The present application claims priority from U.S. provisional application No. 63/376,579, filed on month 21 of 2022, the entire contents of which are hereby incorporated by reference.
Detailed Description
Various publications, articles, and patents are cited or described throughout the background and specification, each of which is incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is intended to provide a context for the present invention. Such discussion is not an admission that any or all of these matters form part of the prior art base with respect to any of the inventions disclosed or claimed.
Unless defined otherwise, all 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. Otherwise, certain terms used herein have the meanings set forth in the specification. All patents, published patent applications, and publications cited herein are hereby incorporated by reference as if fully set forth herein.
It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.
Unless otherwise indicated, any numerical value, such as the% sequence identity or the% sequence identity range described herein, is to be understood as modified in all instances by the term "about. Thus, a numerical value typically includes ±10% of the value. For example, a dose of 10mg includes 9mg to 11mg. As used herein, a numerical range, unless the context clearly indicates otherwise, includes all possible subranges, all individual values within the range, including integers within such range and fractions of the values.
As used herein, the connection term "and/or" between a plurality of recited elements is understood to encompass both single options and combined options. For example, where two elements are connected by an "and/or," a first option refers to the first element being applicable without the second element. The second option refers to the second element being applicable without the first element. A third option refers to the first element and the second element being adapted to be used together. Any of these options is understood to fall within the meaning and thus meet the requirements of the term "and/or" as used herein. Parallel applicability of more than one option is also understood to fall within the meaning and thus meet the requirements of the term "and/or".
Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The term "comprising" as used herein may be replaced with the term "containing" or "including" or sometimes with the term "having" as used herein.
As used herein, "consisting of" excludes any elements, steps, or ingredients not specified in the claim elements. As used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claims. Whenever used herein in the context of one aspect or embodiment of the present invention, any of the foregoing terms "comprising," "including," "comprising," and "having" may be substituted with the terms "consisting of or" consisting essentially of to alter the scope of the present disclosure.
As used herein, "nucleobase" may include unmodified, natural or modified nucleobases. "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). "modified nucleobases" include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C≡C-CH3) uracil and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-sulfanyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halo, in particular 5-bromo, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaadenine and 8-deaza, 3-deaza and 3-deaza. Additional modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido [5,4-b ] [1,4] benzoxazin-2 (3H) -one), phenothiazine cytidine (1H-pyrimido [5,4-b ] [1,4] benzothiazin-2 (3H) -one), G-clamp such as substituted phenoxazine cytidine (e.g., 9- (2-aminoethoxy) -H-pyrimido [5,4-b ] [1,4] benzoxazin-2 (3H) -one), carbazole cytidine (2H-pyrimido [4,5-b ] indol-2-one), pyrido-indole cytidine (H-pyrido [3,2,5] pyrrolo [2,3-d ] pyrimidin-2-one). Modified nucleobases can also include those in which the purine or pyrimidine base is substituted with other heterocycles, for example, 7-deazaadenine, 7-deazaguanine, 2-aminopyridine, and 2-pyridone.
"Alkyl" refers to an optionally substituted saturated straight or branched hydrocarbon group. Examples include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, n-pentyl, and n-hexyl. "cycloalkyl" refers to a cyclic alkyl group of 3 to 10 carbon atoms having a single ring or multiple rings and includes, for example, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, and the like. In some embodiments, optionally substituted alkyl is substituted with one or more halogens (such as F).
As used herein, "heterocycle" refers to a saturated, unsaturated, or aromatic ring system containing 3 to 18 atoms of at least one N, O, S or P. In some embodiments, the heterocycle comprises one N, O or S. In some embodiments, the heterocycle comprises two N, O or S.
As used herein, "protecting group" refers to the chemical modification of a functional group to obtain chemoselectivity in a subsequent chemical reaction. Examples of protecting groups include those disclosed, for example, in Greene Wuts, protective Groups in Organic Synthesis.
The term "activating group" includes moieties that increase the ability of the group to form a covalent bond with another molecule. The activating group comprises a phosphite-triester, a phosphotriester, an H-phosphonate or preferably a phosphoramidite group on at least one oxygen atom of the sugar moiety. Preferably, the activating group is on the C-3 'or C-5' oxygen of the nucleic acid monomer. Typically, the activating group is on the C-3 'oxygen of the nucleic acid monomer for synthesis of the probe in the 3'. Fwdarw.5 'direction, the oligonucleotide being attached to the support via the 3' end. The activating group is on the C-5 'oxygen of the nucleic acid monomer for synthesis of the probe in the 5'. Fwdarw.3 '("reverse") direction, the oligonucleotide being attached to the support via the 5' end.
To assist the reader of this application, the specification has been divided into individual paragraphs or chapters, or directed to various embodiments of this application. These divisions should not be considered as breaking apart the main content of a paragraph or section or embodiment from the main content of another paragraph or section or embodiment. Rather, those skilled in the art will appreciate that the present description has broad application and encompasses all combinations of individual chapters, paragraphs and sentences that may be envisioned. The discussion of any embodiment is intended to be exemplary only, and is not intended to suggest that the scope of the disclosure, including the claims, is limited to these examples.
Compounds of the present disclosure
In a general aspect, the present disclosure relates to compounds represented by formula (Ia) or (Ib):
Wherein X is selected from 3-to 5-membered cycloalkyl, -CHCH-, 3-to 5-membered heterocycle and-CHR3CHR3 -, Y is selected from O or NR ', R' is a counter ion, H or a protecting group, Z is selected from H, a counter ion, an activating group and an oligonucleotide, A and the attached dotted line are optional and when present, A is selected from O, S and CR4R4, B is a nucleobase, each R is independently selected from an oligonucleotide, a counter ion, H and a protecting group such as C1-C5 alkyl and POM or C1-C5 alkyl, each R1 and R2 is independently selected from H, F, OH and optionally substituted O-alkyl, or R1 and R2 form optionally substituted oxycyclobutene, each R3 is independently selected from C1-C3 alkyl, each R4 is independently selected from H, F and C1-C5 alkyl, provided that when A is O, then X is not-CHCH-, and when X is cyclopropyl, then at least one of A or Y is not O. In some embodiments, when a is present and X is selected from 3-to 5-membered cycloalkyl, -CHCH-, 3-to 5-membered heterocycle, and-CHR3CHR3 -, then R1 and R2 are not each H.
In some embodiments, the compounds of formulas (Ia) and (Ib) are represented by:
In some embodiments, the compounds of formulas (Ia) and (Ib) are represented by:
In some embodiments, the compound of formula (Ia) is represented by:
in some embodiments, X is cyclobutyl. In some embodiments, cyclobutyl is represented by:
Wherein the dashed lines represent the points of attachment to adjacent atoms. In some embodiments, X is-CHR3CHR3 -, and R3 is methylene. In some embodiments, X is selected from the following:
Wherein the dashed lines represent the points of attachment to adjacent atoms. In some embodiments, X is a 3-to 5-membered heterocycle having the structure:
wherein the curve contains one or more heteroatoms (e.g., N, O or S). In some embodiments, X is a 3-to 5-membered heterocycle selected from the group consisting of:
Wherein the dashed lines represent the points of attachment to adjacent atoms. In some embodiments, Y is O. In some embodiments, Y is O and X is not cyclopropyl.
In some embodiments, Z is an activating group. In some embodiments, Z is phosphoramidite or a functionally similar moiety. In some embodiments, the activating group is represented by:
wherein the dashed lines represent the points of attachment to adjacent atoms.
In some embodiments, Z is an oligonucleotide. In some embodiments, the oligonucleotide is the antisense strand of RNA, preferably the antisense strand of siRNA. In some embodiments, the 5' end of the oligonucleotide is linked to Y.
In some embodiments, a is O. In some embodiments, a is O and X is not cyclopropyl. In some embodiments, a is CH2 or CHF.
In some embodiments, R1 and R2 are independently selected from H, F, OH and optionally substituted O-alkyl, provided that when X is selected from 3-to 5-membered cycloalkyl, -CHCH-, 3-to 5-membered heterocycle, and-CHR3CHR3 -, then R1 and R2 are not each H, or R1 and R2 form optionally substituted oxetane. In some embodiments, substituted O-alkyl groups include-OMe, -OEt, -CH2CH2OCH3 (or MOE)、-CF2CH2OCH3、-CH2CF2OCH3、-CH2CH2OCF3、-CF2CF2OCH3、-CH2CF2OCF3、-CF2CH2OCF3、-CF2CF2OCF3、-CHFCH2OCH3、-CHFCHFOCH3、-CHFCH2OCFH2、-CHFCH2OCHF2 and-CH2CHFOCH3, etc.
In some embodiments, B is uracil. In some embodiments, R1 is H and R2 is OMe, OEt, MOE or F. In some embodiments, R is a protecting group, such as POM, C1-C5 alkyl (e.g., et), and Z is an activating group. In some embodiments, R is H and Z is an oligonucleotide.
In some embodiments, the compound is selected from:
Or a compound of the table, wherein the nucleobase is thymine or cytosine.
In some embodiments, R2 is selected from H, F, OH and optionally substituted O-alkyl, such as OMe, OEt, MOE, and the like. In some embodiments, R2 is —ome. In some embodiments, each R is independently selected from an oligonucleotide, a counter ion, H, and a protecting group, such as POM or C1-C5 alkyl. In some embodiments, R is a protecting group, such as POM or C1-C5 alkyl (e.g., et), and Z is an activating group, such as phosphoramidite or a functionally similar moiety. In some embodiments, the activating group is represented by:
In some embodiments, R is H and Z is an oligonucleotide.
Examples
The following examples are provided to illustrate but not limit the invention of the present disclosure. Those skilled in the art will recognize that the following procedure may be modified using methods known to those of ordinary skill in the art.
Terminology
The following abbreviations are used herein:
m=2' -O-methyl modification
F=2' -fluoro modifications
VP = vinyl phosphonate
(Invabasic) =reverse abasic
Ps=phosphorothioate
TEG-chol=cholesterol-triethylene glycol
Ecc) =carbocycle, e refers to the geometric isomer of vinyl phosphonate
VP (ecc) mu=carbocycle mU e-vinylphosphonate
DMP (RS) =r, S-dimethylphosphonate
DMP (SR) =s, R-dimethylphosphonate
DMP (RR) =r, R-dimethylphosphonate
DMP (SS) =s, S-dimethylphosphonate
P (ccb) =cis-cyclobutylphosphonate
P (tcb) =trans-cyclobutylphosphonate
(UNAA) =should be (UNA-A)
(UNA-A) =adenosine unlocking nucleic acid
(J15 AdaC) =j 2-c15AdaC =2' -O- [15- (adamantyl-1) pentadecyl ] cytidine example 1 Synthesis of dimethyl phosphonate
Scheme 1. Synthesis of dimethyl phosphonate
Scheme 2 Synthesis of Cyclobutyl phosphonate
EXAMPLE 2 Synthesis of phosphonate
Example 2-1
To a solution of potassium tert-butoxide (65.5 g,583.8mmol,3.0 eq.) in tert-butyl methyl ether (1300 mL) was added sec-butyllithium (450 mL,583.8mmol,3.0 eq.) dropwise under an inert atmosphere of argon and stirred at-78℃for 2.5 h. To the resulting solution was added dropwise, with stirring at-78 ℃, a solution of lithium bromide (101.5 g,1167.5mmol,6.0 eq.) in tetrahydrofuran (1000 mL). The resulting solution was stirred at-15 ℃ for 30 minutes. The resulting solution was cooled to-78 ℃. To the resulting solution was added a solution of copper (I) -dimethyl sulfide bromide (60 g,291.9mmol,1.5 eq.) in 6-O- (triisopropylsilyl) -d-galactal cyclic carbonate (420 mL) at-78 ℃. The resulting solution was stirred at-78 ℃ for 1 hour. To the resulting solution was added a solution of (3 ar,6 ar) -2, 2-dimethyl-3 a,6 a-dihydro-4H-cyclopenta [ d ] [1,3] dioxol-4-one (30 g,194.6mmol,1.0 eq.) in tetrahydrofuran (300 mL) at-78 ℃. The resulting solution was stirred at-30 ℃ for 30 minutes. The reaction mixture was diluted with 2000mL of t-butyl methyl ether. The reaction mixture was quenched by the addition of 100mL of acetic acid and methanol (v/v=1:1). The organic layer was washed with 3×3000mL of mixed ammonium chloride and aqueous ammonia solution (ph=9). The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was applied to a silica gel column with petroleum ether/ethyl acetate (100:1-50:1). 35g (74% yield) of white solid was obtained 21.MS m/z[M+NH4+]+(ESI):260.30.1H NMR(300MHz,DMSO-d6)δ4.60(d,J＝5.4Hz,1H),4.11(d,J＝5.4,1.1Hz,1H),3.49(dd,J＝8.7,2.7Hz,1H),3.35-3.29(m,1H),2.61(dd,J＝17.7,8.9Hz,1H),2.49-2.42(m,1H),1.95-1.84(m,1H),1.33(s,3H),1.27(s,3H),1.06(s,9H).
To a solution of 21 (20 g,123.96mmol,1.0 eq.) cerium (III) chloride heptahydrate (46.172 g,123.96mmol,1.0 eq.) in methanol (180 mL) was slowly added borohydride (7.034 g,185.94mmol,1.5 eq.) under an inert atmosphere of argon at 0 ℃. The resulting solution was stirred at room temperature for 4 hours. The reaction mixture was quenched by the addition of water. The resulting solution was extracted with 300mL ethyl acetate. The organic layers were combined and washed with 300mL brine. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The crude product was applied to a silica gel column with petroleum ether/ethyl acetate (50:1-20:1). 18g (89% yield) of 21 were obtained as a pale yellow liquid.
To a solution of pyrimidine-2, 4 (1H, 3H) -dione (100 g,892.85mmol,1.0 eq.) in 840mL acetonitrile and 160mL pyridine at 0deg.C under an inert atmosphere of argon was added benzoyl chloride (114.05 mL,982.14mmol,1.1 eq.). The resulting solution was stirred at room temperature for 5 hours. The reaction mixture was quenched with 200mL of 1N hydrochloric acid, diluted with 500mL of dichloromethane, and washed with 3X 200mL of saturated aqueous sodium chloride. The organic layers were combined and dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was applied to a silica gel column with petroleum ether/ethyl acetate (10:1-1:1). 60g (31% yield) 19 were obtained as a white solid.
To a solution of 19 (60 g,277.78mmol,1.0 eq.) 1, 8-diazabicyclo [5.4.0] undec-7-ene (48 mL) in acetonitrile (600 mL) was added benzyl chloromethyl ether (52 g,333.33mmol,1.2 eq.) under an inert atmosphere of argon. The resulting solution was stirred at room temperature for 2 hours. The reaction was then concentrated under vacuum. The residue was extracted with ethyl acetate (250 mL) and washed with 1×200mL water, 1×200mL saturated aqueous sodium chloride solution. The organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was dissolved in 600mL of sodium methoxide (2M sodium methoxide in methanol) and stirred at room temperature under an inert atmosphere of argon for 1 hour. The reaction mixture was then evaporated to dryness. The residue was applied to a silica gel column with petroleum ether/ethyl acetate (2:1-1:3). 55g (85% yield) of 20 were obtained as a white solid. MS M/z [ M-H ]- (ESI): 231.00.
Diethyl azodicarboxylate (50.85 g,292.0mmol,2.5 eq) was slowly added dropwise to a solution of 22 (28.5 g,116.8mmol,1.0 eq), 20 (40.6 g,175.2mmol,1.5 eq), triphenylphosphine (76.6 g,292.0mmol,2.5 eq) in tetrahydrofuran (1000 mL) under an inert atmosphere of argon at-78 ℃. The resulting solution was stirred at-78 ℃ for 1 hour, then slowly warmed to room temperature, and stirred for 16 hours. The resulting mixture was concentrated under vacuum. The residue was purified by flash prep HPLC using column, C18 silica gel, mobile phase, water and acetonitrile (30% acetonitrile reached 100% in 20 min), UV 254nm. The fractions were diluted with dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure. 36.5g (68% yield) of 23 are obtained as a yellow oil. MS M/z [ M+H ]+(ESI):459.30.1 H NMR (300 MHz, chloroform) -d)δ7.32(t,J＝3.3Hz,5H),7.20(d,J＝7.9Hz,1H),5.71(d,J＝7.8Hz,1H),5.39-5.15(m,3H),5.03(dt,J＝6.8,3.5Hz,1H),4.61(d,J＝3.2Hz,2H),4.52(t,J＝6.6Hz,1H),3.62(dd,J＝8.7,4.6Hz,1H),3.37-3.25(m,J＝8.3,5.9Hz,1H),2.33-2.03(m,3H),1.52(s,3H),1.28(s,3H),1.17(s,9H).
To a solution of 23 (35 g,76.4mmol,1.0 eq.) in ethyl acetate (350 mL) was added 10% pd/C (wt/wt = 10%,3.5 g), trifluoroacetic acid (35 mL) in sequence at room temperature under an inert atmosphere of hydrogen. The resulting solution was stirred at room temperature for 4 hours. The solution was filtered and concentrated under reduced pressure. The residue was dissolved in 350mL trifluoroacetic acid/water=1:1 at room temperature. The resulting solution was stirred at 50 ℃ under an inert atmosphere of argon for 2 hours. The solution was concentrated under reduced pressure. The residue was dissolved in 1N ammonium hydroxide in methanol (200 mL). The resulting mixture was concentrated in vacuo. The residue was purified by flash prep HPLC using column, C18 silica gel, mobile phase, water and acetonitrile (0% acetonitrile up to 30% in 15 min), UV 254nm. The fractions were concentrated under reduced pressure. 15.7g (85% yield) of-5 were obtained as a pale yellow oil. MS M/z [ M+Na ]+ (ESI): 265.00.
To a solution of 5 (17 g,70.24mmol,1.0 eq.) in N, N-dimethylformamide (170 mL) was added diphenyl carbonate (22.57 g,105.37mmol,1.5 eq.) followed by sodium bicarbonate (590.38 mg,7.02mmol,0.1 eq.) under an inert atmosphere of argon. The reaction mixture was heated at 150 ℃ for 3 hours until the solution turned dark red. The reaction mixture was cooled to room temperature and slowly poured into 3000mL of diethyl ether with stirring. The precipitate was collected by filtration, redissolved in methanol, decolorized with activated carbon and filtered. The filtrate was concentrated in vacuo. 10.3g (73% yield) of 6 are obtained as yellow solid. MS M/z [ M-H ]- (ESI): 223.00.
To a solution of 6 (14.7 g,65.62mmol,1.0 eq.) in methanol (150 mL) was added trimethyl borate (13.6 g,131.25mmol,2.0 eq.), trimethoxymethane (6.96 g,65.6mmol,1.0 eq.), sodium bicarbonate (1.1 g,13.1mmol,0.2 eq.) in this order. The reaction mixture was stirred in a sealed tube at 150 ℃ for 16 hours. The reaction mixture was cooled to room temperature and concentrated in vacuo. The residue was purified by flash prep HPLC using column, C18 silica gel, mobile phase, water and acetonitrile (0% acetonitrile 40% in 20 min), UV 254nm. The fractions were concentrated under reduced pressure. 7.7g (45% yield) of 7 were obtained as a pale yellow oil. MS M/z [ M-H ]- (ESI): 255.00.
To a solution of 7 (5 g,19.53mmol,1.0 eq.) in pyridine (50 mL) was added 4,4' -dimethoxytrityl chloride (6.94 g,20.5mmol,1.05 eq.) under an inert atmosphere of argon at 0 ℃. The resulting solution was stirred at room temperature for 2 hours. The reaction mixture was quenched with 10mL of methanol, diluted with 200mL of dichloromethane, and washed with 2X 150mL of saturated aqueous sodium bicarbonate and 2X 150mL of saturated aqueous sodium chloride, respectively. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by flash prep HPLC using column, C18 silica gel, mobile phase, water and acetonitrile (30% acetonitrile reached 100% in 20 min), UV 254nm. The fractions were concentrated under reduced pressure. 8.4g (77% yield) of a pale yellow solid was obtained 31.MS m/z[M-H]-(ESI):557.10.1H NMR(300MHz,DMSO-d6)δ11.13(s,1H),7.45-7.35(m,3H),7.35-7.18(m,7H),6.93-6.84(m,4H),5.57(d,J＝7.5Hz,1H),5.10-4.97(m,1H),4.37(d,J＝7.3Hz,1H),4.14-3.99(m,1H),3.90(t,1H),3.74(s,6H),2.89(t,J＝7.9Hz,1H),3.23-3.15(m,3H),1.96-1.69(m,2H),1.35-1.08(m,1H),0.83(t,J＝7.3Hz,1H).
To a solution of 31 (8.4 g,15.05mmol,1.0 eq.) in N, N-dimethylformamide (85 mL) was added imidazole (2.55 g,37.59mmol,2.5 eq.) followed by tert-butyldimethylsilyl chloride (5.66 g,37.59mmol,2.5 eq.) under an inert atmosphere of argon at room temperature. The resulting solution was stirred at room temperature for 2 hours. The reaction mixture was diluted with 500mL of methylene chloride and washed with 2X 200mL of saturated aqueous sodium bicarbonate solution and 1X 200mL of saturated aqueous sodium chloride solution, respectively. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash prep HPLC using column, C18 silica gel, mobile phase, water and acetonitrile (30% acetonitrile reached 100% in 20 min), UV 254nm. The fractions were concentrated under reduced pressure. 8.6g (81% yield) of 32 were obtained as a pale yellow oil. MS M/z [ M-H ]- (ESI): 671.30.
Triethylsilane (5.2 g,44.64mmol,3.0 eq.) was added to a solution of 32 (10.0 g,14.88mmol,1.0 eq.) in dichloromethane dissolved in 100ml 6% dichloroacetic acid under an inert atmosphere of argon. The resulting solution was stirred at room temperature for 1 hour. The reaction mixture was diluted with 200mL of methylene chloride and washed with 2X 200mL of saturated aqueous sodium bicarbonate solution, 1X 100mL of water and 1X 200mL of saturated aqueous sodium chloride solution, respectively. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash prep HPLC using column, C18 silica gel, mobile phase, water and acetonitrile (30% acetonitrile reached 100% in 20 min), UV 254nm. The fractions were concentrated under reduced pressure. 4.2g (76%) of a white solid are obtained 31.MS m/z[M-H]-(ESI):369.15.1H NMR(300MHz,DMSO-d6)δ11.09(d,J＝5.8Hz,1H),7.42(dd,J＝7.6,5.8Hz,1H),5.57(dd,J＝7.6,1.5Hz,1H),5.05(td,J＝9.7,5.5Hz,1H),4.52(t,J＝5.2Hz,1H),4.26(dd,J＝6.7,5.4Hz,1H),4.02(t,J＝5.4Hz,1H),3.49-3.59(m,J＝10.6,5.3Hz,1H),3.33-3.27(m,1H),3.17(s,3H),1.90-2.03(m,J＝7.5Hz,1H),1.86-1.56(m,2H),0.88(s,9H),0.06(d,6H).
To a solution of 9 (2.5 g,6.74mmol,1.0 eq.) in 25mL of dichloromethane was added Dess-Martin periodate (4.0 g,10.12mmol,1.5 eq.) under an inert atmosphere of argon at 0 ℃. The resulting solution was stirred at room temperature for 1 hour. The reaction mixture was diluted with 300mL of methylene chloride and washed with 1X 200mL of saturated aqueous sodium thiosulfate solution, 1X 200mL of saturated aqueous sodium bicarbonate solution, 2X 100mL of water and 1X 200mL of saturated aqueous sodium chloride solution, respectively. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. 2.1g (84%) of 10 are obtained as a white solid. MS M/z [ M-H ]- (ESI): 367.05. It was used in the next step without further purification.
To a solution of methylenebis (phosphonic acid) tetraethyl ester (2.13 g,7.41mmol,1.3 eq.) in anhydrous tetrahydrofuran (20 mL) was added potassium tert-butoxide (2.13 g,5.70mmol,1.35 eq.) at 0 ℃. The reaction mixture was stirred at 0 ℃ for 10 minutes, then warmed to room temperature and continued for 30 minutes. A solution of 10 (2.10 g,5.70mmol,1.0 eq.) in 20mL dry tetrahydrofuran was then added dropwise at 0deg.C. The resulting solution was stirred at 0 ℃ for 3 hours. The reaction mixture was diluted with 200mL of dichloromethane and washed with 2x 200mL of saturated aqueous sodium bicarbonate solution. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash prep HPLC using column, C18 silica gel, mobile phase, water and acetonitrile (30% acetonitrile reached 100% in 20 min), UV 254nm. The fractions were diluted with an equal volume of dichloromethane. The organic phase layer was separated. The aqueous phase was extracted with 3X 20mL of dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. The solid was filtered off. The filtrate was concentrated at 25 ℃. 2g (68%) of a pale yellow solid was obtained 12.MS m/z[M+H]+(ESI):503.20.1H NMR(300MHz,DMSO-d6)δ11.18(s,1H),7.45(d,J＝7.6Hz,1H),6.65-6.48(m,J＝21.9,17.1,7.6Hz,1H),5.90-5.81(m,1H),5.61(d,J＝7.5Hz,1H),5.06-4.95(m,1H),4.40(dd,J＝9.8,5.8Hz,1H),4.00-3.89(m,J＝8.3,7.0,1.3Hz,4H),3.81(dd,J＝5.9,2.6Hz,1H),3.21(s,3H),2.75(s,1H),1.93-.73(m,2H),1.22(t,J＝7.0Hz,6H),0.87(s,9H),0.03(s,6H).31P NMR(121MHz,DMSO-d6)δ17.62.
11 (2.00 G,5.15mmol,1.0 eq.) is dissolved in 20mL formic acid and H2 O (v/v=1:1) under an inert atmosphere of argon. The mixture solution was stirred at room temperature for 1 hour. The resulting solution was concentrated under vacuum. The residue was purified by flash prep HPLC using column, C18 silica gel, mobile phase, water and acetonitrile (15% acetonitrile reaching 35% in 20 min), UV 254nm. The fractions were concentrated under reduced pressure. 1.3g (84%) of a white solid was obtained 12.MS m/z[M+H]+(ESI):389.25.1H NMR(300MHz,DMSO-d6)δ11.17(d,1H),7.48-7.38(m,1H),6.76-6.55(m,1H),5.87-5.72(m,1H),5.60(d,1H),5.02(td,1H),4.74(d,1H),4.17-4.07(m,1H),4.02-3.90(m,4H),3.88-3.83(m,1H),3.26(s,3H),2.75-2.58(m,1H),1.94-1.72(m,2H),1.23(td,6H).31P NMR(121MHz,DMSO-d6)δ18.08.
To a solution of 12 (1.7 g,4.38mmol,1.0 eq.) in dichloromethane (170 mL) was added in order propylamino) phosphinyloxy propionitrile (1.72 g,5.69mmol,1.3 eq.) 4, 5-dicyanoimidazole (569 mg,4.88mmol,1.1 eq.). The resulting solution was stirred at room temperature for 40 minutes. The resulting solution was diluted with 500mL of dichloromethane and washed with 1X 300mL of sodium bicarbonate and 1X 300mL of brine, respectively. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The crude product was purified by flash prep HPLC using conditions (INTELFLASH-1) of column, C18 silica gel, mobile phase, water and acetonitrile (30% acetonitrile reached 100% in 10 min and 100% for 5 min), detector, UV 254nm. The fractions were diluted with an equal volume of dichloromethane. The organic phase layer was separated. The aqueous phase was extracted with 3X 50mL of dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. The solid was filtered off. The filtrate was concentrated at 25 ℃ to give the product, which was dried at 25 ℃ under reduced pressure for 4 hours. 1.85g of a white solid was obtained 0.MS m/z[M-H]-(ESI):587.10.1H NMR(300MHz,DMSO-d6)δ11.14(s,1H),7.42(dd,J＝7.6,1.1Hz,1H),6.67-6.49(m,1H),5.95-5.69(m,1H),5.59(d,J＝7.6Hz,1H),5.10-4.98(m,1H),4.53-4.29(m,1H),4.02-3.83(m,5H),3.80-3.66(m,2H),3.63-3.47(m,2H),3.22(d,J＝3.0Hz,3H),2.85(s,1H),2.79-2.71(m,2H),1.96-1.76(m,2H),1.24-1.17(m,6H),1.15-1.11(m,12H).31P NMR(121MHz,DMSO)δ147.53,146.78,17.69,17.50.
Example 2-2
To a solution of (3 ar,5s,6R,6 ar) -5- ((R) -2, 2-dimethyl-1, 3-dioxol-4-yl) -2, 2-dimethyltetrahydrofurano [2,3-d ] [1,3] dioxol-6-ol (400 g,1.54mol,1.0 eq.) in 4L tetrahydrofuran was added sodium hydride (55.2 g,2.31mol,1.5 eq.) under an inert atmosphere of nitrogen at 0 ℃. The resulting solution was stirred at 0 ℃ for 20 minutes. Then, 2- (bromomethyl) naphthalene (509.7 g,2.31mol,1.5 eq.) was added dropwise with stirring at 0 ℃. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was quenched with saturated aqueous ammonium chloride. The resulting mixture was diluted with 4L of ethyl acetate and washed with 2X 4L of water and 2X 4L of saturated aqueous sodium chloride solution, respectively. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was applied to a silica gel column with ethyl acetate/petroleum ether (1:5-1:3). 490g (80%) of 41 are obtained as yellow oil. MS M/z [ M+Na ]+ (ESI): 423.08.
41 (490 G,1.23mol,1.0 eq.) was dissolved in acetic acid (80% aqueous solution, 4900 mL) under an inert atmosphere of nitrogen and stirred overnight at room temperature. The resulting mixture was concentrated under reduced pressure and diluted with 5000mL of ethyl acetate. The resulting mixtures were washed with 3X 1500mL of saturated aqueous sodium bicarbonate solution, respectively. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was applied to a silica gel column with ethyl acetate/petroleum ether (1:5-1:1). 390g (85%) 42 are obtained as a yellow oil. MS M/z [ M+Na ]+ (ESI): 383.21.
Imidazole (146.8 g,2.16mol,4.0 eq.) and tert-butyldiphenylchlorosilane (156 g,0.55mol,1.05 eq.) were added sequentially to a mixture solution of 42 (195 g,0.54mol,1.0 eq.) in 2000mL of dichloromethane under an inert atmosphere of argon at 0 ℃. The reaction mixture was warmed to room temperature and stirred at room temperature for 4 hours. The resulting solution was diluted with 5000mL of methylene chloride and washed with 3X 1500mL of water and 3X 1500mL of saturated aqueous sodium chloride solution, respectively. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was applied to a silica gel column with ethyl acetate/petroleum ether (1:10-1:5). 190g (60%) of a yellow oil are obtained 43.MS m/z[M+Na]+(ESI):621.25.1H NMR(300MHz,DMSO-d6)δ7.90-7.85(m,1H),7.79-7.71(m,3H),7.65-7.56(m,4H),7.50-7.45(m,2H),7.43-7.33(m,5H),7.29-7.22(m,2H),5.72(d,J＝3.7Hz,1H),5.06(d,J＝4.8Hz,1H),4.78-4.72(m,2H),4.57(d,J＝11.9Hz,1H),4.19-4.15(m,1H),4.13-3.98(m,1H),3.87-3.94(m,1H),3.83-3.77(m,1H),3.57(d,J＝6.5Hz,1H),1.47(s,3H),1.30(s,3H),0.94(s,9H).
Dimethyl sulfoxide (78.19 g,0.95mol,3.0 eq.) was added dropwise to a solution of oxalyl chloride (120.84 g,0.953mol,1.5 eq.) in dichloromethane (2000 mL) with stirring at-78 ℃. The resulting solution was stirred at-78 ℃ for 30 minutes, and a solution of 43 (120 g,158.86mmol,1.0 eq.) in dichloromethane (500 mL) was added dropwise with stirring at-78 ℃. The resulting solution was allowed to react for an additional 1.5 hours at-78 ℃ with stirring. Then, triethylamine (72.24 g,714.75mmol,4.5 eq.) was added dropwise with stirring at-78 ℃. The resulting solution was allowed to react for an additional 2 hours at-78 ℃ with stirring. The resulting solution was diluted with 500mL of dichloromethane. The resulting mixture was washed with 1X 500mL of saturated aqueous sodium bicarbonate solution and 1X 500mL of saturated aqueous sodium chloride solution, respectively. The mixture was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was applied to a silica gel column with ethyl acetate/petroleum ether (1:10-1:6). 80g (85%) of 44 are obtained as a yellow oil. MS M/z [ M+Na ]+ (ESI): 619.57.
To a solution of methyltriphenylphosphonium bromide (96.2 g, 0.399 mol,2.0 eq) in tetrahydrofuran (500 mL) under an inert atmosphere of nitrogen was added sodium hydride (6.5 g, 0.399 mol,2.0 eq). The resulting solution was stirred at room temperature for 30 minutes. A solution of 44 (80 g,134.7mmol,1.00 eq.) in tetrahydrofuran (500 mL) was added. The resulting solution was allowed to react at room temperature for an additional 3 hours with stirring. The reaction was then quenched by addition of saturated aqueous ammonium chloride. The resulting solution was extracted with 4000mL ethyl acetate and the organic layers were combined. The resulting mixture was washed with water and saturated aqueous sodium chloride solution. The mixture was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was applied to a silica gel column with ethyl acetate/petroleum ether (1:20 to 1:5). 63g (80%) of 45 are obtained as a yellow oil. MS M/z [ M+Na ]+ (ESI): 617.60.
Borane-dimethylsulfide complex (10 m,42.1mL,2.0 eq.) is added to a solution of 45 (125 g,210.4mmol,1.0 eq.) in tetrahydrofuran (1250 mL) at 0 ℃. The resulting solution was stirred at room temperature for 2 hours. Sodium hydroxide (630 mL of a 2N aqueous solution, 6.0 eq) was then added at room temperature, and hydrogen peroxide (30%, 143.1g,6.0 eq) was added dropwise with stirring at room temperature. The resulting solution was allowed to react at room temperature for an additional 2 hours with stirring. The resulting solution was extracted with 2×2000mL dichloromethane and the organic layers were combined. The mixture was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was applied to a silica gel column with ethyl acetate/petroleum ether (1:20-1:5). 73g (56%) of 46 are obtained as a yellow oil. MS M/z [ M+Na ]+ (ESI): 635.62.
To a solution of hydrogen trifluoride (381.6 g,2.37mol,10.0 eq.) in 730mL of anhydrous tetrahydrofuran at room temperature under an inert atmosphere of argon was added triethylamine (391.5 g,2.37mol,10.0 eq.). The resulting solution was stirred at room temperature for 10 minutes. A solution of 46 (145 g,236.54mmol,1.0 eq.) in 730mL dry tetrahydrofuran was added to the resulting solution. The reaction mixture was stirred at room temperature for 12 hours and diluted with 3000mL of dichloromethane, washed with 2×1000mL of saturated aqueous sodium bicarbonate and 2×1000mL of saturated aqueous sodium chloride, respectively. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by flash prep HPLC using column, C18 silica gel, mobile phase, water (0.1% fa) and acetonitrile (10% acetonitrile reached 100% in 12 min), UV 254nm. The fractions were diluted with dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure. 70g (80%) of 47 were obtained as a pale yellow oil. MS M/z [ M+Na ]+ (ESI): 397.15.1H NMR (300 MHz, chloroform) -d)δ7.93-7.80(m,4H),7.57-7.46(m,3H),5.77(d,J＝3.9Hz,1H),5.00(d,J＝11.4Hz,1H),4.77-4.64(m,2H),4.23(m,1H),3.92-3.73(m,5H),2.29(s,3H),2.03-1.87(m,1H),1.64(s,3H),1.40(s,3H).
To a solution of 47 (70 g,187.17mmol,1.0 eq.) in 700mL of anhydrous pyridine was added 4-methylbenzenesulfonyl chloride (107.12 g,561.51mmol,3.0 eq.) under an inert atmosphere of nitrogen at 0 ℃. Then, the reaction mixture was stirred at room temperature for 12 hours. The reaction was quenched by addition of water and concentrated under reduced pressure. The reaction mixture was diluted with 3000mL of dichloromethane and washed with 1X 1000mL of saturated sodium bicarbonate and 2X 1000mL of saturated sodium chloride, respectively. The organic phase was dried over anhydrous sodium sulfate. The solid was filtered off and concentrated under reduced pressure. The crude product was purified by flash prep HPLC using conditions (INTELFLASH-1) of column, C18 silica gel, mobile phase, water (0.04% nh4HCO3) and acetonitrile (10% acetonitrile 100% in 15 min and 100% for 5 min), detector, UV 254nm. 90g (70%) of 48 are obtained as a pale yellow solid. [ M+Na+ ]+ (ESI): 705.81
To a solution of diethyl ((phenylthio) methyl) phosphonate (22.62 g,87.00mmol,2.00 eq.) in 150mL of anhydrous tetrahydrofuran, butyllithium (2 m,43.50mL, 2.00 eq.) was added dropwise under stirring under nitrogen inert atmosphere at-78 ℃. The resulting solution was stirred at-30 ℃ for 2 hours. A solution of 48 (30 g,43.99mmol,1.00 eq.) in 150mL of tetrahydrofuran was added dropwise with stirring at-78 ℃. The resulting solution was stirred at room temperature for 12 hours. The resulting solution was extracted with ethyl acetate, and the organic layers were combined. The organic layer was washed with 1X 500mL of water and 2X 500mL of saturated sodium chloride, respectively. The mixture was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by flash prep HPLC using conditions (INTELFLASH-1) of column, C18 silica gel, mobile phase, water (0.04% nh4HCO3) and acetonitrile (30% acetonitrile 100% in 10min and 100% for 5 min), detector, UV 254nm. 12.5g (50%) of 201 are obtained as a pale yellow solid. [ M+H+ ]+ (ESI): 599.66
To a solution of 201 (15 g,25.08mmol,1.0 eq.) in 300mL of toluene was added 2,2' -azobis (2-methylpropanenitrile) (3.29 g,20.07mmol,0.4 eq.) and tributylstannane (14.6 g,50.16mmol,2.0 eq.) under an inert atmosphere of nitrogen at room temperature. The resulting solution was stirred at 110 ℃ for 1 hour. The resulting mixture was concentrated under reduced pressure. The crude product was purified by flash prep HPLC using column, C18 silica gel, mobile phase, water (0.04% nh4HCO3) and acetonitrile (30% acetonitrile reached 100% in 10 min and 100% for 5 min), detector, UV 254nm. 8.5g (65%) of a pale yellow solid was obtained 91.[M+H+]+(ESI):491.15.1H NMR(400MHz,DMSO-d6)δ7.96-7.82(m,4H),7.57-7.43(m,3H),5.80-5.71(m,1H),4.85-4.72(m,2H),4.64(d,J＝12.2Hz,1H),4.02-3.78(m,5H),3.51-3.44(m,1H),2.65-2.58(m,2H),2.21-1.85(m,4H),1.48(d,J＝5.0Hz,3H),1.31(d,J＝3.1Hz,3H),1.19(m,6H).31P NMR(162MHz,DMSO)δ33.08,30.36.
To a solution of 91 (17 g,34.69mmol,1.0 eq.) in 170mL of acetic acid under an inert atmosphere of nitrogen was added acetoacetate (35.4 g,346.94mmol,10.0 eq.) and sulfuric acid (679.9 mg,6.94mmol,0.2 eq.). The resulting solution was stirred at room temperature for 2 hours. The reaction was then quenched by addition of ice water. The resulting solution was extracted with ethyl acetate. The resulting mixture was washed with water and saturated aqueous sodium chloride solution. The mixture was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by flash prep HPLC using column, C18 silica gel, mobile phase, water and acetonitrile (20% acetonitrile reached 100% in 10 min and 100% for 5 min), detector, UV 254nm. 14g (75%) of 82 are obtained as a pale yellow solid. MS M/z [ M+H ]+ (ESI): 535.72.
To a solution of uracil (5.87 g,52.43mmol,2.0 eq.) in 140mL of acetonitrile at room temperature under an inert atmosphere of nitrogen was added N, O-bis (trimethylsilyl) acetamide (23.95 g,117.98mmol,4.5 eq.). The resulting solution was stirred at 60 ℃ for 0.5 hours. 82 (14 g,26.22mmol,1.00 eq.) was added thereto at 0 ℃, followed by dropwise addition of Quan Lv stannane (8.86 g,34.08mmol,1.3 eq.) under stirring at 0 ℃. The resulting solution was allowed to react for an additional 1 hour with stirring at 60 ℃. The reaction mixture was cooled to 0 ℃ and quenched by the addition of 100mL of saturated aqueous sodium bicarbonate. The resulting solution was extracted with 2×300mL of ethyl acetate, and the organic layers were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by flash prep HPLC using column, C18 silica gel, mobile phase, water (0.04% nh4HCO3) and acetonitrile (45% acetonitrile 100% in 10min and 100%5 min), detector, UV 254nm. 9g (60%) of a white solid was obtained 83.MS m/z[M+H]+(ESI):587.10.1H NMR(300MHz,DMSO-d6)δ11.43(s,1H),7.93(d,J＝8.7Hz,3H),7.84(s,1H),7.68(d,J＝13.4Hz,1H),7.54(d,J＝9.3Hz,2H),7.47(d,J＝8.5Hz,1H),5.79(dd,J＝12.3,3.8Hz,1H),5.68(d,J＝8.0Hz,1H),5.49-5.42(m,1H),4.68(d,J＝2.7Hz,2H),4.11(t,J＝6.3Hz,1H),3.98-3.90(m,5H),2.78-2.56(m,2H),2.10(s,4H),2.04-1.98(m,1H),1.24(d,J＝7.0Hz,2H),1.21-1.14(m,6H).31P NMR(121MHz,DMSO)δ32.76,30.94,30.15,30.14.
To a solution of 83 (9 g,15.36mmol,1.0 eq.) in 90mL of ethanol at 0deg.C under an inert atmosphere of nitrogen was added sodium methoxide (1.24 g,23.04mmol,1.5 eq.). The resulting solution was stirred at room temperature for 2 hours. The mixture was then adjusted to ph=7 by Amberlite IR-120 (h+). The resulting mixture was filtered and the filter cake was washed with 3X 50mL ethanol. The filtrate was concentrated under reduced pressure. The 9g crude product was used in the next step without further purification. MS M/z [ M+H ]+ (ESI): 549.32.
Sodium hydride (737.28 mg,30.72mmol,2.0 eq.) was added to a solution of 84 (15.36 mmol,1.0 eq.) in 70mL tetrahydrofuran under an inert atmosphere of nitrogen at 0 ℃. The mixture was stirred at 0 ℃ for 30 minutes. A solution of methyl iodide (4.33 g,30.72mmol,2.0 eq.) in 20mL tetrahydrofuran was added dropwise at 0deg.C. The resulting solution was stirred at room temperature for 2 hours. The reaction was quenched with 50mL of water. The resulting solution was extracted with 200mL ethyl acetate and the organic phases were combined. The organic phase was washed with water, then dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by flash prep HPLC using conditions (INTELFLASH-1) of column, C18 silica gel, mobile phase, water (0.04% nh4HCO3) and acetonitrile (20% acetonitrile 100% in 25 min and 100% for 5 min), detector, UV 254nm. 5g (60%) of 416 are obtained as a white solid, in two steps. MS M/z [ M+H ]+ (ESI): 559.41.
To a solution of 416 (6.0 g,14.33mmol,1.0 eq.) in 72mL dichloromethane and 8mL water was added 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone (4.6 g,20.06mmol,1.4 eq.) under an inert atmosphere of nitrogen at room temperature. The resulting solution was stirred at room temperature for 1 hour. The reaction mixture was diluted with 40mL of water and extracted with 200mL of dichloromethane. The aqueous phase was concentrated under reduced pressure. The crude product was purified by flash prep HPLC using column, C18 silica gel, mobile phase, water (0.04% nh4HCO3) and acetonitrile (5% acetonitrile reached 50% in 15 min and 100% for 5 min), detector, UV 254nm. A mixture of 3g (60%) was obtained as a white solid and isolated by SFC. 1.5g of a white solid was obtained 417.MS m/z[M+H]+(ESI):419.00.31P NMR(162MHz,DMSO)δ30.27.1H NMR(400MHz,DMSO-d6)δ11.39(d,J＝2.3Hz,1H),7.60(d,J＝8.0Hz,1H),5.77-5.74(m,1H),5.66(dd,J＝8.1,2.2Hz,1H),5.17(s,1H)4.07-3.89(m,4H),3.89-3.79(m,2H),3.68(dd,J＝7.4,5.1Hz,1H),3.34(s,3H),2.69-2.61(m,2H),2.15-1.97(m,4H),1.20(t,J＝7.1Hz,6H).
1.2G of 417S (for white solid was obtained 100).MS m/z[M+H]+(ESI):419.00.31P NMR(162MHz,DMSO)δ32.801H NMR(400MHz,DMSO-d6)δ11.39(d,J＝2.2Hz,1H),7.57(d,J＝8.1Hz,1H),5.79-5.76(m,1H),5.67(dd,J＝8.0,2.2Hz,1H),5.17(s,1H),4.07-3.90(m,4H),3.90-3.77(m,3H),3.34(s,3H),2.63(q,J＝8.8,7.4Hz,2H),2.26-2.02(m,4H),1.22(td,J＝7.1,0.8Hz,6H).
To a solution of 417 (1.5 g,3.6mmol,1.0 eq.) in 15mL dichloromethane under an inert atmosphere of argon at room temperature was added 2-cyanoethyl N, N' -tetraisopropyl diamino phosphate (1.41 g,4.7mmol,1.3 eq.) and 4, 5-dicyanoimidazole (467.3 mg,3.9mmol,1.1 eq.) in sequence. The reaction mixture was stirred at room temperature for 1 hour. The reaction solution was diluted with 100mL of methylene chloride and washed with 1X 20mL of saturated aqueous sodium bicarbonate solution and 1X 20mL of saturated aqueous sodium chloride solution, respectively. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated until no residual solvent remained under reduced pressure. The residue was purified by flash prep HPLC using column, C18 silica gel, mobile phase, water (0.04% nh4HCO3) and acetonitrile (20% acetonitrile reached 100% in 10 min, 100% acetonitrile held for 5 min). The organic phase layer was separated. The aqueous phase was extracted with 3X 50mL of dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. The solid was filtered off. The filtrate was concentrated at 25 ℃ to give the product, which was dried at 25 ℃ under reduced pressure for 6 hours. 1.38g (64%) of 2-0 are obtained as a white solid. MS M/z [ M-H ]-(ESI):617.15.1 H NMR (300 MHz, chloroform) -d)δ9.24(s,1H),7.40-7.36(m,1H),5.82-5.72(m,2H),4.14-4.03(m,5H),4.01-3.81(m,3H),3.76-3.56(m,3H),3.51(m,3H),2.80-2.59(m,4H),2.33-2.22(m,4H),1.33-1.28(m,6H),1.22-1.17(m,12H).31P NMR(121MHz,CDCl3)δ149.84,149.66,29.89,29.55.
To a solution of 417 (1.2 g,2.9mmol,1.0 eq.) in 20mL dichloromethane under an inert atmosphere of argon at room temperature was added 2-cyanoethyl N, N, N ', N' -tetraisopropyl-diaminophosphate (1.1 g,3.8mmol,1.3 eq.) and 4, 5-dicyanoimidazole (376.4 mg,3.2mmol,1.1 eq.) in sequence. The reaction mixture was stirred at room temperature for 1 hour. The reaction solution was diluted with 20mL of methylene chloride and washed with a saturated aqueous sodium bicarbonate solution and a saturated aqueous sodium chloride solution, respectively. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated until no residual solvent remained under reduced pressure. The residue was purified by flash prep HPLC using column, C18 silica gel, mobile phase, water (0.04% nh4HCO3) and acetonitrile (20% acetonitrile reached 100% in 10 min, 100% acetonitrile held for 5 min). The organic phase layer was separated. The aqueous phase was extracted with 3X 50mL of dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. The solid was filtered off. The filtrate was concentrated at 25 ℃ to give the product, which was dried at 25 ℃ under reduced pressure for 4 hours. 990.9mg (70%) of 2-100 are obtained as a white solid. MS M/z [ M-H ]-(ESI):617.25.1 H NMR (300 MHz, chloroform) -d)δ9.28(s,1H),7.30-7.23(m,1H),5.85(dd,J＝7.7,3.2Hz,1H),5.77(dd,J＝8.1,1.2Hz,1H),4.19-4.05(m,5H),4.02-3.55(m,6H),3.51(d,J＝11.7Hz,3H),2.85-2.71(m,1H),2.69-2.58(m,3H),2.56-2.18(m,4H),1.37-1.27(m,6H),1.25-1.11(m,12H).31PNMR(121MHz,CDCl3)δ149.70,149.69,32.96,32.65.
Examples 2 to 3
To a solution of (3R, 4S, 5R) -5- (hydroxymethyl) tetrahydrofuran-2, 3, 4-triol (600 g,3.997mol,1.0 eq.) in 6L methanol was slowly added sulfuric acid (39.19 g,0.4mol,0.1 eq.) under an inert atmosphere of argon at 0 ℃. The reaction mixture was allowed to warm to room temperature and stirred overnight. The mixture was adjusted to pH 7 with sodium bicarbonate, then filtered and concentrated under reduced pressure to give 21 (520 g, crude) as a yellow oil. MS M/z [ M+H ]+ (ESI): 165.07. The crude product was used in the next step without further purification.
To a solution of 21 (260 g, 1.284 mol,1.0 eq.) in 2.6L pyridine was added 1, 3-dichloro-1, 3-tetraisopropyl disiloxane (549.5 g,1.742mol,1.1 eq.) under an inert atmosphere of argon at 0 ℃. The mixture was stirred at room temperature for 1 hour. The resulting solution was extracted with 3×4L ethyl acetate, and the organic layers were combined. The organic layer was washed with 2X 500mL of water and 2X 4L of saturated sodium chloride, respectively. The mixture was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was dissolved in 100mL ethyl acetate and evaporated to dryness. The residue was applied to a silica gel column with ethyl acetate/petroleum ether (1:5-1:3). 195g (33%) of 22 were obtained as a yellow oil. MS M/z [ M+H ]+ (ESI): 407.23.
Sodium hydride (23.1 g,0.959mol,2.0 eq.) was added to a solution of 22 (195 g,0.48mol,1.0 eq.) in a mixture of 2L 1, 3-dimethyl-2-imidazolidinone and 1L methyl iodide under an inert atmosphere of argon. The resulting solution was stirred at room temperature for 40 minutes. The resulting solution was diluted with dichloromethane and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (silica gel, ethyl acetate/petroleum ether=1:5 to 1:1) to give 23 (390 g, crude) as a yellow oil. MS M/z [ M+H ]+ (ESI): 421.25.H NMR (400 MHz, chloroform -d)δ4.77(s,1H),4.50(m,1H),4.01(m,2H),3.94-3.84(m,1H),3.61(d,J＝4.3Hz,1H),3.59(s,3H),3.34(s,3H),1.15-1.02(m,28H).. This crude product was used in the next step without further purification.
To a solution of hydrogen trifluoride (3 kg,18.541mol,10.0 eq.) in 5L anhydrous tetrahydrofuran was added triethylamine (3.7 kg,37.082mol,20.0 eq.) under an inert atmosphere of argon at room temperature. The resulting solution was stirred at room temperature for 10 minutes. A solution of 23 (780 g, 1.850 mol,1.0 eq.) in 1L anhydrous tetrahydrofuran was added to the resulting solution. The reaction mixture was stirred at room temperature overnight and diluted with 3L of dichloromethane, washed with 2×1L of saturated aqueous sodium bicarbonate and2×1L of saturated aqueous sodium chloride, respectively. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (silica gel, ethyl acetate/petroleum ether=1:5 to 3:1) to give 24 (280 g) as a yellow oil. MS M/z [ M+H ]+ (ESI): 179.11.
To a solution of crude 24 (140 g,0.786mol,1.0 eq.) in 1.4L pyridine was added 4,4' - (chloro (phenyl) methylene) bis (methoxybenzene) (292.8 g,0.865mol,1.1 eq.) under an inert atmosphere of argon at 0 ℃. The mixture solution was stirred at room temperature for 12 hours. The resulting solution was diluted with 3000mL of dichloromethane and washed with 2 x 1000mL of water. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was applied to a silica gel column with ethyl acetate/petroleum ether (1:50-1:1). 230g (61%) of 25 are obtained as a yellow oil. MS M/z [ M+H ]+ (ESI): 481.23.
To a solution of 25 (460 g,0.957mol,1.00 eq.) in 4.6L of N, N-dimethylformamide were added tert-butylchlorodiphenylsilane (289.4 g,1.053mol,1.1 eq.) and imidazole (162.9 g,2.393mol,2.5 eq.) under an inert atmosphere of nitrogen at room temperature. The resulting solution was stirred at room temperature for 12 hours. The organic layer was washed with water and extracted with 3×4L ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was applied to a silica gel column with ethyl acetate/petroleum ether (1:20-1:5). 520g (72%) 26 as yellow oil. MS M/z [ M+H ]+ (ESI): 719.35.
Triethylsilane (500 mL) was added to a solution of 26 (520 g,166.910mmol,1.0 eq.) in 5.2L dichloromethane at room temperature. Trifluoroacetic acid (500 mL) was then added at 0 ℃ and the resulting solution was stirred at room temperature for 2 hours. The reaction was quenched by the addition of 5L saturated sodium bicarbonate and extracted with 3×2L dichloromethane. The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was applied to a silica gel column with ethyl acetate/petroleum ether (1:10-5:1). The fractions of the desired product were concentrated under reduced pressure. 260g (82%) of a yellow oil were obtained 27.MS m/z[M+NH4]+(ESI):434.35.H NMR(400MHz,DMSO-d6)δ7.81-7.34(m,10H),4.79(d,J＝1.5Hz,1H),4.65(s,1H),4.16(m,1H),3.91(m,1H),3.44(dd,J＝11.6,3.6Hz,1H),3.22(m,1H),3.19(s,3H),3.11(s,3H),3.03(m,1H),1.03(s,9H).
To a solution of oxalyl chloride (47.3 g,372.6mmol,2.0 eq.) in 1.3L dichloromethane was added dimethyl sulfoxide (43.7 g,558.9mmol,3.0 eq.) under an inert atmosphere of nitrogen at-78 ℃. The resulting solution was stirred at-78 ℃ for 0.5 hours, then a solution of 27 (75 g,186.3mmol,1.0 eq.) in 750mL dichloromethane was added dropwise with stirring at-78 ℃. The resulting solution was stirred at-78 ℃ for 3 hours. Triethylamine (84.8 g,838.4mmol,4.5 eq.) was then added dropwise with stirring at-78 ℃ over 0.5 hours. The resulting solution was diluted with 2000mL of dichloromethane. The resulting mixture was washed with 1X 1L of saturated aqueous sodium bicarbonate solution and 1X 1L of saturated aqueous sodium chloride solution, respectively. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The above procedure was repeated 2 times to obtain 120g (crude) 28 as a yellow oil. The crude product was used in the next step without further purification. MS M/z [ M+H ]+ (ESI): 415.21.
To a solution of 28 (120 g,289.5mmol,1.0 eq.) in 1.2L tetrahydrofuran was added methyl magnesium bromide (103.549 g,868.4mmol,3.0 eq.) under an inert atmosphere of nitrogen at-78 ℃. The resulting solution was stirred at-78 ℃ for 2 hours. The resulting solution was diluted with 3L of dichloromethane and washed with 2X 1000mL of water. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was applied to a silica gel column with ethyl acetate/petroleum ether (1:10-5:1). The fractions of the desired product were concentrated under reduced pressure. 86g (62%) of 3-1 were obtained as a yellow oil. MS M/z [ M+H ]+ (ESI): 431.25.
To a solution of oxalyl chloride (50.7 g,399.4mmol,2.0 eq.) in 860mL of dichloromethane at-78℃under an inert atmosphere of nitrogen was added dimethyl sulfoxide (46.8 g,599.12mmol,3.0 eq.). The resulting solution was stirred at-78 ℃ for 0.5 hours. And 3-1 (86 g,199.7mmol,1.00 eq.) was added. The resulting solution was stirred at-78 ℃ for 3 hours. Then, triethylamine (90.9 g,898.7mmol,4.5 eq.) was added dropwise with stirring at-78 ℃. The resulting solution was stirred at-78 ℃ for 0.5 hours. The resulting solution was diluted with 1L of dichloromethane and washed with 3X 600mL of water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was applied to a silica gel column with ethyl acetate/petroleum ether (1:50-3:1). The fractions of the desired product were concentrated under reduced pressure. 72g (80%) of a yellow oil were obtained 3-2.MS m/z[M+H]+(ESI):429.22.H NMR(300MHz,DMSO-d6)δ7.71-7.30(m,10H),4.94(d,J＝1.2Hz,1H),4.36(m,1H),4.27(d,J＝6.8Hz,1H),3.12(s,3H),3.01(m,1H),1.99(d,J＝4.9Hz,3H),1.01(s,9H).
3-2 (72 G,168.0mmol,1.0 eq.) was added to 720mL of a solution of N, N-dimethylformamide dimethyl acetal under an inert atmosphere of nitrogen. The resulting solution was stirred at 110 ℃ for 17 hours, then diluted with 1L dichloromethane and washed with 2 x 600mL water. The resulting mixture was concentrated under reduced pressure. The residue was applied to a silica gel column with ethyl acetate/petroleum ether (1:50-5:1). The fractions of the desired product were concentrated under reduced pressure. 60g (70%) of 4-1 are obtained as a yellow oil. MS M/z [ M+H ]+ (ESI): 484.26.
To a solution of 4-1 (30 g,62.026mmol,1.0 eq.) in 100mL acetic acid and 200mL 1.4-dioxane at room temperature under an inert atmosphere of nitrogen was added diethyl phosphonate (17.1 g,124.1mmol,2.0 eq.) manganese triacetate dihydrate (49.9 g,186.1mmol,3.0 eq.). The resulting solution was stirred at 80 ℃ for 20 minutes. The resulting solution was extracted with 3X 500mL ethyl acetate and the organic layers were combined. The organic layer was washed with 2X 200mL of water and 2X 200mL of saturated sodium chloride, respectively. The mixture was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The above procedure was repeated 2 times. The crude product was purified by flash prep HPLC using conditions (INTELFLASH-1) of column, C18 silica gel, mobile phase, water and acetonitrile (30% acetonitrile reached 100% in 10 min and 100% for 5 min), detector, UV 254nm. 38g (52%) of 4-52 are obtained as a yellow oil. MS M/z [ M+H ]+ (ESI): 565.25.
To a solution of 4-52 (38 g,67.3mmol,1.0 eq.) in 380mL 1, 1-dimethoxy-N, N-dimethylamine. The resulting solution was stirred at 80 ℃ for 12 hours. The resulting solution was diluted with 500mL of dichloromethane and washed with 2×300mL of water. The crude product was purified by flash prep HPLC using conditions (INTELFLASH-1) of column, C18 silica gel, mobile phase, water (0.04% nh4HCO3) and acetonitrile (15% acetonitrile 100% in 25 min and 100% for 5 min), detector, UV 254nm. 27g (62%) of 4-53 are obtained as a yellow solid. MS M/z [ M+H ]+ (ESI): 620.30.
Hydroxylamine hydrochloride (9.1 g,130.7mmol,3.0 eq.) and pyridine (34.5 g,435.6mmol,10.0 eq.) were added to a solution of 4-53 (27 g,43.56mmol,1.0 eq.) in 270mL ethanol at room temperature under an inert atmosphere of nitrogen. The final reaction mixture was stirred at 60 ℃ for 1 hour. The resulting solution was diluted with 400mL of dichloromethane and washed with 2X 200mL of water. The crude product was purified by flash prep HPLC using conditions (INTELFLASH-1) of column, C18 silica gel, mobile phase, water and acetonitrile (30% acetonitrile reached 100% in 10 min and 100% for 5 min), detector, UV 254nm. 16g (59.2%) of 4-4 were obtained as a yellow oil. MS M/z [ M-H ]- (ESI): 588.25.
To a solution of 4-4 (16 g, 42.3995 mmol,1.0 eq.) in 160mL acetic acid and 80mL acetic anhydride at 0deg.C with stirring was added sulfuric acid (133.1 mg, 1.356 mmol,0.05 eq.). The resulting solution was stirred at room temperature for 1 hour, then diluted with 300mL of dichloromethane and washed with 2×100mL of water. The crude product was purified by flash prep HPLC using conditions (INTELFLASH-1) of column, C18 silica gel, mobile phase, water (0.04% nh4HCO3) and acetonitrile (15% acetonitrile 100% in 25 min and 100% for 5 min), detector, UV 254nm. 12g (68%) of 4-24 as a yellow oil was obtained. MS M/z [ M+H ]+ (ESI): 618.24.
To a solution of uracil (0.544 g,4.86mmol,1.5 eq.) in 80mL of acetonitrile was added N, O-bis (trimethylsilyl) acetamide (2.305 g,11.3mmol,3.5 eq.) under an inert atmosphere of nitrogen at room temperature. The resulting solution was stirred at 60 ℃ for 0.5 hours. The reaction mixture was cooled to 0 ℃ and 4-4 (2 g,3.24mmol,1.0 eq.) was added. Then, trimethylsilyl triflate (1.44 g,6.48mmol,2.0 eq.) was added dropwise with stirring at 0 ℃. The resulting solution was allowed to react at 60 ℃ for 1 hour with stirring. The resulting solution was diluted with dichloromethane and washed with water. The procedure described above was repeated 6 times. The crude product was purified by flash prep HPLC using conditions (INTELFLASH-1) of column, C18 silica gel, mobile phase, water (0.04% nh4HCO3) and acetonitrile (30% acetonitrile 100% in 10min and 100% for 5 min), detector, UV 254nm. 3.28g (24%) of 4-5 are obtained as an off-white solid. MS M/z [ M+H ]+ (ESI): 670.25.
To a solution of hydrogen trifluoride (7.9 g,49.0mmol,10.0 eq.) in 20mL of anhydrous tetrahydrofuran was added triethylamine (9.9 g,97.9mmol,20.0 eq.) under an inert atmosphere of argon at room temperature. The resulting solution was stirred at room temperature for 10 minutes. A solution of 4-5 (3.28 g,4.9mmol,1.0 eq.) in 12mL anhydrous tetrahydrofuran was added to the resulting solution. The reaction mixture was stirred at room temperature overnight and diluted with 200mL of dichloromethane, washed with 2×200mL of saturated aqueous sodium bicarbonate and 2×200mL of saturated aqueous sodium chloride, respectively. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by flash prep HPLC using conditions (INTELFLASH-1) of column, C18 silica gel, mobile phase, water (0.04% nh4HCO3) and acetonitrile (30% acetonitrile 100% in 10 min and 100% for 5 min), detector, UV 254nm. 1.3g (62%) of a yellow oil is obtained 4-6.MS m/z[M+H]+(ESI):432.15.H NMR(400MHz,DMSO-d6)δ11.50-11.29(m,1H),8.89(s,1H),7.76(d,J＝8.1Hz,1H),6.45(d,J＝5.0Hz,1H),6.05(d,J＝6.0Hz,1H),5.66(m,1H),5.52(d,J＝6.1Hz,1H),4.68(m,1H),4.16(t,J＝4.8Hz,1H),4.07(m,3.7Hz,4H),3.36(s,3H),1.24(m,6H).
To a solution of 4-6 (1 g,2.318mmol,1.0 eq.) in 10mL of dichloromethane was added 2-cyanoethyl N, N, N ', N' -tetraisopropyl-diaminophosphate (838.6 mg,2.782mmol,1.2 eq.) and 4, 5-dicyanoimidazole (273.8 mg,2.318mmol,1.0 eq.) at room temperature. The resulting solution was stirred at room temperature for 1 hour. The reaction solution was diluted with 300mL of methylene chloride and washed with 2X 100mL of saturated aqueous sodium hydrogencarbonate solution and 1X 100mL of saturated aqueous sodium chloride solution, respectively. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated until no residual solvent remained under reduced pressure. The crude product was purified by flash prep HPLC using conditions (INTELFLASH-1) of column, C18 silica gel, mobile phase, water (0.04% nh4HCO3) and acetonitrile (30% acetonitrile 100% in 15min and 100% for 5 min), detector, UV 254nm. The fractions were diluted with an equal volume of dichloromethane. The organic phase layer was separated. The aqueous phase was extracted with 3X 50mL of dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. The solid was filtered off. The filtrate was concentrated at 25 ℃. 840.5mg (57%) of a white solid was obtained 4-0.MS m/z[M-H]-(ESI):630.20.H NMR(300MHz,DMSO-d6)δ11.43(s,1H),8.90(s,1H),7.65(m,1H),6.48(d,J＝4.4Hz,1H),5.80-5.56(m,2H),4.95(m,1H),4.24(m,1H),4.16-3.95(m,4H),3.82-3.63(m,2H),3.62-3.40(m,2H),3.37(d,J＝3.5Hz,3H),2.76(m,2H),1.32-1.16(m,7H),1.09(t,J＝6.8Hz,8H),0.96(d,J＝6.8Hz,3H).P NMR(121MHz,DMSO)δ150.00,149.96,7.37,7.35.
Examples 2 to 4
To a solution of L-ascorbic acid (125 g,0.71mol,1.0 eq.) in 1L water was slowly added calcium carbonate (125 g,1.25mol,1.76 eq.) over 30 minutes under an inert atmosphere of argon. Hydrogen peroxide (250 ml,30% aqueous solution) was added dropwise to the resulting solution at 0 ℃ over 1 hour with stirring. The reaction mixture was allowed to warm to room temperature and stirred overnight. The reaction mixture was then filtered and the filter cake was washed with 2x 100mL of water. The filtrate was treated with activated carbon (25 g) and then heated to 70 ℃. The hot suspension was filtered and the solid material was washed with 2x 50mL of water. The filtrates were combined, crystallized by adding 2 volume equivalents of methanol while stirring at 4 ℃ for 16 hours. The solid material was filtered, washed with 2x 50mL methanol and dried under high vacuum at 40 ℃. 88.1g (75%) 1 were obtained as a white solid. No MS signal.1H NMR(400MHz,D2 O): delta 3.98 (d, j=2.2 hz, 1H), 3.88-3.72 (m, 1H), 3.62-3.49 (m, 2H).
To a solution of 1 (100 g,0.30mol,1.0 eq.) in 500mL of anhydrous acetonitrile at room temperature under an inert atmosphere of argon was added sequentially anhydrous oxalic acid (28.8 g,0.32mol,1.0 eq.) and p-toluenesulfonic acid monohydrate (1.0 g,0.01 eq.). The mixture was stirred under reflux for 3 hours. The hot mixture was cooled to room temperature and filtered. The filter cake was washed with 50mL of acetonitrile and the combined filtrates were evaporated under reduced pressure. The residue was dissolved in 100mL ethyl acetate and evaporated to dryness. 50.1g (70%) 2 are obtained as a white solid. MS M/z [ M+H ]+ (ESI): 119. It was used in the next step without further purification.
To a solution of 2 (66.5 g,0.56mol,1.0 eq.) in 1200mL dichloromethane and 135mL anhydrous pyridine at 0 ℃ under an inert atmosphere of argon was added benzoyl chloride (72.0 mL,0.62mol,1.1 eq.) dropwise. The resulting solution was stirred at 0 ℃ for 30 minutes. The reaction mixture was quenched with 1N HCl and washed with 3X 200mL saturated aqueous sodium chloride. To the organic layer was added 2 volume equivalents of hexane over 1 hour and stirred at 0 ℃ for 16 hours. The desired product was collected by filtration and dried under vacuum. Obtain a white solid 75.1g(60％)3.MS m/z[M+H]+(ESI):223.1H NMR(400MHz,DMSO-d6):δ8.08-7.98(m,2H),7.78-7.67(m,1H),7.59-7.51(m,2H),6.12(d,J＝17.9Hz,1H),5.73(d,J＝7.9Hz,1H),4.74-4.71(m,1H),4.55-4.44(m,1H),4.09-4.03(m,1H).
To a solution of 3 (16.5 g,74.3mmol,1.0 eq.) in 160mL of dichloromethane at 0deg.C under an inert atmosphere of argon was added, in sequence, 4-dimethylaminopyridine (60 mg,6.0mmol,0.01 eq.), imidazole (10.2 g,150mmol,2.0 eq.) and tert-butyldiphenylchlorosilane (21.4 g,75.0mmol,1.05 eq.). The reaction mixture was warmed to room temperature and stirred overnight. The resulting solution was evaporated under reduced pressure. The residue was dissolved in 300mL of hexane and washed with 1X 100mL of 1N HCl, 3X 100mL of water and 3X 100mL of saturated aqueous sodium chloride solution, respectively. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. It was used in the next step without further purification. MS M/z [ M+H ]+ (ESI): 461.
To a solution of crude 4 (35.0 g,76mmol,1.0 eq.) in 150mL of 1, 2-dimethoxyethane was added dropwise diisobutylaluminum hydride (100 mL,100mmol,1M in toluene) at-78℃under an inert atmosphere of argon over 10 minutes. The mixture solution was stirred at-78 ℃ for 30 minutes. The reaction was monitored by TLC and used in the next step without further purification.
To the solution of 5 was added dropwise a preformed solution containing acetic anhydride (35 mL,367mmol,5.0 eq.) and 4-dimethylaminopyridine (14.0 g,115mmol,1.5 eq.) in 40mL dichloromethane at-78℃under an inert atmosphere of argon. After stirring for 10 minutes, the reaction mixture was warmed to room temperature and stirred for 2 hours. The mixture solution was diluted with 200mL of hexane and poured into 200mL of cold 1NHCl aqueous solution. The organic layer was washed with 3X 100mL of water, 3X 100mL of saturated aqueous sodium bicarbonate solution, and 3X 100mL of saturated aqueous sodium chloride solution, respectively. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by flash prep HPLC using column, C18 silica gel, mobile phase, water (0.1% fa) and acetonitrile (20% acetonitrile 100% in 10min, 100% acetonitrile 8 min), UV 254nm. The fractions were diluted with dichloromethane and dried over anhydrous sodium sulfate. The solid was filtered off and the filtrate was concentrated under reduced pressure. 21.1g (56%) of a colorless oil was obtained in three steps 6.MS m/z[M+H]+(ESI):505.1H NMR(300MHz,DMSO-d6):δ7.89-7.82(m,2H),7.71-7.62(m,5H),7.57-7.28(m,8H),6.44-6.09(m,1H),5.48-5.25(m,1H),4.76-4.59(m,1H),4.19-3.83(m,2H),2.14(s,2H),1.86(s,1H),1.04(d,J＝15.2Hz,9H).
Uracil (3.3 g,29.7mmol,1.5 eq.) and N, O-bis (trimethylsilyl) acetamide (8.1 g,39.6mmol,2.0 eq.) were added sequentially to a solution of 6 (10.0 g,59.4mmol,1.0 eq.) in 50mL of anhydrous acetonitrile at room temperature under an inert atmosphere of argon. The mixture was stirred at 60 ℃ for 30 minutes. Trimethylsilyl triflate (7.0 g,31.7mmol,1.5 eq.) was then added dropwise and stirred at 60 ℃ for an additional 2 hours. The reaction mixture was cooled to room temperature, diluted with 200mL of ethyl acetate and poured into 100mL of cold saturated aqueous sodium bicarbonate solution. The organic layer was washed with 3X 100mL of water and 3X 100mL of saturated aqueous sodium chloride solution, respectively. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was used directly in the next step without further purification. MS M/z [ M+H ]+ (ESI): 557.
To a solution of hydrogen trifluoride (28.9 g,17.9mmol,10.0 eq.) in 50mL of anhydrous tetrahydrofuran was added triethylamine (36.4 g,35.9mmol,20.0 eq.) under an inert atmosphere of argon at room temperature. The resulting solution was stirred at room temperature for 10 minutes. A solution of 7 (10.0 g,53.9mmol,1.0 eq.) in 50mL dry tetrahydrofuran was added to the resulting solution. The reaction mixture was stirred at room temperature overnight and diluted with 300mL of dichloromethane, washed with 2×100mL of saturated aqueous sodium bicarbonate and 2×100mL of saturated aqueous sodium chloride, respectively. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by flash prep HPLC using column, C18 silica gel, mobile phase, water (0.1% fa) and acetonitrile (10% acetonitrile reached 100% in 12 min), UV 254nm. The fractions were diluted with dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure. 5.0g (80%) of a pale yellow solid was obtained, in two steps 8.MS m/z[M+H]+(ESI):319.1H NMR(400MHz,DMSO-d6):δ11.35(d,J＝2.2Hz,1H),8.05-7.94(m,2H),7.76(d,J＝8.2Hz,1H),7.73-7.65(m,1H),7.54-7.32(m,2H),5.92-5.75(m,2H),5.67-5.46(m,1H),5.26(d,J＝1.8Hz,1H),4.36-4.25(m,1H),4.14-3.89(m,2H).
To a solution of 8 (10.0 g,31.4mmol,1.0 eq.) in 80mL of anhydrous dichloromethane was added 2,4, 6-collidine (22.8 g,188.1mmol,6.0 eq.) and 4,4' -dimethoxytrityl chloride (26.6 g,78.7mmol,2.5 eq.) in sequence at room temperature under an inert atmosphere of argon. The resulting solution was stirred at room temperature for 24 hours. The reaction mixture was quenched with 10mL of methanol, diluted with 500mL of dichloromethane, and washed with 2×200mL of saturated aqueous sodium bicarbonate and 2×200mL of saturated aqueous sodium chloride, respectively. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was dissolved in 400mL of ammonia (7M NH3 in methanol) and stirred under an inert atmosphere of argon at room temperature for 24 hours. The reaction mixture was concentrated under reduced pressure. The residue was purified by flash prep HPLC using column, C18 silica gel, mobile phase, water (0.04% nh4HCO3) and acetonitrile (15% acetonitrile 100% acetonitrile in 10 min, 100% acetonitrile 6 min) with UV 254nm. The fractions were diluted with dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure. 12.1g (75%) of 81 were obtained as a pale yellow oil. MS M/z [ M-H ]- (ESI): 515.
To a solution of 81 (10.0 g,19.4mmol,1.0 eq.) in 100mL of anhydrous N, N-dimethylformamide was added imidazole (3.9 g,57.3mmol,3.0 eq.) followed by tert-butyldimethylsilyl chloride (8.8 g,58.1mmol,3.0 eq.) under an inert atmosphere of argon at room temperature. The resulting solution was stirred at room temperature for 12 hours. The reaction mixture was diluted with 500mL of methylene chloride and washed with 2X 200mL of saturated aqueous sodium bicarbonate solution and 1X 200mL of saturated aqueous sodium chloride solution, respectively. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was dissolved in 120mL of dichloromethane, trifluoroacetic acid (21.7 g,190.5mmol,10.0 eq.) and triethylsilane (11.1 g,95.2mmol,5.0 eq.) were added sequentially at room temperature and stirred under an inert atmosphere of argon at room temperature for 1 hour. The reaction mixture was diluted with 400mL of methylene chloride and washed with 2X 150mL of saturated aqueous sodium bicarbonate and 1X 150mL of saturated aqueous sodium chloride, respectively. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash prep HPLC using column, C18 silica gel, mobile phase, water (0.1% fa) and acetonitrile (15% acetonitrile 100% in 10 min, 100% acetonitrile 6 min), UV 254nm. The fractions were diluted with dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure. 4.7g (75%) of a pale yellow solid was obtained 82.MS m/z[M-H]-(ESI):515.1H NMR(300MHz,DMSO-d6):δ11.29(s,1H),7.62(d,J＝8.2Hz,1H),5.58-5.55(m,2H),5.43(d,J＝2.6Hz,1H),4.17-3.94(m,4H),0.86(s,9H),0.10(d,J＝4.0Hz,6H).
To a solution of 82 (4.0 g,11.9mmol,1.0 eq.) in 40mL dry tetrahydrofuran was added 4-methylbenzenesulfonic acid (diethoxyphosphoryl) methyl ester (7.7 g,23.9mmol,2.0 eq.) under an inert atmosphere of argon at room temperature. Sodium hydride (1.9 g,47.7mmol,4.0 eq.) was added to the reaction mixture at 0 ℃ and stirred under an inert atmosphere of argon at the same temperature for 3 hours. The reaction was quenched with 6mL of acetic acid (1M) and diluted with 150mL of ethyl acetate, washed with 1X 50mL of H2 O and 1X 50mL of saturated aqueous sodium chloride, respectively. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was applied to a silica gel column with ethyl acetate/petroleum ether (1:10-5:1). The fractions of the desired product were concentrated under reduced pressure. 2.5g (60%) of a pale yellow oil are obtained 53.MS m/z[M+H]+(ESI):479.1H NMR(400MHz,DMSO-d6):δ11.32(s,1H),7.53(d,J＝8.1Hz,1H),5.60(d,J＝1.1Hz,1H),5.51(d,J＝8.1Hz,1H),4.35-4.28(m,2H),4.07-3.84(m,8H),1.24-1.20(m,6H),0.87(s,9H),0.13(d,J＝2.5Hz,6H).31P NMR(162MHz,DMSO):δ20.74.
A solution of 53 (3.2 g,6.7mmol,1.0 eq.) in 32mL formic acid and H2 O (v/v=1:1) was stirred at room temperature under an inert atmosphere of argon for 24 hours. The resulting solution was concentrated under vacuum. The crude product was diluted with 300mL ethyl acetate and washed with 2X 50mL water and 1X 50mL saturated aqueous sodium bicarbonate, respectively. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash prep HPLC using column, C18 silica gel, mobile phase, water and acetonitrile (15% acetonitrile reaching 35% in 20 min), UV 254nm. The fractions were diluted with dichloromethane, dried over anhydrous sodium sulfate and concentrated under reduced pressure. 1.9g (80%) of a pale yellow oil is obtained 14.MS m/z[M+H]+(ESI):365.1H NMR(300MHz,DMSO-d6):δ11.31(s,1H),7.55(d,J＝8.1Hz,1H),5.94(d,J＝4.1Hz,1H),5.68(d,J＝1.4Hz,1H),5.53(d,J＝8.1Hz,1H),4.28(d,J＝10.2Hz,1H),4.17(s,1H),4.09-3.97(m,6H),3.95-3.84(m,2H),1.23-1.09(m,6H).31P NMR(121MHz,DMSO):δ20.84.
To a solution of 14 (2.2 g,6.0mmol,1.0 eq.) in 20mL of dichloromethane under an inert atmosphere of argon at room temperature were added 2-cyanoethyl N, N, N ', N' -tetraisopropyl-diaminophosphate (2.4 g,7.8mmol,1.3 eq.) and 4, 5-dicyanoimidazole (785 mg,6.6mmol,1.1 eq.) in sequence. The reaction mixture was stirred at room temperature for 1 hour. The reaction solution was diluted with 200mL of methylene chloride and washed with 2X 100mL of saturated aqueous sodium bicarbonate solution and 1X 100mL of saturated aqueous sodium chloride solution, respectively. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated until no residual solvent remained under reduced pressure. The residue was purified by flash prep HPLC using column, C18 silica gel, mobile phase, water (0.04% nh4HCO3) and acetonitrile (20% acetonitrile reached 100% in 10 min, 100% acetonitrile held for 5 min). The fractions were diluted with 500mL dichloromethane. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. 2.2g (70%) of 100 are obtained as a pale yellow oil. MS M/z [ M+H ]+(ESI):565.1 H NMR (300 MHz, acetonitrile -d3):δ9.05(s,1H),7.57-7.54(m,1H),5.88-5.83(m,1H),5.57-5.47(m,1H),4.55-4.41(m,1H),4.39-4.29(m,1H),4.23-4.00(m,6H),3.95-3.76(m,4H),3.73-3.57(m,2H),2.75-2.65(m,2H),1.31-1.27(m,6H),1.22-1.19(m,12H).31P NMR(121MHz,CD3CN):δ151.05,150.14,19.82,19.71.
Examples 2 to 5
To a solution of 1 (120.0 g,461.5 mmol) and KOH (31.0 g,553.6 mmol) in DMF (500.0 mL) was added BnBr (118.0 g,690.1 mmol) at 0℃over 30 min, which was then stirred at room temperature overnight. LC-MS and TLC monitoring 1 were completely consumed. The reaction was dissolved in water and the pH was adjusted to ph=9 by the gradual addition of solid NaHCO3, which was then extracted by EA (700.0 ml×3). The organic layer was washed with water (1.0 l×2) and anhydrous NaCl, dried over anhydrous Na2SO4 and evaporated in vacuo to give crude product 2 (150.0 g, crude) as a white oil. ESI-MS: m/z 334.1.
The crude product 2 (150.0 g, crude) was dissolved in 70% acoh in H2 O (700.0 mL) at room temperature, then stirred overnight at room temperature. TLC showed that 2 was completely consumed. The reaction was quenched with solid aqueous NaHCO3 and extracted by EA (700.0 ml×3). The organic layer was washed with water (1.0 l×2) and aqueous NaCl, dried over anhydrous Na2SO4 and evaporated in vacuo to give the crude product, which was purified by column chromatography (SiO2, PE/ea=2:1 to 0:1) to give 3 (110.0 g,90.5% purity, 76.8% yield) as a pale yellow solid ).ESI-MS:m/z 333.1;1HNMR(400MHz,CDCl3):δ7.36(m,5H),5.76(d,J＝3.72Hz,1H),4.78(d,J＝11.28Hz,1H),4.60(t,J＝4.04Hz,1H),4.56(d,J＝11.28Hz,1H),4.11(m,1H),4.00(m,1H),3.93(m,1H),3.67(m,2H),2.55(s,2H),1.59(s,3H),1.36(s,3H).
To a stirred solution of 3 (110.g, 350.0 mmol) in DCM (1.2L) was added a solution of NaIO4 (153.0 g,710 mmol) in H2 O (600.0 mL) at room temperature, which was then stirred at room temperature for 1.0H. TLC monitored that the work was complete. The reaction was dissolved by water and extracted with DCM (700.0 ml×3). The organic layer was washed with water (1.0 l×2) and aqueous NaCl, dried over anhydrous Na2SO4 and concentrated under reduced pressure to give 4 (92.0 g, crude ).ESI-MS:m/z 262.1;1HNMR(400MHz,CDCl3):δ9.60(d,J＝1.76Hz,1H),7.35(m,5H),5.81(d,J＝3.44Hz,1H),4.74(d,J＝12.12Hz,1H),4.60(m,2H),4.48(dd,J＝1.68Hz and 9.24hz, 1H), 3.85 (q, j=4.28 hz 1H), 1.60 (s, 3H), 1.37 (s, 3H) as a pale yellow oil.
To a solution of 4 (85.0 g, crude) in THF (300.0 mL) at 0 ℃ was added dropwise a solution of MeLi in diethoxymethane (1.6 m,287.0 mL) over 0.5 hours, and then the reaction was stirred at 0 ℃ for 2.5 hours. TLC showed 4 was completely consumed. The reaction was quenched by aqueous NH4 Cl and extracted by EA (400.0 ml×3). The organic layer was washed with water (1.0 L×2) and aqueous NaCl, dried over anhydrous Na2SO4 and evaporated in vacuo to give 5 (86.6 g, crude) as a pale yellow oil ).ESI-MS:m/z 278.1;1HNMR(400MHz,CDCl3):δ7.36(m,5H),5.73(d,J＝3.6Hz,1H),4.76(m,1H),4.58(m,2H),4.02(m,1H),3.91(m,1H),3.81(m,1H),1.59(s,3H),1.36(m,3H),1.25(dd,J＝6.6Hz,19.2Hz,3H).
A mixture of 5 (86.6 g, crude) and 2-iodoxybenzoic acid (165.0 g,589.3 mmol) in ACN (1.0L) was stirred at 80℃for 2 hours, then TLC showed complete consumption of 5. It was then filtered and the organic layer was concentrated to give the crude product which was purified by column chromatography (SiO2, PE/ea=10:1 to 8:1) to give 6 (48.6 g,90% purity, 56.5% yield) as a yellow solid ).ESI-MS:m/z 276.1;1HNMR(400MHz,CDCl3)δ7.36(m,5H),5.82(d,J＝3.56Hz,1H),4.77(d,J＝11.96Hz,1H),4.60(m,2H),4.52(d,J＝9.16Hz,1H),3.77(m,1H),2.19(s,3H),1.60(s,3H),1.37(s,3H).
To a stirred solution of diethyl ethylphosphonate (20.7 g,124.7 mmol) in anhydrous THF (600.0 mL) at-70℃was added n-BuLi (1.6M, 86.0 mL) dropwise and stirred at-70℃for 0.5 h. At-70 ℃,6 (40.0 g,137.0 mmol) in THF (300.0 mL) was added over 20 minutes and stirred at-70 ℃ for 2 hours. LCMS showed 6 was completely consumed. It was quenched by aqueous NH4 Cl and extracted by EA (600 ml×3). The organic layer was washed with water (600.0 ml×2) and aqueous NaCl, dried over anhydrous Na2SO4 and filtered, and then the organic layer was concentrated. The crude product was purified by column chromatography (SiO2, PE/ea=1:1) to give 7 (33.6 g,73.3mmol,93% purity) as a yellow solid ).ESI-MS:m/z 459.2[M+H]+;1HNMR(400MHz,CDCl3)δ7.34(m,5H),5.77(m,1H),4.83-4.71(m,1H),4.64-4.52(m,2H),4.23-3.86(m,7H),1.75(m,1H),1.58(t,J＝7.6Hz,3H),1.30(m,15H).31P-NMR(162MHz,CDCl3)δ＝33.74,32.93,32.19,31.53.
To a solution of 7 (33.6 g,73.4 mmol) in pyridine (200.0 mL) was added SOCl2 (86.5 g,73.3 mmol) over 30 min at ice bath, which was stirred at room temperature for 30 min. LCMS showed 7 was completely consumed. The mixture was quenched by aqueous NaHCO3 (200.0 mL), extracted by EA (400.0 ml×2) and washed by aqueous NaCl. After drying over anhydrous Na2SO4 and filtration, the organic layer was concentrated to give the crude product, which was purified by column chromatography (SiO2, PE/ea=1:1) to give a pale yellow solid 8(25.0g,56.8mmol).ESI-MS:m/z 441.2[M+H]+;1HNMR(400MHz,CDCl3)δ7.34(m,5H),5.78(t,J＝3.32Hz,1H),5.43(m,1H),5.31(m,J＝5.36Hz 1H),4.75(m,1H),4.56(m,3H),4.07(m,4H),3.69(m,1H),2.81(m,1H),1.60(s,3H),1.30(m,12H);31P-NMR(162MHz,CDCl3)δ＝30.33,29.97.
Pd/C (3.8 g, 15%) was added to a solution of 8 (25.5 g,56.0 mmol) in CH3 OH (200.0 mL) at room temperature, which was stirred under an atmosphere of N2 at room temperature for 30 minutes, then filtered. Another portion of Pd/C (3.8 g, 15%) was added and stirred at 50℃for 3 hours. LCMS showed 8 was completely consumed. After filtration, the organic layer was concentrated to give a crude product, which was purified by column chromatography to give 9 (15.0 g,34.0mmol,58.6% yield) as a pale yellow oil ).ESI-MS:m/z 443.5[M+H]+;1HNMR(400MHz,CDCl3)δ7.35(m,5H),5.69(m,1H),4.81-4.73(m,1H),4.60-4.49(m,2H),4.14-4.01(m,5H),3.56-3.48(m,1H),2.47-2.10(m,2H),1.58(m,3H),1.37-1.23(m,15H).31P-NMR(162MHz,CDCl3)δ＝34.76,34.17,33.95,33.55.
A solution of 9 (15.0 g,34.0 mmol) in 60% AcOH/H2 O (75.0 mL) was stirred at 100℃for 3 hours. TLC showed that 9 was completely consumed and then concentrated in vacuo to give the crude intermediate. The crude intermediate and DMAP (828.0 mg,6.8 mmol) were dissolved in anhydrous pyridine (60.0 mL) and then Ac2 O (20.8 g,203.9 mmol) was added at 0deg.C over 10 minutes. It was stirred at room temperature for 2 hours, then LCMS monitored for its completion. The mixture was quenched by aqueous NaHCO3 (100.0 mL) and extracted by EA (100.0 ml×2). The organic layer was washed with aqueous NaCl solution and dried over anhydrous Na2SO4. After filtration, the organic layer was concentrated to give a crude product, which was purified by column chromatography to give 10 (13.8 g,28.4mmol,83.7% yield) as a yellow oil ).ESI-MS:m/z 509.2[M+Na]+;1HNMR(400MHz,CDCl3)δ7.36-7.23(m,5H),6.36-6.07(m,1H),5.36-5.04(m,1H),4.65-4.52(m,1H),4.49-4.42(m,1H),4.34-4.84(m,6H),2.45-2.18(m,1H),2.13-2.04(m,6H),1.95-1.69(m,1H),1.35-0.95(m,12H).31PNMR(162MHz,CDCl3)δ＝34.43,34.17,34.15,33.56,33.44,33.39,32.61.
A solution of uracil (4.1 g,36.3 mmol) and BSA (15.9 g,77.7 mmol) in anhydrous ACN (80.0 mL) was stirred at 50deg.C until the mixture was clear. A solution of 10 (11.8 g,24.3 mmol) in anhydrous ACN (40.0 mL) was then added, and TMSOTF (5.5 g,24.8 mmol) was added dropwise over 30 minutes at room temperature. The mixture was stirred at room temperature for 5 hours. LMCS shows that 10 is completely consumed. The mixture was quenched by aqueous NaHCO3 (100.0 mL) and extracted by EA (200.0 ml×2). The organic layer was washed with aqueous NaCl solution and dried over anhydrous Na2SO4. After filtration, the organic layer was concentrated to give a crude product, which was purified by column chromatography (SiO2, PE: ea=1:2) to give 11 (10.3 g,19.1mmol,78.8% yield) as a yellow solid ).ESI-MS:m/z 539.1[M+H]+;1HNMR(400MHz,CDCl3)δ9.94-9.86(m,1H),7.37-7.27(m,6H),5.77-5.71(m,2H),5.45-5.29(m,1H),4.60-4.46(m,2H),4.32-4.26(m,1H),4.14-4.03(m,5H),2.38-2.34(m,1H),2.22-2.16(m,1H),2.11-2.08(m,3H),1.38-1.02(m,12H).31PNMR(162MHz,CDCl3)δ＝34.16,33.56,33.29,33.03,32.44.
A solution of 11 (10.3 g,19.1 mmol) in CH3NH2 (100.0 mL,30% MeOH solution) was stirred at room temperature for 1 hour. LCMS showed 11 was completely consumed. The mixture was then concentrated in vacuo to give 12 (11.0 g, crude product) ).ESI-MS:m/z 497.2[M+H]+;31PNMR(162MHz,CDCl3)δ＝34.18,33.17,33.02,33.03,32.40.
To a stirred solution of 12 (9.1 g, crude) and DBU (8.4 g,55.2 mmol) in anhydrous DMF (50.0 mL) at 0℃was added benzyl chloromethyl ether (5.7 g,36.4 mmol) over 10 minutes. The mixture was then stirred at room temperature overnight. LCMS showed 12 was completely consumed. The mixture was then quenched by aqueous NaHCO3 (50.0 mL) and extracted by EA (100.0 ml×2). The organic layer was washed with aqueous NaCl solution and dried over anhydrous Na2SO4. After filtration, the organic layer was concentrated to give a crude product, which was purified by column chromatography (SiO2, PE: ea=1:1) to give 13 (7.0 g,11.4mmol,59.4% yield) ).ESI-MS:m/z 617.4[M+H]+;1H NMR(400MHz,CDCl3)δ9.94-9.86(m,1H),7.38-7.17(m,11H),5.83-5.74(m,2H),5.66-5.43(m,3H),4.78-4.55(m,4H),4.37-4.29(m,1H),4.22-3.93(m,6H),3.10-2.99(m,1H),2.45-2.11(m,1H),2.04-1.93(m,3H),1.65(m,1H),1.34-1.03(m,12H).31PNMR(162MHz,CDCl3)δ＝34.15,33.17,33.03,33.46.
A solution of 13 (7.0 g,10.2 mmol), naI (767.0 mg,5.1 mmol) and Ag2 O (3.6 g,15.3 mmol) in MeI (30.0 mL) was stirred at 40℃for 1 hour, LCMS showed that 13 was completely consumed. After filtration, the organic layer was concentrated to give a crude product, which was purified by column chromatography (SiO2, PE: ea=1:1) to give 14-P1 (3.9 g,6.2 mmol) as a yellow solid and 14-P2(3.1g,4.9mmol).ESI-MS:m/z 631.3[M+H]+;14-P1：31PNMR(162MHz,CDCl3)δ＝33.03,32.85;14-P2：31PNMR(162MHz,CDCl3)δ＝34.27,33.55.
A solution of 14-P1 (3.9 g,6.2 mmol) in CF3 COOH (20.0 mL) was stirred at 80℃for 1 hour, LCMS showed that 14 was completely consumed. After concentration, the crude product was purified by column chromatography (SiO2, PE: ea=0:1) to give the mixture product (2.5 g,93% purity, 96.2% yield). The mixture was separated by SFC to give isomer 1 of 15 (500.0 mg,1.2mmol,20.0% yield) and isomer 2 of 15 (760 mg,1.8mmol,30.4% yield) as white solids. A solution of 14-P2 (3.1 g,4.9 mmol) in CF3 COOH (20.0 mL) was stirred at 80℃for 1 hour, LCMS showed that 14-P2 was completely consumed. After concentration, the crude product was purified by column chromatography (SiO2, PE: ea=0:1) to give the mixture product (1.6 g,91% purity, 77.4% yield). The mixture was separated by SFC to give isomer 3 of 15 (130.0 mg,0.3mmol,8.1% yield) and isomer 4 of 15 (800 mg,1.9mmol,50.0% yield) as white solids.
15 Isomer 1：1HNMR(400MHz,CDCl3)δ8.98(s,1H),7.33(d,J＝8.12Hz,1H),5.76(m,2H),4.24(t,J＝7.9Hz,1H),4.11(m,5H),3.89(d,J＝5.6Hz,1H),3.59(s,3H),2.38-2.27(m,1H),2.15-2.04(m,1H),1.35-1.18(m,12H).31PNMR(162MHz,CDCl3)δ＝33.29;ESI-MS:m/z 421.2[M+H]+
15 Isomer 2：1HNMR(400MHz,CDCl3)δ8.69(s,1H),7.40(d,J＝8.1Hz,1H),5.72-5.69(m,2H),4.04-3.97(m,5H),3.83(q,J＝6.2Hz,1H),3.72(m,1H),3.52(s,3H),2.27-2.16(m,1H),2.03-1.88(m,1H),1.26-1.17(m,12H).31PNMR(162MHz,CDCl3)δ＝33.56;ESI-MS:m/z 421.2[M+H]+
15 Isomer 3：1HNMR(400MHz,CDCl3)δ8.69(s,1H),7.40(d,J＝8.1Hz,1H),5.83-5.76(m,2H),4.20-4.05(m,4H),3.91-3.88(m,1H),3.74-3.71(m,2H),3.61(s,3H),2.46-2.31(m,2H),1.35-1.32(t,J＝7.04Hz,6H),1.19-1.12(m,6H).31PNMR(162MHz,CDCl3)δ＝33.51;ESI-MS:m/z 421.2[M+H]+
15 Isomer 4：1HNMR(400MHz,CDCl3)δ8.89(s,1H),7.33(d,J＝8.04Hz,1H),5.82-5.70(m,2H),4.11-4.02(m,4H),3.88(m,1H),3.72(s,1H),3.63(t,J＝6.2Hz,9.28Hz,1H),3.52(s,3H),2.38-2.23(m,2H),1.27-1.01(m,12H).31PNMR(162MHz,CDCl3)δ＝34.18;ESI-MS:m/z 421.1[M+H]+
Synthesis of A
To a stirred solution of isomer 1 (500 mg,1.2 mmol) and DCI (126.4 mg,1.1 mmol) of 15 in anhydrous DCM (5.0 mL) under an atmosphere of N2 was added CEP [ N (iPr)2]2 (430.0 mg,1.4 mmol). The mixture was stirred at 30 ℃ for 1 hour. LCMS showed complete consumption of isomer 1 of 15. The solution was then diluted with DCM (10 mL) and washed with H2 O (10 mL. Times.3). After drying over anhydrous Na2SO4, the organic layer was concentrated under reduced pressure and the residue was purified by flash prep HPLC with (INTELFLASH-1) column, C18 silica gel, mobile phase, CH3CN/H2O(0.5％NH4HCO3) =1/1, increasing to CH3CN/H2O(0.5％NH4HCO3) =1/0 over 20min, collecting the eluted product at CH3CN/H2O(0.5％NH4HCO3) =6/1, detector, UV 254nm. Obtained a (500 mg,0.8mmol,95% purity, 65% yield) as a white solid ).ESI-MS:m/z 619.2[M-H]-;1HNMR(400MHz,CDCl3)δ9.10(s,1H),7.30(m,J＝8.4Hz,1H),5.82(t,J＝4.4Hz,1H),5.76(d,J＝8.4Hz,1H),4.34(m,1H),4.10(m,5H),3.84(m,5H),3.48(m,5H),3.50(m,3H),2.72-2.66(m,2H),2.20(m,1H),2.04-1.97(m,1H),1.34-1.17(m,24H);31P NMR(162MHz,CDCl3)δ＝149.59,33.43,34.03.
Synthesis of B
To a stirred solution of isomer 2 (760 mg,1.8 mmol) and DCI (192.2 mg,1.6 mmol) of 15 in anhydrous DCM (5.0 mL) under N2 atmosphere was added CEP [ N (iPr)2]2 (653.6 mg,2.1 mmol). The mixture was stirred at 30 ℃ for 1 hour. LCMS showed complete consumption of isomer 2 of 15. The solution was then diluted with DCM (10 mL) and washed with H2 O (10 mL. Times.3). After drying over anhydrous Na2SO4, the organic layer was concentrated under reduced pressure and the residue was purified by flash prep HPLC with (INTELFLASH-1) column, C18 silica gel, mobile phase, CH3CN/H2O(0.5％NH4HCO3) =1/1, increasing to CH3CN/H2O(0.5％NH4HCO3) =1/0 over 20min, collecting the eluted product at CH3CN/H2O(0.5％NH4HCO3) =6/1, detector, UV 254nm. B (500 mg,0.8mmol,95% purity, 45% yield) was obtained as a white solid ).ESI-MS:m/z 619.2[M-H]-;1HNMR(400MHz,CDCl3)δ9.40(s,1H),7.45(m,1H),5.77(m,2H),4.27-3.60(m,11H),3.49(d,J＝14Hz,3H),2.66(m,2H),2.38-2.30(m,1H),2.01(m,1H),1.33-1.15(m,24H).31PNMR(162MHz,CDCl3)δ＝149.35,32.76,32.73.
Synthesis of C
To a stirred solution of isomer 3 (130 mg,0.3 mmol) and DCI (32.9 mg,0.3 mmol) of 15 in anhydrous DCM (2.0 mL) under N2 atmosphere was added CEP [ N (iPr)2]2 (111.8 mg,0.4 mmol). The mixture was stirred at 30 ℃ for 1 hour. LCMS showed complete consumption of isomer 3 of 15. The solution was then diluted with DCM (10 mL) and washed with H2 O (10 mL. Times.3). After drying over anhydrous Na2SO4, the organic layer was concentrated under reduced pressure and the residue was purified by flash prep HPLC with (INTELFLASH-1) column, C18 silica gel, mobile phase, CH3CN/H2O(0.5％NH4HCO3) =1/1, increasing to CH3CN/H2O(0.5％NH4HCO3) =1/0 over 20 min, collecting the eluted product at CH3CN/H2O(0.5％NH4HCO3) =6/1, detector, UV 254nm. C (110 mg,0.2mmol,95% purity, 57% yield) was obtained as a white solid ).ESI-MS:m/z 619.2[M-H]-;1HNMR(400MHz,CDCl3)δ8.50(s,1H),7.31(m,1H),5.84(m,1H),5.75(d,J＝8.4Hz,1H),4.16-3.59(m,11H),2.70(m,2H),2.40-2.18(m,2H),1.41-1.12(m,24H).31PNMR(162MHz,CDCl3)δ＝150.21,149.66,33.61,33.01.
Synthesis of D
To a stirred solution of isomer 4 (800 mg,1.9 mmol) and DCI (202.3 mg,1.7 mmol) of 15 in anhydrous DCM (5.5 mL) under N2 atmosphere was added CEP [ N (iPr)2]2 (688.0 mg,2.3 mmol). The mixture was stirred at 30 ℃ for 1 hour. LCMS showed complete consumption of isomer 4 of 15. The solution was then diluted with DCM (10 mL) and washed with H2 O (10 mL. Times.3). After drying over anhydrous Na2SO4, the organic layer was concentrated under reduced pressure and the residue was purified by flash prep HPLC with (INTELFLASH-1) column, C18 silica gel, mobile phase, CH3CN/H2O(0.5％NH4HCO3) =1/1, increasing to CH3CN/H2O(0.5％NH4HCO3) =1/0 over 20min, collecting the eluted product at CH3CN/H2O(0.5％NH4HCO3) =6/1, detector, UV 254nm. Obtained D (760 mg,1.23mmol,95% purity, 64% yield) as a white solid ).ESI-MS:m/z 619.2[M-H]-;1HNMR(400MHz,CDCl3)δ9.40(s,1H),7.45(m,1H),6.03(t,J＝4.8Hz,1H),5.70(m,1H),4.22-4.06(m,5H),3.94-3.61(m,6H),3.48(d,J＝10.8Hz,3H),2.64(m,2H),2.49-2.29(m,2H),1.39-1.29(m,6H),1.24-1.15(m,12H),1.12-1.04(m,6H).31P NMR(162MHz,CDCl3)δ＝150.3,149.90,34.52,34.36.
Example 3 exemplary Synthesis of oxetane VP
1 (Prepared according to T.Jonckers et al J.Med. Chem.; 2016,59,5790-5798) (10.0 g,25.6 mmol), imidazole (34.9 g,512 mmol) and DMAP (0.94 g,7.7 mmol) were dissolved in DMF (100 mL) under an atmosphere of N2. TBSCl (30.9 g,205 mmol) was added separately at 0deg.C. After the addition was complete, the mixture was stirred at 80 ℃ for 16 hours. To the reaction was added saturated aqueous NH4 Cl and the aqueous layer was extracted with EtOAc (3×). The combined organic layers were washed with 5% aqueous licl (2×), dried over Na2SO4 and concentrated in vacuo to give 2 (21.1 g) as crude product, which was used in the next step without further purification.
2 (15.8 G,25.6 mmol) was dissolved in THF (51.7 mL) and distilled water (13.7 mL). The mixture was cooled to 0 ℃ and TFA (13.7 mL) was added dropwise over 15 minutes while maintaining the internal reaction temperature below 2 ℃. The mixture was stirred at 0 ℃ for 1 hour and then at-18 ℃ for 14 hours. The reaction was warmed to 0 ℃ and stirring was continued for 4 hours, followed by stirring at 5 ℃ for 4 hours. The reaction was basified to pH-8 with NH4 OH solution, diluted with water, and the aqueous layer was extracted with EtOAc (3×). The combined organic layers were dried over Na2SO4 and concentrated in vacuo. The crude product was purified by flash chromatography on silica gel (DCM/MeOH, gradient from 100:0 to 98:2) to give 3 (7.6 g, 59%).
3 (3.0 G,6.0 mmol) was dissolved in DCM (100 mL) under an atmosphere of N2. DMP (3.6 g,8.4 mmol) was added at 0deg.C and the mixture was stirred at 0deg.C for 1 hour and then at room temperature for an additional 3 hours. To the mixture was added 50mL of a mixture of 30% aqueous Na2S2O3 and saturated aqueous NaHCO3 (1:1), and the aqueous layer was extracted with EtOAc (3X). The combined organic layers were dried over Na2SO4 and concentrated in vacuo to give 4 (4.4 g) as crude product, which was used in the next step without further purification.
NaH (60% dispersion in mineral oil, 720mg,18 mmol) was suspended in THF (10 mL) under an atmosphere of N2, and the mixture was cooled to-78 ℃. A solution of 5 (9.5 g,14.5 mmol) in THF (11 mL) was added and the mixture stirred at-78℃for 20 min. To this mixture was added dropwise a solution of 4 (3.0 g,6 mmol) in THF (21 mL) at-78 ℃ over a period of 30 minutes, and stirring was continued for 100 minutes at the same temperature. The mixture was warmed to 0 ℃ and stirred for 1 hour, followed by stirring for an additional 90 minutes at room temperature. The crude reaction mixture was poured into 100mL of saturated aqueous NH4 Cl and the product was extracted into 100mL ethyl acetate (2X). The organic layer was washed with brine and dried over anhydrous Na2SO4. The solution was then concentrated under reduced pressure and the residue was purified by flash chromatography on silica gel (heptane: etOAc, gradient from 100:0 to 0:100) to give 6 (2.7 g, 56%) as a 90:10 mixture of E: Z isomers.
6 (2.7 G,2.7 mmol) was dissolved in MeCN (26 mL) and distilled water (8.1 mL). CAN (7.5 g,13.7 mmol) was added and the mixture stirred at room temperature for 24 hours. The reaction was basified to pH 6-7 with NH4 OH solution, poured into brine solution (50 mL) and extracted with EtOAc (3×). The combined organic layers were dried over Na2SO4 and concentrated in vacuo. The crude product was purified by flash chromatography on silica gel (DCM/MeOH, gradient from 100:0 to 90:10) to give 7 (823 mg, 52%).
7 (552 Mg,1.7 mmol) and 4, 5-dicyanoimidazole (DCI, 166mg,1.4 mmol) were dissolved in anhydrous DCM (9.2 mL) under an atmosphere of N2. 2-cyanoethyl-N, N, N ', N' -tetraisopropyl phosphoramidate (0.684 mL,2.2 mmol) was added and the mixture stirred at room temperature for 16 hours. Another portion of 2-cyanoethyl-N, N, N ', N' -tetraisopropyl phosphoramidate (0.158 mL,0.5 mmol) was added and the mixture was stirred at room temperature for 3 hours. The organic layer was poured into water and washed with water (2×) and brine (1×). The organic layer was dried over Na2SO4 and concentrated in vacuo. The crude product was purified by flash chromatography on silica gel (heptane with 0.15% TEA/EtOAc with 0.15% TEA, gradient from 100:0 to 0:100) to give 8(532mg,41％).1HNMR(400MHz,DMSO-d6)δppm 1.12-1.16(m,18H),1.17-1.24(m,12H),2.52-2.78(m,H),2.79-2.89(m,2H),3.60-3.79(m,3H),3.79-3.91(m,1H),4.16-4.29(m,1H),4.32-4.56(m,3H),5.56-5.67(m,5H),5.88-5.94(m,1H),6.05-6.21(m,1H),6.67-6.97(m,1H),7.57-7.68(m,1H),11.43-11.50(m,1H).31P NMR(162MHz,DMSO-d6)δppm 16.4,17.2,148.7,150.7LCMS： methods A, rt:1.24,775.5[ M+H ]+
To a 5L 3-neck round bottom flask was added chlorodimethylphenylsilane (183.90 g,1077.25 mmol) and imidazole (173.35 g,2546.24 mmol) and DCM (3000 mL) at room temperature. 9 (200 g,979.32 mmol) was added dropwise to the above mixture over 2 hours at 0 ℃. The resulting mixture was stirred at room temperature overnight. The reaction was quenched with water at room temperature. The resulting mixture was extracted with CH2Cl2 (2X 2L). The combined organic layers were washed with brine (1×2L) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography on silica eluting with PE/ea=10:1 to give 10 (260 g, 78%).
LiBH4 (50.19 g,2304.45 mmol) and THF (6500 mL) were added to a 10L 4-neck round bottom flask at room temperature. 10 (260 g,768.15 mmol) was added dropwise to the above mixture over 1.5 hours at 0 ℃. The resulting mixture was stirred at room temperature overnight. The reaction was quenched at 0 ℃ by addition of saturated NH4 Cl (aq) (3L). The resulting mixture was extracted with CH2Cl2 (2X 1L). The combined organic layers were washed with brine (1×2L) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography on silica eluting with PE/EA (1:1) to give 11 (120 g, 61%).
To a 10L 4-necked round bottom flask was added 11 (120 g,471.69 mmol), DCM (6000 mL), pyridine (37.68 g,476.41 mmol) and DMAP (5.76 g,47.17 mmol) at room temperature. Ac2 O (48.64 g,476.41 mmol) was added dropwise to the above mixture over 1 hour at 0deg.C. The resulting mixture was stirred at room temperature overnight. The reaction was quenched with NaHCO3 (aqueous) at room temperature. The resulting mixture was extracted with CH2Cl2 (2X 2L). The combined organic layers were washed with brine (1×2L) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography on silica eluting with PE/EA (1:1) to give 12 (64 g, 46%).
Uracil (70 g,624.50 mmol), acetonitrile (210 mL) and pyridine (123.50 g,1.56 mol) were added to a 1L 3-neck round bottom flask at room temperature. Benzyl chloroformate (234.38 g,1.37 mol) was added dropwise to the above mixture at 0℃over 1.5 hours. The resulting mixture was stirred at room temperature overnight. The resulting mixture was extracted with EtOAc (2X 500 mL). The combined organic layers were washed with brine (1×500 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure.
The crude product was recrystallized from MeOH (200 mL) to give 13 (80 g, 59%).
To a 3L 3-neck round bottom flask was added 12 (64 g,215.89 mmol), PPh3 (124.58 g,474.97 mmol) and THF (1600 mL) at room temperature. 13 (93.35 g,431.79 mmol) and DIAD (96.04 g,474.97 mmol) were added dropwise to the above mixture over 2 hours at-10 ℃. The resulting mixture was stirred at room temperature for an additional 3 hours. The reaction was quenched by the addition of water/ice (1L) at 0 ℃. The resulting mixture was extracted with EtOAc (2X 1L). The combined organic layers were washed with brine (1×1L) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography on silica eluting with PE/EA (1:1) to give 14 (80 g, 75%).
To a 2L 3-neck round bottom flask was added 14 (80 g,161.74 mmol) and THF (800 mL) at room temperature. Hydrazine hydrate (12.15 g,242.61 mmol) was added dropwise to the above mixture over 30 minutes at 0 ℃. The resulting mixture was stirred at room temperature for an additional 3 hours. The resulting mixture was concentrated under reduced pressure. The residue was purified by flash chromatography on silica eluting with PE/EA (1:1) to give 15 (55 g, 87%).
To a 500mL 3-necked round bottom flask was added 15 (55 g,140.84 mmol) and THF (550 mL) at room temperature. TBAF (36.82 g,140.84 mmol) was added dropwise to the above mixture over 0.5 hours at 0deg.C. The resulting mixture was stirred at room temperature for an additional 3 hours. The resulting mixture was extracted with CH2Cl2 (2X 1L). The combined organic layers were washed with brine (1×1L) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. 16 (40 g, crude) was obtained and used in the next step without further purification.
To a 1L 3-neck round bottom flask was added 16 (40 g,156.09 mmol), imidazole (31.42 g,468.27 mmol) and THF (400 mL) at room temperature. T-butyl (chloro) diphenylsilane (85.81 g,312.18 mmol) was added dropwise to the above mixture at 0℃for 1 hour. The resulting mixture was stirred at room temperature for an additional 3 hours. The resulting mixture was extracted with EtOAc (2X 400 mL). The combined organic layers were washed with brine (1×400 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography on silica eluting with PE/EA (1:1) to give 17 (52 g, 67%).
To a 1L 3-neck round bottom flask was added 17 (52 g,105.12 mmol) and methanol (500 mL) at room temperature. NaOH (2M, 100 mL) was added dropwise to the above mixture over 1 hour at 0deg.C. The resulting mixture was stirred at room temperature for an additional 3 hours. The resulting mixture was extracted with EtOAc (2X 200 mL). The combined organic layers were washed with brine (1×200 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by trituration with MeOH (100 mL) and the solid collected by filtration to give 18 (40.2 g, 84%).
18 (2.4 G,5 mmol) was dissolved in DCM (75 mL) under an atmosphere of N2. DMP (3.0 g,7 mmol) was added at 0deg.C and the mixture was stirred at room temperature for an additional 2 hours. To the mixture was added 50mL of a mixture of 30% aqueous Na2S2O3 and saturated aqueous NaHCO3 (1:1), and the aqueous layer was extracted with EtOAc (3X). The combined organic layers were dried over Na2SO4 and concentrated in vacuo to afford 19 as crude product, which was used in the next step without further purification.
NaH (60% dispersion in mineral oil, 440mg,18 mmol) was suspended in THF (10 mL) under an atmosphere of N2, and the mixture was cooled to-40 ℃. Tetraethylmethylenediphosphonate (3.0 ml,12 mmol) was added and the mixture was stirred at-40 ℃ for 30 min. To this mixture was added dropwise a solution of 19 (2.3 g,5 mmol) in THF (25 mL) at-40 ℃ over a period of 10 minutes, and the mixture was stirred for 100 minutes while warming the mixture to-10 ℃. The crude reaction mixture was poured into water and the product was extracted into ethyl acetate (3×). The organic layer was dried over anhydrous Na2SO4 and concentrated in vacuo. The crude product was purified by flash chromatography on silica gel (heptane/EtOAc: etOH (3:1), gradient from 100:0 to 0:100) to give 20 as an inseparable mixture with tetraethyl methylenebisphosphonate (2.1 g, 35%).
20 (2.1 G,1.8 mmol) was dissolved in THF (31 mL) under N2, TBAF (1M in THF, 2.3mL,2.3 mmol) was added at room temperature, and the mixture was stirred for 16h. The solvent was removed in vacuo and the crude product was purified by flash chromatography on silica gel (DCM/MeOH, gradient from 100:0 to 94:6) to give 21a and 21b (562 mg, 91%) as racemates. Purification was carried out via preparative SFC (stationary phase: CHIRALPAK DIACEL AD X250 mm, mobile phase: CO2,EtOH+0.4iPrNH2). Each of 21a and 21b was purified again via preparative SFC (stationary phase: torus Diol 30×150mm, mobile phase: CO2,MeOH+0.4iPrNH2) to give 21a (192 mg, 31%). 21b was further purified via preparative SFC (stationary phase: CHIRALPAK DIACEL AD X250 mm, mobile phase: CO2,EtOH+0.4iPrNH2) to give 21b (163 mg, 27%).
7 (192 Mg,0.55 mmol) and 4, 5-dicyanoimidazole (DCI, 56mg,0.47 mmol) were dissolved in anhydrous DCM (3 mL) under an atmosphere of N2. 2-cyanoethyl-N, N, N ', N' -tetraisopropyl diamino phosphate (0.23 mL,0.72 mmol) was added and the mixture stirred at room temperature for 16 hours. The mixture was concentrated in vacuo and the crude product was purified by flash chromatography on silica gel (heptane with 0.15% tea/EtOAc with 0.15% tea, gradient from 100:0 to 0:100, then DCM/MeOH, gradient from 100:0 to 95:5) to give 22a (205 mg, 68%).1 H NMR (400 MHz, acetonitrile -d3)δppm 1.14–1.22(m,12H),1.23-1.30(m,6H),1.78–1.90(m,2H),2.50–2.65(m,2H),2.65–2.72(m,2H),3.56–3.78(m,4H),3.78–3.94(m,2H),3.94–4.10(m,5H),5.49–5.57(m,1H),5.72–5.87(m,1H),6.59–6.80(m,1H),7.27–7.37(m,1H),8.79–9.00(m,1H).31P NMR(162MHz, acetonitrile-d3) delta ppm 16.9,17.2,146.8,147.8.LCMS, method A, rt 1.00,545.2[ M-H ]-,1.02,545.2[M-H]-
21B (163 mg,0.47 mmol) and 4, 5-dicyanoimidazole (DCI, 47mg,0.40 mmol) were dissolved in anhydrous DCM (2.6 mL) under an N2 atmosphere. 2-cyanoethyl-N, N, N ', N' -tetraisopropyl diamino phosphate (0.19 mL,0.61 mmol) was added and the mixture was stirred at room temperature for 16 hours. The mixture was concentrated in vacuo and the crude product was purified by flash chromatography on silica gel (heptane with 0.15% tea/EtOAc with 0.15% tea, gradient from 100:0 to 0:100, then DCM/MeOH, gradient from 100:0 to 95:5) to give 22a (196 mg, 76%).1 H NMR (400 MHz, acetonitrile -d3)δppm 1.16-1.22(m,12H),1.23-1.30(m,6H),1.79-1.91(m,2H),2.48-2.62(m,2H),2.64-2.71(m,2H),3.56-3.76(m,4H),3.79-3.94(m,2H),3.94-4.09(m,5H),5.50-5.58(m,1H),5.71-5.85(m,1H),6.58-6.79(m,1H),7.28-7.39(m,1H),8.81-9.03(m,1H).31P NMR(162MHz, acetonitrile-d3) delta ppm 16.9,17.2,146.8,147.8.LCMS, method A, rt 0.96,545.3[ M-H ]-,0.99,545.3[M-H]-
Ac2 O (3.1 mL,32.8 mmol) was added to a solution of compound 23 (9.00 g,32.8 mmol) in pyridine (90.0 mL) at 10℃to 25 ℃. The mixture was stirred at 0 ℃ for 2 hours. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by flash chromatography on silica gel (PE/EtOAc, gradient from 100:0 to 0:100) to give compound 13 (18.0 g,86% yield).
To a solution of compound 24 (18.0 g,56.9 mmol) in DCM (100 mL) was added imidazole (11.6 g,171 mmol). A solution of TBSCl (20.9 mL,170 mmol) in 25.0mL of dichloromethane was then slowly added at 25 ℃. The mixture was stirred at 25 ℃ for 12 hours. The reaction mixture was quenched by the addition of H2 O400 mL at 0℃and then extracted with 600mL (200 mL. Times.3) of ethyl acetate. The combined organic layers were washed with brine 200mL, dried over Na2SO4, filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography on silica gel (PE/EtOAc, gradient from 100:0 to 80:20) to give compound 25 (21.0 g,85% yield).
To a solution of compound 25 (21.0 g,48.7 mmol) in MeOH (150 mL) was added K2CO3 (13.5 g,97.5 mmol). The mixture was stirred at 25 ℃ for 0.5 hours. The reaction mixture was quenched by the addition of H2 O350 mL at 0℃and then extracted with 600mL (200 mL. Times.3) of ethyl acetate. The combined organic layers were washed with brine 200mL, dried over Na2SO4, filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography on silica gel (PE/EtOAc, gradient from 100:0 to 0:100) to give compound 25 (21.0 g,85% yield). The crude product was further purified by reverse phase SFC (column: DAICEL CHIRALPAK AS (250 mm. Times.50 mm,10 μm); mobile: [ Neu-ETOH ]; B%:20% -20%, min) to give 26 (14.5 g, 84%).
26 (1.9 G,5.0 mmol) was dissolved in DCM (50 mL) under an atmosphere of N2. DMP (3.3 g,7.8 mmol) was added and the mixture was stirred at room temperature for 3 hours. To the mixture was added 50mL of a mixture of 30% aqueous Na2S2O3 and saturated aqueous NaHCO3 (1:1), and the aqueous layer was extracted with EtOAc (3X). The combined organic layers were dried over Na2SO4 and concentrated in vacuo to give 27 (2.5 g) as crude product, which was used in the next step without further purification.
NaH (60% dispersion in mineral oil, 588mg,15 mmol) was suspended in THF (17 mL) under an atmosphere of N2, and the mixture was cooled to-78 ℃. A solution of 5 (7.7 g,14.5 mmol) in THF (9 mL) was added and the mixture stirred at-78℃for 60 min. To this mixture was added dropwise a solution of 27 (3.0 g,6 mmol) in THF (17 mL) at-78 ℃ over a period of 15 minutes, and stirring was continued for 1 hour at the same temperature. The mixture was warmed to 0 ℃ and stirred for 1 hour, followed by stirring at room temperature for another 1 hour. The crude reaction mixture was poured into 50mL of saturated aqueous NH4 Cl and the product was extracted into 50mL ethyl acetate (3×). The organic layer was washed with brine and dried over anhydrous Na2SO4. The solution was then concentrated under reduced pressure and the residue was purified by flash chromatography on silica gel (heptane: etOAc, gradient from 100:0 to 0:100) to yield 28 (1.4 g, 40%).
To a mixture of 28 (608 mg,0.9 mmol) and distilled water (15 mL) was added formic acid (15 mL). The reaction was stirred at room temperature for 20 hours. The solvent was removed in vacuo and the crude product was purified by flash chromatography (DCM/MeOH, gradient from 100:0 to 95:5) to give 29 (402 mg, 79%).
7 (402 Mg,0.7 mmol) and 4, 5-dicyanoimidazole (DCI, 70mg,0.6 mmol) were dissolved in anhydrous DCM (3.9 mL) under an N2 atmosphere. 2-cyanoethyl-N, N, N ', N' -tetraisopropyl diamino phosphate (0.29 mL,0.9 mmol) was added and the mixture stirred at room temperature for 16 hours. The organic layer was poured into water and washed with water (2×) and brine (1×). The organic layer was dried over Na2SO4 and concentrated in vacuo. The crude product was purified by flash chromatography on silica gel (heptane with 0.15% tea/EtOAc with 0.15% tea, gradient from 100:0 to 0:100) to give 30 (163 mg, 30%) and another fraction of 30 (279 mg,36%,70% purity).1 H NMR (400 MHz, acetonitrile -d3)δppm 1.15-1.22(m,30H),2.63-2.71(m,2H),3.43-3.49(m,3H),3.56-3.68(m,2H),3.68-3.79(m,1H),3.79-3.88(m,1H),3.92-4.00(m,1H),4.09-4.22(m,1H),4.29-4.39(m,1H),5.57-5.71(m,5H),5.99-6.15(m,2H),6.79-7.00(m,1H),7.79-7.85(m,1H),8.82-9.07(m,1H).31P NMR(162MHz, acetonitrile-d3) delta ppm 15.7,15.9,149.9,150.3.LCMS, method A, rt 1.23,779.4[ M+H ]+,1.24,779.4[M+H]+.
Example 4-example of unlocking vinyl phosphonate Synthesis
LCMS method:
CPG (25. Mu. Mol,30 mg) with the synthesized n-1 oligonucleotide with 5' -DMTr was placed in a 1mL syringe provided with a filter and a lid.
Step 1. Fill the syringe with 3% dichloroacetic acid in DCM and hold at ambient temperature for 20 minutes. The solvent was removed and CPG was washed 6 times with 0.6mL acetonitrile, 3 times with anhydrous DCM, and then dried in vacuo for 30 minutes.
Step 2. Fill the syringe with 200. Mu.L of a 0.1M solution of phosphonate amide ester in anhydrous acetonitrile and 200. Mu. L0.25M 5- (ethylsulfanyl) -1H-tetrazole in acetonitrile. The syringe was closed and gently shaken at ambient temperature for 40 minutes. The solvent was removed and the second run repeated. The solvent was removed and CPG was washed 6 times with 0.6mL acetonitrile. A solution of 0.05M iodine in pyridine/water 9:1 (v: v) was added and maintained for 15 minutes, and repeated twice. CPG was washed 6 times with 0.6mL of acetonitrile, 3 times with anhydrous DCM, and then dried in vacuo for 30 minutes. Aliquots of CPG (2 mg) were taken, treated with AMA at 60℃for 10 min, and the oligonucleotides obtained were checked for MW by LCMS. If the coupling does not proceed to completion, step 2 is repeated.
Step 3. Anhydrous CPG in a syringe is treated with 250. Mu.L of a 3.5% trimethylsilyl iodide solution in acetonitrile/pyridine 50:1 (v/v) for 20 minutes at ambient temperature. This treatment was repeated 3 times. The solvent was removed and CPG was washed 6 times with 0.6mL acetonitrile and then treated with a solution of 4% mercaptoethanol in pyridine/triethylamine 1:1 (v: v) at ambient temperature for 20 minutes. The solvent was removed and CPG was washed 6 times with 0.6mL acetonitrile, 3 times with anhydrous DCM, and then dried in vacuo for 30 minutes. Aliquots of CPG (2 mg) were taken, treated with AMA at 60℃for 10 min, and the oligonucleotides obtained were checked for MW by LCMS. If the deprotection does not proceed to completion, step 3 is repeated.
Step 4. CPG in the syringe was treated with 200. Mu.L AMA for 3 hours and filtered. The solid support was washed 5 times with 250 μl of water. The filtrate and washings were combined, diluted to 10mL, and the target compound was isolated by ion exchange chromatography. Column GE Source 15Q 250X 10mm. Buffer A is a 20mM sodium phosphate in acetonitrile/water 9:1 (v: v) solution and buffer B is a 1.8M sodium borate in buffer A (pH 7.5). The target compound was eluted at 25% -35% b using a gradient of 5% to 60% over 10 column volumes. The oligonucleotides obtained were desalted and used for annealing.
Step 5. Annealing is achieved by mixing equimolar amounts of sense and antisense strands. The siRNA obtained was analyzed by LCMS under denaturing conditions at 80 ℃.
Examples of synthetic AS:
Examples of synthetic siRNAs
HTRAKD obtained by stabilized phosphoric acid in ARPE-19
The above table shows the percentage of HTRA remaining at 3 days in vitro for different concentrations of the oligonucleotides attached with the recognized phosphate (n=3 samples), the oligonucleotides in 1 and 2 being different, and the oligonucleotides in the remaining samples being identical except for the recognized phosphate.
Novel stable phosphate results KD at week 4 post intravitreal injection
The table above shows the novel stable phosphate results KD at weeks 2 and 4 after intravitreal injection. The oligonucleotides in the sample are identical except for the recognized phosphate.
Additional embodiments include:
1. A compound represented by formula (Ia) or (Ib):
Wherein:
x is selected from 3-to 5-membered cycloalkyl, -CHCH-, 3-to 5-membered heterocycle and-CHR3CHR3 -;
Y is selected from O or NR';
R' is a counter ion, H or a protecting group
Z is selected from H, counter ion, activating group and oligonucleotide;
a is selected from O, S and CR4R4;
B is a nucleobase;
Each R is independently selected from the group consisting of oligonucleotides, counter ions, H, and protecting groups, such as C1-C5 alkyl and POM or C1-C5 alkyl;
Each R1 and R2 is independently selected from H, F, OH and optionally substituted O-alkyl, provided that R1 and R2 are not each H;
Each R3 is independently selected from C1-C3 alkyl;
Each R4 is independently selected from H, F and C1-C5 alkyl;
Provided that when A is O then X is not-CHCH-, and when X is cyclopropyl then at least one of A or Y is not O.
2. The compound of embodiment 1, wherein the compound of formula (Ia) is represented by:
3.a compound according to embodiment 1 or 2 wherein X is cyclobutyl.
4. The compound of embodiment 3, wherein the cyclobutyl is represented by:
wherein the dashed lines represent the points of attachment to adjacent atoms.
5. A compound of embodiment 1 or 2 wherein X is-CHR3CHR3 -and R3 is methylene.
6. The compound of embodiment 5, wherein X is selected from the group consisting of:
wherein the dashed lines represent the points of attachment to adjacent atoms.
7. A compound according to embodiment 1 or 2 wherein X is a 3-to 5-membered heterocycle selected from:
wherein the dashed lines represent the points of attachment to adjacent atoms.
8. The compound of any one of embodiments 1 through 7 wherein Y is O.
9. The compound of any one of embodiments 1 to 8, wherein Z is an activating group.
10. The compound of embodiment 9 wherein the activating group is represented by:
wherein the dashed lines represent the points of attachment to adjacent atoms.
11. The compound of any one of embodiments 1 to 8, wherein Z is an oligonucleotide.
12. The compound of embodiment 11, wherein the oligonucleotide is an antisense strand of RNA, preferably an antisense strand of siRNA.
13. The compound of embodiment 11 or 12, wherein the 5' end of the oligonucleotide is linked to Y.
14. The compound of any one of embodiments 1 through 13 wherein a is O.
15. A compound according to any one of embodiments 1-13, wherein a is CH2 or CHF.
16. The compound of embodiment 15 wherein X is-CHCH-.
17. A compound according to any one of embodiments 1 to 16 wherein B is uracil.
18. The compound of any one of embodiments 1 to 17, wherein R1 is H and R2 is OMe, OEt, MOE or F.
19. The compound of any one of embodiments 1 to 18, wherein R is a protecting group, such as POM, et, and Z is an activating group.
20. The compound of any one of embodiments 1 to 18, wherein R is H and Z is an oligonucleotide.
As will be understood by those skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be readily understood as sufficiently describing and enabling the same range to be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each of the ranges discussed herein can be readily broken down into a lower third, a middle third, an upper third, and the like. Those skilled in the art will also appreciate that all language such as "up to", "at least", "greater than", "less than" and the like include the listed numbers and refer to ranges that can be subsequently broken down into subranges as described above. Finally, as will be appreciated by those skilled in the art, a range includes each individual member.
All publications, patent applications, issued patents, and other documents mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. The definitions contained in the text incorporated by reference are excluded to the extent that they contradict the definitions in this disclosure.
Other embodiments are set forth in the following claims.