 4- (hydroxymethyl) benzoate (24 g) obtained in step (1-2) was dissolved in anhydrous THF, cooled to-78 ℃, pyrophosphoryl chloride (13.8 mL,300 mmol) was added dropwise for 10min, stirred for 4h, and then quenched with water. The pH of the resulting solution was adjusted to 8 with saturated NaHCO 3 solution. To the resulting suspension was added dropwise 37% hcl solution until the solution became clear. After extraction with ethyl acetate (2 x 200 ml), the organic phases were combined and washed with water. The organic phase was dried over sodium sulfate, filtered, concentrated in vacuo, the product suspended in DCM, the filter cake washed with DCM after filtration, and dried under reduced pressure to give Compound S2 (15.5 g,50mol, 50% yield).
1H NMR
1H NMR (600 MHz, CD3OD)δ 8.20-8.16 (m, 2H), 7.72-7.67 (m, 1H), 7.59-7.54 (m, 2H), 7.50 (d,J= 8.5 Hz, 2H), 7.25 (d,J= 8.5 Hz, 2H), 5.05 (d,J= 7.4 Hz, 2H).
(2) Preparation of Compound S3
The method comprises the following specific steps:
The round bottom flask was evacuated, filled with argon and the flask mouth capped with a septum. Anhydrous THF (320 mL), PCl 3 (8 mL,92 mmol) were added, cooled to 0deg.C, DIPEA (32 mL,184 mmol) was added, anhydrous i-Pr 2 NH (24 mL,174 mmol) was added dropwise, and stirred for 1h. DIPEA (32 mL,184 mmol) and 9-fluorenylmethanol (35.8 g,194 mmol) were added again, warmed to room temperature and stirred overnight. The resulting suspension was filtered, the filtrate was concentrated under reduced pressure, diluted with DCM, chromatographed on silica gel, and rinsed rapidly with hexane/ethyl acetate/triethylamine (volume ratio 100:5:1). The intermediate obtained was concentrated under reduced pressure and dried under vacuum to give compound S3 (24 g,46mmol, yield 50%) which was stored under argon at-20 ℃.
1H NMR
1H NMR (600 MHz, CDCl3)δ 7.76-7.73 (m, 4H), 7.67-7.64 (m, 4H), 7.40-7.35 (m, 4H), 7.31-7.26 (m, 4H), 4.18 (t,J= 6.9 Hz, 2H), 4.01 (dt,J= 9.9, 6.8 Hz, 2H), 3.81 (dt,J= 9.9, 7.3 Hz, 2H), 3.66 (hept,J= 6.8 Hz, 2H), 1.16 (d,J= 6.8 Hz, 12H).
(3) Preparation of Compound 1-a
The method comprises the following specific steps:
To a round bottom flask equipped with a stirrer was added compound S3 (8 g,15.4 mmol) and anhydrous MeCN (40 mL) to give a suspension, to which was added compound S2 (4 g,12.8 mmol) and triethylamine (1.8 mL,12.8 mmol) in this order, stirred for 2min, 5-phenyl-1H-tetrazole (2.8 g,19.2 mmol) was added and stirred under argon atmosphere for 1H. Tert-butanol peroxide (4.6 mL,25.6 mmol) was then added and stirred for an additional 1h. The resulting turbid liquid was filtered, and ethyl acetate was added thereto for dilution. The filtrate was separated by column chromatography on silica gel, washed with ethyl acetate (500 mL), the eluent was changed to MeOH/DCM (volume ratio 1:15), chromatography was continued, and then concentrated under reduced pressure, washed and dried to give compound 1-a (6.2 g, 57.6% yield). The compound 1-a can be stored for several months at the temperature of minus 20 ℃ without obvious loss of purity.
1H NMR
1H NMR (600 MHz, CDCl3)δ8.86-8.78 (m, 2H), 8.18-8.16 (m, 2H), 7.68 (ddt,J= 8.7, 7.6, 1.0 Hz, 4H), 7.65-7.62 (m, 1H), 7.52-7.49 (m, 6H), 7.37-7.29 (m, 6H), 7.20 (tdd,J= 7.4, 4.7, 1.2 Hz, 4H), 7.09-7.06 (m, 2H), 4.97 (d,J= 8.0 Hz, 2H), 4.32-4.23 (m, 4H), 4.14 (t,J= 7.1 Hz, 2H), 3.12 (hept,J= 6.5 Hz, 2H), 1.21 (d,J= 6.5 Hz, 12H).
EXAMPLE 2 preparation of Compounds 1-b
(1) Preparation of Compound S4
The method comprises the following specific steps:
Phosphorus pentasulfide (60 g,264 mmol), toluene (500 mL), 4-bromothiophene (101 g, 284 mmol) were mixed and purged with nitrogen for 2min. Triethylamine (78 mL,554 mmol) was added over 30min and stirred overnight at room temperature. The filtrate was collected by filtration and concentrated under vacuum. Methanol (360 mL) and heptane (360 mL) were added and stirred for 15min. Water (300 mL) was then added for 30 minutes. After completion of the addition of water, the mixture was filtered after 1 hour, and the filter cake was washed and dried in vacuo at 50 ℃ for 15 hours to give compound S4 (white solid, 104g, 69% yield).
1H NMR
1H NMR (400 MHz, Chloroform-d):δ 7.57 (dd,J= 8.5, 2.1 Hz, 4H), 7.52–7.45 (m, 4H), 3.29–3.19 (m, 6H), 1.38 (td,J= 7.3, 3.6 Hz, 9H).
(2) Preparation of Compound S5
The method comprises the following specific steps:
Hydrogenation of (+) -cis-limonene oxide (25 mL) was performed in EtOH (100 mL) with Pd/C (250 mg) as catalyst, stirred overnight at room temperature, and the product was filtered and concentrated to give 1-methyl-4-isopropyl-1, 2-epoxycyclohexane.
Compound S4 (50 g,87.2 mmol), 1-methyl-4-isopropyl-1, 2-epoxycyclohexane (18.2 g,130.8 mmol), dibutyl phosphate (16.5 mL,87.2 mmol), dichloroacetic acid (11 mL,130.8 mmol) was dissolved in CHCl 3 solution (200 mL). The reaction mixture was heated to 60℃and reacted for 1h. The crude reaction mixture was concentrated and part of the solvent was removed. Water (125 mL) and heptane (125 mL) were added, cooled to 5℃and stirred for 30 min. The crude product was collected by filtration and then washed with water and hexane to give the intermediate. The intermediate was mixed with DCM (200 mL), allowed to stand for demixing, the aqueous layer was discarded, the remaining solution was collected and concentrated, heptane (80 mL) was added for concentration, the product was collected by filtration and washed with a small amount of hexane to give compound S5 (white solid, 26g, yield 68%).
1H NMR
1H NMR (400 MHz, Chloroform-d):δ 7.62-7.46 (m, 4H), 3.53 (dt,J=12.9, 3.4 Hz, 1H),2.04 (ddq,J=13.1, 3.2, 1.7 Hz, 1H), 1.85-1.77 (m, 1H), 1.72-1.56 (m, 8H), 1.47 (dd,J=15.2, 8.3 Hz, 1H), 1.29-1.19 (m, 1H), 0.91 (dd,J=12.2, 6.5 Hz, 6H).
(3) Preparation of Compound 1-b
The method comprises the following specific steps:
SeO 2 (5.1 g,45.7 mmol) and compound S5 (20 g,45.7 mmol) were added to DCM (300 mL), stirred for 2h, seO 2 (5.1 g,45.7 mmol) was added again and stirred overnight. The mixture was filtered and washed with DCM. Heptane (480 mL,24 v) was added for extraction, cooled to 0 ℃, cold NaH 2PO4 (10% water, 120 mL) was added to the organic layer, then cold bleach (8%, 120 mL) was added, after mixing for 10-20 min (note: when the organic layer became colorless, otherwise secondary washing was performed), the organic layer was washed with NaH 2PO4 (10% water, 40 mL) and the volatiles were removed by filtration over MgSO 4, the product was dissolved in heptane (80 mL), and after filtration washed with heptane to give compound 1-b (white solid, 9.5g, yield 49%).
1H NMR
1H NMR (600 MHz, Chloroform-d):δ 7.54-7.48 (m, 4H), 3.21 (dd,J=13.0, 3.9 Hz, 1H),1.95 (dq,J=13.5, 1.9 Hz, 1H), 1.78-1.72 (m, 1H), 1.61-1.51 (m, 2H), 1.49 (td,J=12.7,12.0, 5.5 Hz, 1H), 1.39 (dt,J=10.8, 5.2 Hz, 1H), 1.12 (ddd,J=13.3, 10.9, 6.4 Hz, 1H), 0.85(dd,J=11.8, 6.6 Hz, 6H)
EXAMPLE 3 preparation of Compound 2-1
The method comprises the following specific steps:
To a round bottom flask with a septum on the neck was added compound 1-a (85 mg,0.1 mmol), anhydrous acetonitrile (1.5 mL) and DBU (60. Mu.L, 0.4 mmol), and after stirring for 10min molecular sieves (3 a, 100 mg), compound 1-b (0.13 mmol) and stirring for 30min at room temperature. Thereafter, compound 1-c (59.4 mg,0.2 mmol) was added, and a further portion of DBU (80. Mu.L, 0.6 mmol) was added and stirred for 4h. After the completion of the reaction, the resulting mixture was subjected to filtration, concentration under reduced pressure, and stirring at 30℃for 16 hours with 5mL of aqueous ammonia, the reaction mixture was diluted with water and washed with ethyl acetate to obtain an aqueous phase and an oil phase. The aqueous phase was concentrated at 30℃or less under reduced pressure and then separated by adding 850 mL centrifuge tubes, each of which was preloaded with an acetone solution (30 mL) containing 0.2M NaClO 4. A suspension was obtained and centrifuged at 1000 x g for 3 minutes. The supernatant was discarded, the solid residue was washed 2 times with acetone, combined after adding a small amount of water for dissolution, the product was collected by column chromatography, the solvent was evaporated in vacuo (temperature. Ltoreq.30℃), the product was dissolved in water and lyophilized to give compound 2-1 (white solid, 64.9mg, yield 69%).
1H NMR
1H NMR (600 MHz, D2O)δ 7.79 (s, 1H), 6.28 (t,J=6.9 Hz, 1H), 4.60 (dt,J=6.7, 3.5 Hz, 1H), 4.29-4.20 (m, 3H), 3.49(s, 2H).2.54-2.45 (m, 2H), 1.95 (s, 3H).
EXAMPLE 4 preparation of Compound 3-1
The method comprises the following specific steps:
To a round bottom flask with a septum on the neck was added compound 1-a (1.69 g,2.0 mmol), anhydrous acetonitrile (20 mL) and DBU (1.20 mL,8.0 mmol), and after stirring for 10min molecular sieves (3 a, 2.0 g), compound 1-b (2.6 mmol) were added and stirring was performed at room temperature for 30min. Thereafter, compound 2-c (4.0 mmol) was added and a further portion of DBU (1.8 mL,12 mmol) was added and stirred for 4h. After the completion of the reaction, the resulting mixture was subjected to filtration and concentration under reduced pressure. 5mL of aqueous ammonia was added and the mixture was stirred at 30℃for 16 hours, diluted with water and washed with ethyl acetate to give an aqueous phase and an oil phase. The aqueous phase was concentrated to 20mL under reduced pressure at 30℃or less, and then separated by adding to 8 50mL centrifuge tubes, each of which was preloaded with an acetone solution (40 mL) containing 0.2M NaClO 4. A suspension was obtained and centrifuged at 1000 x g for 3 minutes. The supernatant was discarded, the solid residue was washed 2 times with acetone, combined after adding a small amount of water for dissolution, the product was collected by column chromatography, the solvent was evaporated in vacuo (temperature. Ltoreq.30℃), the product was dissolved in water and lyophilized to give compound 3-1 (white solid, 1.2g, yield 65%).
1H NMR
1H NMR (600 MHz, D2O)δ 8.04 (d,J=7.6 Hz, 1H), 6.33 (t,J=6.6 Hz, 1H), 6.13 (d,J=7.6 Hz, 1H), 4.63 (dt,J=6.6, 3.5 Hz, 1H), 4.25-4.20 (m, 3H), 3.50 (s, 2H),2.42 (ddd,J=13.9, 6.6, 4.1 Hz, 1H), 2.33 (dt,J=13.9, 6.6 Hz, 1H).
EXAMPLE 5 preparation of Compound 4-1
The method comprises the following specific steps:
To a round bottom flask capped with a septum was added compound 2-a (185 mg,0.6 mmol), anhydrous acetonitrile (4.0 mL) and DBU (180 μl,1.2 mmol), the flask was sealed with polytetrafluoroethylene and evacuated and filled with argon. Stirring until the starting material was completely dissolved (about 5 min). Molecular sieve (3 a, 120 mg), compound 1-b (0.4 mmol) was then added and stirred at room temperature for 30min. Thereafter, the compound 1-c (0.2 mmol) was added, and a portion of DBU (120. Mu.L, 0.8 mol) was added thereto, followed by stirring for 90 minutes. After completion of the reaction, the resulting mixture was filtered and concentrated under reduced pressure to 0.5mL. 5mL of aqueous ammonia was added and the mixture was stirred at 30℃for 16 hours, diluted with water and washed with ethyl acetate to give an aqueous phase and an oil phase. The aqueous phase was concentrated to 20mL under reduced pressure at 30℃or less, and then separated by adding to 8 50mL centrifuge tubes, each of which was preloaded with an acetone solution (40 mL) containing 0.2MNaClO 4. A suspension was obtained and centrifuged at 1000 x g for 3 minutes. The supernatant was discarded, the solid residue was washed 2 times with acetone, combined after adding a small amount of water for dissolution, the product was collected by column chromatography, the solvent was evaporated in vacuo (temperature. Ltoreq.30℃), the product was dissolved in water and lyophilized to give compound 4-1 (white solid, 97.2mg, yield 64%).
1H NMR
1H NMR (600 MHz, D2O)δ 7.79 (s, 1H), 6.28 (t,J=6.9 Hz, 1H), 4.60 (dt,J=6.7, 3.5 Hz, 1H), 4.29-4.20 (m, 3H), 3.50 (s, 2H).2.54-2.45 (m, 2H), 1.95 (s, 3H).
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (2)
1. A method for preparing a nucleotide analogue of formula (I), comprising:
Contacting a compound shown in a formula (II), a first deprotection reagent and a molecular sieve with a compound shown in a formula (III), and contacting the obtained product with a compound shown in a formula (IV) and a second deprotection reagent to obtain a compound shown in a formula (I);
Wherein B is cytosine, thymine or uracil;
R 1 is a bisphosphate group or a triphosphate group;
R 2 is azidomethyl;
r 3 is ；
R 4 is benzoyl;
R 6 is selected from Br;
r 7 is selected from H;
The first deprotection reagent and the second deprotection reagent are independently selected from at least one of DBU, et 3 N, DABCO, TMG, KOtBu, bartons and Hunigs, phosphazene;
Formula (I)
Formula (II)
Formula (III)
Formula (IV).
2. The preparation method according to claim 1, wherein the molar ratio of the compound represented by formula (II), the compound represented by formula (III) to the compound represented by formula (iv) is (1-4): (1-4): (1-4).
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1501940A (en) * | 2000-08-21 | 2004-06-02 | 印斯拜尔药品股份有限公司 | Compositions and methods for inhibiting platelet aggregation |
| DE102014112055A1 (en) * | 2014-08-22 | 2016-02-25 | Universität Hamburg | Di- and triphosphate prodrugs |
| WO2021207085A1 (en) * | 2020-04-06 | 2021-10-14 | The Scripps Research Institute | Synthetic elaboration of native dna by rass (sendr) |
| WO2023043794A1 (en) * | 2021-09-14 | 2023-03-23 | Terran Biosciences Inc. | Processes of preparing psilocin and psilocybin |
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| US8481712B2 (en) * | 2001-01-22 | 2013-07-09 | Merck Sharp & Dohme Corp. | Nucleoside derivatives as inhibitors of RNA-dependent RNA viral polymerase |
| US7105499B2 (en) * | 2001-01-22 | 2006-09-12 | Merck & Co., Inc. | Nucleoside derivatives as inhibitors of RNA-dependent RNA viral polymerase |
| WO2007030227A2 (en) * | 2005-07-29 | 2007-03-15 | The Board Of Governors For Higher Education, | Modified oligonucleotides containing diphosphodiester internucleotide linkages |
| CA2885369A1 (en) * | 2012-09-28 | 2014-04-03 | Tufts University | Uridine diphosphate derivatives, prodrugs, compositions and uses thereof |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1501940A (en) * | 2000-08-21 | 2004-06-02 | 印斯拜尔药品股份有限公司 | Compositions and methods for inhibiting platelet aggregation |
| DE102014112055A1 (en) * | 2014-08-22 | 2016-02-25 | Universität Hamburg | Di- and triphosphate prodrugs |
| CN106661075A (en) * | 2014-08-22 | 2017-05-10 | 汉堡大学 | Di- and triphosphate prodrugs |
| WO2021207085A1 (en) * | 2020-04-06 | 2021-10-14 | The Scripps Research Institute | Synthetic elaboration of native dna by rass (sendr) |
| WO2023043794A1 (en) * | 2021-09-14 | 2023-03-23 | Terran Biosciences Inc. | Processes of preparing psilocin and psilocybin |
Non-Patent Citations (2)
| Title |
|---|
| Prodrugs of the Phosphoribosylated Forms of Hydroxypyrazinecarboxamide Pseudobase T-705 and its De- Fluoro-Analogue T-1105 as Potent Influenza Virus Inhibitors;Johanna Huchting等;《Journal of Medicinal Chemistry》;20180615;第1-78页 * |
| Synthesis of a Bioreversibly Masked Lipophilic Adenosine Diphosphate Ribose Derivative;Marta Rachwalak等;《ChemBioChem.》;20170724;第18卷;第1616-1626页 * |
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