EA045540B1 - MODIFIED NUCLEOSIDE AMIDE PHOSPHITES - Google Patents

Description

Cross-references to related applications

This application claims benefit from U.S. Provisional Patent Application No. 62/558,763, filed September 14, 2017, which is incorporated herein by reference in its entirety.

Prerequisites for creating the invention

Oligonucleotide synthesis is critical to provide access to specially designed oligonucleotides of the desired sequence. To produce the desired oligonucleotide, building blocks (monomers) are sequentially linked to the growing oligonucleotide chain in the order necessary to create the product sequence.

During recent years, there has been increased interest in modified oligonucleotides having modifications, for example at the 2' and/or 3' position, which are used for, for example, therapeutic purposes. Although synthetic methods exist for constructing modified oligonucleotides, there is a need for additional synthetic methods to synthesize a wide range of modified oligonucleotides. To meet the need for new modified oligonucleotides, new highly purified monomeric nucleosides are needed that can be synthesized in higher yields and in larger quantities.

The monomers described herein address this need by providing new monomers useful for the synthesis of modified oligonucleotides.

Summary of the invention

The present description relates to compounds and compositions containing nucleoside 5'amidophosphite monomers, as well as methods for their preparation and use.

The present description relates to compounds having the structure represented by formula (I):

N(R ¹ ) ₂ r ² op °Α- ^Ό ν ^Β \___ \ (I),

X represents NH;

Y is -OCF3, -O(CR ⁴ 2)aCR ⁴ ₃ , -O(CR ⁴ 2)bOCR43 or -O(CR ⁴ 2)b-CR ⁴ =CR ⁴ 2;

Z represents H;

B is a nucleotide base selected from the group consisting of adenine (A), guanine (G), thymine (T), cytosine (C), uracil (U), 5-methylcytosine (5-me-C), 6- N-benzoyladenine (A ^Bz ), 4-N-benzoylcytosine (C ^Bz ) and 2-N-isobutyrylguanine (G ^1Bu );

each R ¹ is independently C1-6 alkyl or C3-6 cycloalkyl;

R ² represents -CH2CH2CN or C1-6 alkyl;

R ³ is H, trityl, monomethoxytrityl (MMTr) or tritolyl;

each R ⁴ is independently H or F;

a is equal to 1 or 2; and b is 1, 2 or 3.

The present description also applies to compounds having the structure represented by formula (II):

XX represents NH;

Z represents H;

B is a nucleotide base selected from the group consisting of adenine (A), guanine (G), cytosine (C), uracil (U), 6-N-benzoyladenine (A ^Bz ), 4-N-benzoylcytosine (CBz) and 2-Nisobutyrylguanine (G ^1Bu );

each R ¹ is independently C1-6 alkyl or C3-6 cycloalkyl;

R ² represents -CH2CH2CN or C1-6 alkyl;

R ³ is H, trityl, monomethoxytrityl (MMTr) or tritolyl;

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A represents -(CR'R') _1-2 -; and each R' is independently H or Me.

In some embodiments, X is NH, Y is OEt or Omethoxyethoxy, and Z is H.

In some embodiments, R1 is _C2-3 alkyl. In some embodiments, R ¹ is isopropyl.

In some embodiments, PG is trityl. In some embodiments, PG is monomethoxytrityl (MMTr) or trilyl when X is NH.

In some embodiments, B is selected from adenine (A), guanine (G), thymine (T), cytosine (C), uracil (U), and 5-methylcytosine (5-me-C) or a protected variant thereof. In some embodiments, B is selected from 6-N-benzoyl adenosine (A ^Bz ), 4-N-benzoylcytidine (C ^Bz ), and 2-Nisobutyryl guanosine (G ^{1 Bu} ).

The present description also relates to a composition containing at least one compound of formula (I) or formula (II). In embodiments, the composition further contains one or more other agents selected from the group consisting of reagents, solvents and impurities. In embodiments, the composition contains less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% impurities. In some embodiments, the composition is >95% pure, such as 96, 97, 98, 99% or more.

The present description further relates to methods for producing an oligonucleotide containing at least one modified nucleoside. The method involves reacting a compound of formula (I) with another monomer to form an oligonucleotide.

Detailed description

The present invention is described in more detail in this section.

Connections of this description

The compounds of the present description include one or more compounds of the following formulas (I) and/or (II):

where X represents NH;

Y is -OCF3, -O(CR ⁴ 2)aCR ⁴ 3, -O(CR ⁴ 2)bOCR43 or -O(CR ⁴ 2)b-CR ⁴ =CR ⁴ 2;

Z represents H;

B is a nucleotide base selected from the group consisting of adenine (A), guanine (G), thymine (T), cytosine (C), uracil (U), 5-methylcytosine (5-me-C), 6- N-benzoyladenine (A ^Bz ), 4-N-benzoylcytosine (C ^Bz ) and 2-N-isobutyrylguanine (G ^1Bu );

each R ¹ independently represents C _1-6 alkyl or C _3-6 cycloalkyl;

R ² represents -CH2CH2CN or C _1-6 alkyl;

R ³ is H, trityl, monomethoxytrityl (MMTr) or tritolyl;

each R ⁴ is independently H or F;

a is equal to 1 or 2;

b is equal to 1, 2 or 3,

A represents -(CR'R') _1-2 -;

each R' is independently H or Me.

In some embodiments, X is NH, Y is OEt or Omethoxyethoxy, and Z is H.

In nucleotides of formula (I), Y is -O(CR ⁴ 2)2OCR ⁴ 3. In some embodiments, R ⁴ is H in each instance. In other embodiments, at least one R ⁴ is F, for example 1, 2, 3, 4, 5, 6, or 7 R ⁴ is F. In some embodiments, CR ⁴ 3 contains 1, 2, or 3 F atoms. For example, in embodiments, Y is selected from the group consisting of -OCH2CH2OCH3 (or MOE) , -CF ₂ CH ₂ OCH ₃ , -OCH2CF2CH3, -OCH2ch2CH2OCF3, -OCF2CF2OCH3, -OCH2CF2CF3, -OCF2ch2CH2CH2CF2CF2OCF3, -OCHFCH2OCH3, -OCHFCHFOCH3, -OCHFF3, -OCHFF3 CH2OCFH2, -OCHFCH2OCHF2 and -OCH2chfoch3.

In the nucleotide of formula (I), Y is -OCR ⁴ 3, -O(CR ⁴ 2) _1-3 OCR ⁴ 3 or -O(CR ⁴ 2) _1-2 CR ⁴ ₃ . In some embodiments, Y is -OCR ⁴ 3 or -OCR ⁴ 2CR ⁴ 3. In some variations

- 2045540 each embodiment, R ⁴ is H in each case. In other embodiments, at least one R ⁴ is F, such as 1, 2, 3, 4, or 5 R ⁴ is F. In some embodiments, OCR ⁴ ₃ contains 1, 2 or 3 F atoms. For example, in embodiments, Y is selected from the group consisting of _-OCH3 (or Me), _-OCFH2 , -oChF2, _OCF3 , _-OCH2OCH3 , -OCFH2OCH3, -OCHF2OCH3, -OCF3OCH3, -OCH2OCFH2, -OCH2OCHF2, -OCH2OCF3, -OCFH2OCH3, -OCFH2OCFH2, -OCFH2OCHF2, -OCFH2OCF3, -OCHF2OCH3, -OCHF2OCFH2, -OCHF2OCHF2, -OCHF2OCF3, -O(CR ⁴ 2)3OC R43, -OCH2 CH3 (or Et), -OCFH2CH3, -OCHF2CH3, -OCF3CH3, -OCH2CFH2, -OCH2CHF2, -OCH2CF3, -OCFH2CH3, -OCFH2CFH2, -OCFH2CHF2, -OCFH2CF3, -OCHF2CH3, -OCHF2CFH2, -OCHF2CHF2, -OCHF2CF3, -OCH2CH2CH3, O CF2CH2CH3, OCH2CF2CH3, OCH2CH2CF3, OCF2CF2CH3, OCH2CF2CF3, OCF2CH2CF3, OCF2CF2CF3, OCHFCH2CH3, OCHFCHFOCH3, OCHFCH2CFH2, OCHFCH2CHF2 and OCH2CHFCH3. In some embodiments, Y is -OCH3 (or Me) or OCH2CH3 (or Et).

In some embodiments of formula (I), Y is -O(CR ⁴ 2)b-CR ⁴ =CR ⁴ 2. In some embodiments, b is 1, 2, or 3. In some embodiments, R ⁴ is in each case H. In other embodiments, at least one R ⁴ is F, such as 1, 2, 3, 4, 5, 6 or 7 R ⁴ is F. In some embodiments, CR ⁴ 2 contains 1 or 2 F atoms, for example, the inner R ⁴ 2 contains 1 or 2 F atoms or the terminal R ⁴ ₂ contains 1 or 2 F atoms.

In some embodiments, R ¹ is C _2-3 alkyl (eg, ethyl or isopropyl).

In some embodiments, R ³ is a protecting group (PG). Protecting groups may include an amine or alcohol protecting group, such as a silyl protecting group (eg, tert-butyldimethylsilyl ether (TBMDS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl ether (TIPS), or monomethoxytrityl (MMTr), or 4. 4'dimethoxytrityl (DMTr), or tritolyl, or any other suitable protecting groups such as those described in Wuts, Peter GM, and Theodora W. Greene, Greene's protective groups in organic synthesis (John Wiley & Sons, 2006). In some embodiments By protecting more than one alcohol and/or amine, orthogonal protection of the alcohols and/or amines can be achieved.

In compounds of formula (II) A is -(CR'R')1. ₂ -. In some embodiments, A is -(CR'R')-, in other embodiments, A is -(CR'R') ₂ -. R' in each case is independently H or Me. In some embodiments, one R' is Me and the remaining R' are H. In other embodiments, all R' are H. In some embodiments, X is NH in formula (II). In some embodiments, A is not -(CR'R')1- when R'=H and X=NH.

The natural or unmodified nucleotide base or the modified nucleotide base or a protected variant thereof is not particularly limited. In some embodiments, B is selected from purine bases such as adenine (A) and guanine (G), diaminopurine (DAP), and pyrimidine bases such as thiamine (T), cytosine (C), and uracil (U), and/ or other synthetic and natural nucleotide bases such as 5-methylcytosine (5-me-C), 5hydroxymethylcytosine. In some embodiments, B is a protected nucleotide base, such as protected Y, T, C, 5meC, A, or G. Protecting functional groups are known in the art and are not particularly limited, but include, for example, amine protecting groups (e.g. acetamide protecting groups such as benzamide and isobutyramide). In some embodiments, B is 6-N-benzoyladenosine (A ^Bz ), 4-N-benzoylcytidine (C ^Bz ), 2-N-isobutyrylguanosine (G ^iBu ).

In some embodiments, the connection is selected from the following table.

N(R ¹ h

R ³ OP

R ³

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X γ Z V R ¹ R ² R ³ N.H. OR N U/T/C/5teS/A/G/OAR iPr CH2CH2CN MMTr N.H. OEt n U/T/C/5teS/A/G/OAR iPr CH2CH2CN MMTr N.H. (9-methoxyethyl n U/T/C/5teS/A/G/OAR iPr CH2CH2CN MMTr N.H. OCF ₃ n U/T/C/5teS/A/G/OAR iPr CH2CH2CN MMTr ABOUT OEt n U/T/C/5teS/A/G/OAR iPr CH ₂ CH ₂ CN DMTr ABOUT (?-methoxyethyl n U/T/C/5teS/A/G/OAR iPr CH2CH2CN DMTr

*B may be a protected nucleotide base (eg, A ^Bz , C ^Bz , G ^iBu ).

In some embodiments, the connection is selected from the following table.

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Compositions.

The present description also covers compositions containing a compound of the present description and one or more reagents, solvents or impurities.

In some embodiments, compositions containing a compound of the present disclosure have a purity of 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99. 9%, 99.95% or more.

Methods of application.

The compositions of the present disclosure can be used as synthetic precursors for oligonucleotides containing modified nucleosides, such as those described in US Provisional Patent Applications Nos. 62/420801, 62/394737, 62/394738, and 62/394739, each of which is incorporated herein by reference . Modified oligonucleotides can be synthesized by methods known in the art, for example on the ABI-394 synthesizer using a 93-step cycle written with modifications regarding the unblocking, binding and waiting steps. The solid support may be 3'-NHTr-5'-LCAA-CPG. Each oligonucleotide can be synthesized separately using the methods described herein.

Methods of obtaining.

The compositions of the present disclosure can be synthesized using synthesis procedures such as those described in the examples below.

Some embodiments include 2'-O-alkylation of the starting material, such as:

- 8 045540 where PG represents a protecting group, such as trityl, and BP represents a protected nucleotide base, protected, for example, PMB. The compound is alkylated, followed by deprotection of PGO and its reprotection with, for example, a Bz functional group, followed by deprotection of the nucleotide base and subsequent conversion of N3 to N-R3.

Some embodiments include protecting a compound, such as described in the previous paragraph, in which the nucleotide base is a C-6-OBn purine. In some embodiments, this reduces depurination during deprotection of, for example, the 5'-O-DMTr and C-2-NH-MMTr group under acidic conditions. Other embodiments include protecting the exocyclic amino group in a compound, such as described in the previous paragraph, in which the nucleotide base is a purine, using a bulky protecting group, such as a 4-monomethoxytrityl group, to achieve 2-O-alkylation with high efficiency.

Some embodiments include protecting a compound, such as described in the previous paragraph, in which the nucleotide base is a pyrimidine, to provide 2'-Oalkylation by protecting the pyrimidine with a PMB functionality.

Definitions.

It should be understood that terminology used herein is for the purpose of describing specific embodiments only and should not be construed as limiting the scope of the present invention. Unless otherwise stated, the following definitions apply.

The term pharmaceutically acceptable refers to a material that is not biologically or otherwise undesirable, for example, the material may be included in a pharmaceutical composition administered to a patient such that it does not cause any undesirable biological effects or interact adversely with any other components of the composition in which it is included. When the term pharmaceutically acceptable is used to refer to a pharmaceutical carrier or excipient, it is implied that the carrier or excipient satisfies required toxicological and manufacturing testing standards or is included in the US Food and Drug Administration's Inactive Ingredients Guide.

The term modified nucleoside means a nucleoside having an independently modified sugar functionality and/or a modified nucleotide base.

The term unmodified or natural nucleotide bases includes the purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). The term modified nucleotide bases includes other synthetic and natural nucleotide bases 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-halogenuracil and cytosine, 5-propynyl (-C=C-CH ₃ ) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6 -azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogen, 8-amino, 8thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halogen, especially 5 -bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-Fadenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3 -deazaguanine, and 3-deazaadenine. Additional modified nucleotide bases include tricyclic pyrimidines such as phenoxyazine 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-clamps such as substituted phenoxazine cytidine (e.g. 9-(2-am-oelhoxy)-H-pyrimido[5,4-b][1,4] benzoxazin-2(3H)-one), carbazolcytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindolcytidine (H-pyrido[3,2,5]pyrrolo[2,3-d]pyrimidine2 -He).

Modified nucleotide bases may also include those in which the purine or pyrimidine base is replaced by other heterocycles, for example 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.

In some embodiments, the modified nucleotide base is selected from the group consisting of 5-methylcytosine, 2,6-diaminopurine, 5-methyluracil, and G-clamp. In some embodiments, the G-clamp is

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It should be noted that, as used herein and in the claims, the use of the singular form includes the plural unless the context clearly indicates otherwise. It should be further noted that the claims may be drafted to exclude any optional element. Thus, this statement should be used as an a priori basis for the use of exclusionary terminology such as exclusively, only, etc., in connection with the enumeration of the elements of the claims or the application of a negative limitation.

The term about will be understood by those skilled in the art, and its meaning will depend to some extent on the context in which it is applied. When using a period whose magnitude is not clear to those of ordinary skill in the art from the context in which it is applied, about will mean up to plus or minus 10% of the specific period. Specific ranges are presented herein with numerical values preceded by the term about. As used herein, the term about is used to literally indicate the exact number that it precedes, as well as any number that is close to or approximately equal to the number that the term precedes. In determining whether a number is close to or approximately equal to a precisely specified number, a near or approximately equal uncompensated number may be a number that, in the context of its presentation, provides a substantial equivalent to the precisely specified number.

Where a range of values is given, it is understood that every intermediate value up to a tenth of a lower limit unit (unless the context clearly indicates otherwise) between the upper and lower limits of that range, and every other specified or intermediate value within that specified range, is included in the invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also included in the invention in the presence of any specifically excluded limit within the specified range. If the actual range of values includes one or both limits, ranges excluding either or both limits are also included within the scope of the invention.

The present description is not limited to the described embodiments, since they may be changed. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the present invention, which may only be limited by the appended claims.

As will be apparent to those of ordinary skill in the art upon reading this specification, each of the individual embodiments described and illustrated herein has distinct components and features that can be readily separated from or combined with those of any of several other embodiments without deviations from the scope or essence of the present invention. Any method mentioned can be carried out in the order of the listed events or in any other logically possible order.

All publications and patents cited herein are incorporated herein by reference as if each individual publication or patent were specifically and individually identified as being incorporated by reference, and are incorporated herein by reference for the purpose of disclosure and description of methods and/ or materials in relation to which these publications are cited. The citation of any publication is given to describe it prior to the filing date of the application and should not be construed as invalidating the present invention as to the anticipation of such publication by prior invention. In addition, publication dates shown may differ from actual publication dates and may require independent confirmation.

Examples

The following examples demonstrate certain embodiments of the present disclosure in order to assist one skilled in the art in practicing the invention. Accordingly, the examples are not intended to limit the scope of the present description in any way.

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Examples 1-4.

Properly protected 2'-O-methoxyethyl-3'-aminonucleoside-5'-amidophosphite building blocks (Examples 1, 2, 3 and 4 were prepared after the chemical transformations shown in Schemes 1, 2, 3, 4 and 5).

Scheme 1.

product

Scheme 1 describes the synthesis of 3'-NH-MMTr-2'-O-methoxyethyl amidophosphites based on uracil (such as example 1). The key 3'-azido-2'-methoxyethyl intermediate 3-3 was prepared in low yields via anhydrous intermediate 3-2.

Due to low yield alkylation, 3-1 reacted with BOMCl/DBU to give N-3-protected intermediate 3-4, which was alkylated using 2-bromoethyl methyl ether/Ag ₂ O/NaI/DMF to give 2'-O-methoxyethyl derivative of compound 3-5. Deprotection of N-3-BOM using the hydrogenation condition (Pd/C/H ₂ ) yielded 10-20% of the desired 3'-amine intermediate 3-6a as well as a significant amount of overoxidized by-product 3-6b .

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Scheme 2.

Scheme 2 describes an alternative approach to achieve 2-O-alkylation in high yield. Intermediate 3-1 was treated with PMBCl/DBU/DMF to give N-3-protected intermediate 4-2, which was 2'-O-alkylated using 2bromoethyl methyl ether/Ag ₂ O/NaI/DMF to give 2'-O -methoxyethyl derivative (intermediate 4-3). 5'-de-tritylation of intermediate 4-3 and reprotection with a 5'-hydroxyl group using benzoyl chloride afforded intermediate 4-5.

Deprotection of the RMB group of intermediate 4-5 under mild conditions to obtain intermediate 4-6. The 3'-azide group of intermediate 4-6 was reduced to an amine, which was subsequently immediately protected, for example by reaction with 4-monomethoxytrityl chloride, to give 4-8. Subsequently, the 5'-benzyl ester was cleaved using an alkaline solution followed by phosphitylation using known methods to obtain the desired 2-O-methoxyethoxyuridine amidophosphite monomer 4-10.

Preparation of intermediate compound (4-2). To a solution of 3-1 (45.30 g, 88.56 mmol) in DMF (120.00 mL) was added PMBCl (20.80 g, 132.84 mmol) and DBU (44.61 g, 177.12 mmol) and the mixture was stirred at rt for 2 hours. Water was added and extracted using EA. The organic layer was concentrated and purified on a column to give 4-2 (52.00 g, 82.32 mmol) as a white solid. ESI-LCMS: m/z 632.3 [M+H]+.

Preparation of intermediate compound (4-3). To a solution of 4-2 (50.00 g, 79.15 mmol) in DMF (120.00 mL) was added 2-bromoethyl methyl ether (16.50 g, 118.73 mmol) and Ag ₂ O (18.34 g , 79.15 mmol, 2.57 mL), NaI (5.93 g, 39.58 mmol) was subsequently added. The reaction mixture was stirred at rt for 12 hours. LCMS showed that the reaction was successful. Filter and add water and EA, concentrate and purify the organic layer on a column to give 4-3 (52.00 g, 75.39 mmol) as a colorless oil. ESI-LCMS: m/z 690.4 [M+H] ⁺ .

Preparation of intermediate compound (4-4). Trifluoroacetic acid (TFA) (150.00 mL) was added to a solution of 4-3 (52.00 g, 75.39 mmol) in dichloromethane (DCM) (200.00 mL). The mixture was stirred at room temperature. for 1 hour. The reaction mixture was slowly added to cold NH4OH, extracted using DCM. The organic layer was concentrated and purified to give 4-4 (31.00 g, 69.28 mmol) as a colorless oil. ESI-LCMS: m/z 448.2 [M+H] ⁺ . ¹ H-NMR (DMSO-d ₆ , 400 MHz): δ ppm 8.02 (d, J=8.12 Hz, 1H), 7.26-7.23 (m, 2H), 6, 87-6.84 (m, 2H), 5.87-5.81 (m, 2H), 5.38 (t, J=5.0 Hz, 1H), 4.96-4.85 (m, 2H), 4.36-4.34 (m, 1H), 4.17-4.14 (m, 1H), 4.00-3.97 (m, 1H), 3.83-3.77 ( m, 1H), 3.75-3.72 (m, 1H), 3.71 (s, 3H), 3.70-3.68 (m, 1H), 3.61-3.56 (m, 1H), 3.45-3.43 (m, 2H), 3.18 (s, 3H).

Preparation of intermediate compound (4-5). BzCl (13.14 g, 93.87 mmol) was added to a solution of 4-4 (31.00 g, 69.28 mmol) in pyridine (200.00 mL), and the reaction mixture was stirred at room temperature. for 15 min and concentrated and purified on a column to give 4-5 (35.10 g, 63.8 mmol) as a white solid. ESI-LCMS: m/z 552.2 [M+H] ⁺ .

Preparation of intermediate compound (4-6). Ammonium cerium nitrate (105 g, 191.40 mmol) was added to a solution of 4-5 (35.10 g, 63.8 mmol) in acetonitrile (300.00 ml) and water (100.00 ml), and the reaction mixture was stirred at k.t. for 12 h, and concentrated and extracted using EA. The organic layer was concentrated and purified on a column to give 4-6 (27.5 g, 63.75 mmol) as a yellow solid. ESI-LCMS: m/z 432.2 [M+H] ⁺ .

Preparation of intermediate compound (4-7). Pd/C (3.00 g) was added to a solution of 4-6 (27.50 g, 63.75 mmol) in tetrahydrofuran (THF) (500.00 mL), and the reaction mixture was stirred at room temperature. during

- 12045540 h, filtered and concentrated to give 4-7 (25.00 g, 61.67 mmol) as a yellow solid. ESI-LCMS: m/z 406.2 [M+H]+.

Preparation of intermediate compound (4-8). To a solution of 4-7 (25.00 g, 61.67 mmol) in DCM (300.00 mL) was added MMTrCl (28.49 g, 92.51 mmol) and collidine (14.95 g, 123.34 mmol) , subsequently AgNO ₃ (15.7 g, 92.5 mmol). The reaction mixture was stirred at room temperature. for 1 hour, filtered and washed the organic layer with water, dried over Na ₂ SO ₄ and purified on a silica gel column to give 4-8 (33.00 g, 48.69 mmol) as a yellow solid.

Preparation of intermediate compound (4-9). To solution 4-8 (14.50 g, 21.39 mmol) was added 1 N. NaOH in methanol (200 ml) in water (20 ml), the reaction mixture was stirred at room temperature. for 1 hour and concentrated and extracted using DCM, the organic layer was concentrated and purified on a silica gel column to give 4-9 (11.50 g, 20.05 mmol) as a white solid. 1HNMR (DMSO-d ₆ , 400 MHz): δ ppm 11.26 (s, 1H), 7.95 (d, J=8.4 Hz, 1H), 7.47-7.44 (m , 4H), 7.34-7.17 (m, 8H), 6.82 (d, J=8.8 Hz, 2H), 5.50-5.48 (m, 2H), 5.13 ( t, J=3.6 Hz, 1H), 4.05-3.98 (m, 3H), 3.78 (s, 3H), 3.52-3.49 (m, 1H), 3.34 -3.32 (m, 2H), 3.14 (s, 3H), 3.08-3.04 (m, 1H), 2.89-2.86 (m, 1H), 2.70 (d , J=10.0 Hz, 1H), 1.51 (d, J=4.4 Hz, 1H).

Receipt (4-10). To a solution of 4-9 (11.50 g, 20.05 mmol) in DCM (100.00 mL) was added DMAP (489.85 mg, 4.01 mmol) and DIPEA (10.36 g, 80.19 mmol, 14.01 ml). Subsequently, CEPCl (5.70 g, 24.06 mmol) was added to the solution. The mixture was stirred at room temperature. within 30 min. The reaction was quenched with saturated NaHCO ₃ . The organic layer was washed with brine, dried over Na ₂ SO ₄ and concentrated to give the crude product. The crude product was purified by preparative flash HPLC. The product was dissolved in anhydrous toluene and concentrated three times. Subsequently, the product was dissolved in anhydrous acetonitrile and concentrated three times. The result was 13 g of compound 4-10 as a white solid. MS m/z [M-H] ^- (IER): 772.3; 1H NMR (CDCl3, 400 MHz): 9.01 (s, 1H), 8.07-7.61 (m, 1H), 7.53-7.41 (m, 6H), 7.29-7. 15 (m, 5H), 6.79-6.76 (m, 2H), 5.63-5.57 (m, 2H), 4.27-4.15 (m, 2H), 4.06- 3.95 (m, 1H), 3.85-3.77 (m, 1H), 3.75 (s, 3H), 3.69-3.35 (m, 7H), 3.23 (d, J=4 Hz, 1H), 2.26-2.91 (m, 3H), 2.59 (t, J=6.4 Hz, 1H), 1.75-1.39 (m, 1H), 1.21-1.11 (m, 12H). ^31H NMR (162 MHz, CDCl3): 149.10, 148.26.

Example 2.

NC —( , , ^NHBl

ΆΑ A

ΜΜΤγΗΝ'Ъ'Х'~о 54 ⁴

The amidophosphite compound 2'-O-methoxyethoxy-NH-benzoylcytosine 5-4 was prepared by converting the uridine intermediate 4-8 to the 3'-aminocytidine analogue 5-1 followed by phosphitylation using known protocols to yield the desired amidophosphite monomer 2-O-methoxyethoxycytidine 5-4, as shown in Diagram 3 below.

Scheme 3

Preparation of intermediate compound (5-1). TPSCl (16.49 g, 54.60 mmol) and DMAP (6.67 g, 54.60 mmol) were added to a solution of 4-8 (18.50 g, 27.30 mmol) in acetonitrile (250.00 mL). , TEA (5.52 g, 54.60 mmol, 7.56 ml) was subsequently added to the solution. The reaction mixture was stirred at room temperature. for 5 hours in an N ₂ atmosphere. NH ₄ OH (50.00 ml) was added to the reaction mixture. The mixture was stirred at room temperature. for 12 hours. The solution was concentrated and extracted using EA. The organic layer was washed with brine and dried over Na2SO4. The organic layer was concentrated and purified on a silica gel column to give 5-1 (16.00 g, 23.64 mmol) as a yellow solid.

Preparation of intermediate compound (5-2). To a solution of 5-1 (16.00 g, 23.64 mmol) in pyridine (100.00 mL) was added BzCl (4.96 g, 35.46 mmol) at 0°C. The mixture was stirred at room temperature. for 1 hour. The solution was concentrated and purified on a silica gel column to give 5-2 (17.40 g, 22.28 mmol) as a white solid.

Preparation of intermediate compound (5-3). Compound 5-2 (17.40 g, 22.28 mmol) was added to 180 ml of 1 N. NaOH solution in a mixture of pyridine/MeOH/H ₂ O (65/30/5) at 0°C. The suspension was stirred at 0°C for 15 minutes. The reaction mixture was quenched by adding sat. NH ₄ Cl solution. Solution

- 13 045540 was extracted with EA and the combined organic layers were washed with sat. NaHCO3 solution, saline solution, dried over Na2SO4, filtered and concentrated. The residue was purified by column to give 5-3 (12.50 g, 18.47 mmol) as a white solid. 1H NMR (DMSO-d ₆ , 400 MHz): δ ppm 12.25 (s, 1H), 8.53 (d, J=7.6 Hz, 1H), 8.01 (d, J= 5.2 Hz, 2H), 7.64-7.60 (m, 1H), 7.52-7.42 (m, 6H), 7.31 (d, J=8.8 Hz, 2H), 7.26-7.14 (m, 7H), 6.79 (d, J=8.8 Hz, 2H), 5.55 (s, 1H), 5.23 (t, J=3.6 Hz , 1H), 4.09-3.97 (m, 3H), 3.73 (s, 3H), 3.70-3.66 (m, 1H), 3.38-3.34 (m, 2H ), 3.17 (s, 3H), 3.11-3.05 (m, 1H), 2.96-2.91 (m, 1H), 2.68 (d, J=10.8 Hz, 1H), 1.49 (d, J=4 Hz, 1H).

Receipt (5-4). To a solution of 5-3 (12.50 g, 18.47 mmol) in DCM (100.00 mL) was added DMAP (451.30 mg, 3.69 mmol) and DIPEA (9.55 g, 73.88 mmol, 12.90 mL), CEPCl (5.25 g, 22.16 mmol) was subsequently added. The mixture was stirred at room temperature. within 30 min. The reaction was quenched with saturated NaHCO ₃ . The organic layer was washed with brine, dried over Na ₂ SO ₄ and concentrated to give the crude product. The crude product was purified by preparative flash HPLC. The product was dissolved in anhydrous toluene and concentrated three times. Subsequently, the product was dissolved in anhydrous acetonitrile and concentrated three times. The result was 13 g, yielding 5-4 as a white solid. MS m/z [M-N] ^- (IER): 875.4. 1H-NMR (400 MHz, CDCl ₃ ): δ ppm 8.64-8.20 (m, 2H), 7.90-7.88 (m, 2H), 7.62-7.58 ( m, 1H), 7.53-7.39 (m, 8H), 7.25-7.15 (m, 6H), 6.78-6.74 (m, 2H), 5.69 (d, J=1.72 Hz, 1H), 4.37-4.21 (m, 2H), 4.10-4.03 (m, 1H), 3.90-3.79 (m, 2H), 3 .75 (d, J=1.64 Hz, 3H), 3.68-3.52 (m, 3H), 3.46-3.42 (m, 2H), 3.26 (d, J=1 .2 Hz, 3H), 3.17-2.97 (m, 2H), 2.94-2.87 (m, 1H), 2.672.48 (m, 2H), 1.79-1.51 ( m, 1H),1.26-1.18 (m, 12H). ³¹ iNMR (162 MHz, CDQ3): 148.93, 148.03.

Example 3.

NO -( / NHBz h-p X ir m

ΜΜΤιΉΝ'

6-10 X

The synthesis of the 2'-O-methoxyethyladenosine analogue 6-10 was performed as shown in Scheme 4 below. Treatment of intermediate 6-2 under alkaline conditions (NH ₃ /MeOH) resulted in diol 6-3, which was subsequently protected by 5'- hydroxyl group with TBDPSCl, was converted into intermediate 6-4. Intermediate 6-4. Subsequently, 2'O-alkylation of 6-4 was performed using 2-bromoethyl methyl ether/NaH/DMF to give the 2'-omethoxyethyl derivative 6-5 without protecting the C-6-exocyclic amine 6-4. The method of the invention performed selective alkylation of the 2'-OH group of intermediate 6-4.

Scheme 4.

- 14 045540

The 3'-azide group of intermediate 6-5 was reduced to the amine 6-7, which was subsequently immediately protected, for example by reaction with 4-monomethoxytrityl chloride to give precursor 6-8 after deprotection of 5'-OTBDPS using TBAF/THF . Phosphytylation of 6-9 using known protocols to obtain the desired 2'-O-methoxyethoxyadenine-NH-benzoyl amidophosphite monomer 6-10.

Preparation of intermediate compound (6-2). To a solution of compound 1 (79.50 g, 210.68 mmol) in dry ACN (1.20 L) was added N-(5H-purin-6-yl)benzamide (100.80 g, 421.36 mmol) and bovine serum albumin (BSA) (180.07 g, 884.86 mmol). The resulting suspension was stirred at 50°C until it became clear. Subsequently, the mixture was cooled to -20°C and TMSOTf (93.54 g, 421.36 mmol) was added via syringe. Subsequently, the mixture was stirred at 70°C for 72 hours in an N2 atmosphere, quenched with sat. NaHCO ₃ solution and extracted using DCM. The organic layer was dried over Na ₂ SO ₄ , subsequently the solvent was evaporated and the residue was purified on silica gel to give 6-2 (107.50 g, 192.26 mmol, 91.26% yield) as a yellow solid. ¹ H-NMR (400 MHz, DMSO): δ=11.28 (s, 1H), 8.64 (d, J=6.4 Hz, 2H), 8.05 (d, J=8.0 Hz , 2H), 7.84 (d, J=8.0 Hz, 2H), 7.66 (t, J=7.6 Hz, 1H), 7.56 (t, J=8.0 Hz, 2H ), 7.33 (d, J=8.0 Hz, 2H), 6.37 (d, J=3.6 Hz, 1H), 6.17 (dd, J=6.0 Hz, 1H), 5.09 (t, J=6.8 Hz, 1H), 4.69-4.56 (m, 2H), 4.40-4.38 (m, 1H), 2.39 (s, 3H) , 2.17 (s, 3H). ESILCMS: m/z 557.2 [M+H]+.

Preparation of intermediate compound (6-3). To a solution of compound 6-2 (107.50 g, 192.26 mmol) dissolved in 33 wt% methylamine in ethanol (600.00 ml), the mixture was subsequently stirred at 20°C for 16 h, after which the solvent evaporate, wash with 50% EtOAc in petroleum ether (1.5 L), filter to give 6-3 (52.50 g, 179.64 mmol, 93.44% yield) as a light yellow solid. ESI-LCMS: m/z 293.1 [M+H] ⁺ .

Preparation of intermediate compound (6-4). A solution of compound 6-3 (52.50 g, 179.64 mmol), imidazole (18.32 g, 269.46 mmol) and TBDPS-Cl (54.34 g, 197.60 mmol) in pyridine (500.00 ml) was stirred at 20°C for 2 hours. LCMS showed that 6-3 was consumed. Subsequently quenched with MeOH (30 mL), concentrated to give the crude product, which was purified on silica gel to give 6-4 (72.60 g, 136.81 mmol, 76.16% yield) as a white solid. ¹ H-NMR (400 MHz, DMSO): δ=8.29 (s, 1H), 8.10 (s, 1H), 7.63-7.59 (m, 4H), 7.48-7, 33 (m, 8H), 6.36 (d, J=5.6 Hz, 1H), 5.97 (d, J=4.4 Hz, 1H), 5.10-5.06 (m, 1H ), 4.47 (t, J=5.6 Hz, 1H), 4.14-4.11 (m, 1H), 3.94 (dd, J=11.2 Hz, 1H), 3.83 (dd, J=11.6 Hz, 1H), 0.99 (s, 9H). ESI-LCMS: m/z 531.3 [M+H] ⁺ .

Preparation of intermediate compound (6-5). NaI (19.77 g, 131.91 mmol) and Ag ₂ O (15.29 g, 65.96 mmol), the mixture was stirred at room temperature for 5 hours. Subsequently, the reaction mixture was poured into ice water, extracted using EA, washed with brine and dried with anhydrous Na ₂ SO ₄ The solvent was evaporated and the residue was purified on silica gel to give 6-5 (23.70 g, 40.26 mmol, 61.04% yield) as a white solid and by-product TBDPS (5.20 g, 9. 81 mmol, yield 14.87%) as a white solid. ¹ H-NMR (400 MHz, DMSO): δ=8.31 (s, 1H), 8.11 (s, 1H), 7.63-7.60 (m, 4H), 7.47-7, 44 (m, 2H), 7.40-7.36 (m, 6H), 6.10 (d, J=4.4 Hz, 1H), 5.02 (t, J=4.8 Hz, 1H ), 4.69 (t, J=5.6 Hz, 1H), 4.18-4.14 (m, 1H), 3.95 (dd, J=11.6 Hz, 1H), 3.84 (dd, J=11.6 Hz, 1H), 3.78-3.75 (m, 2H), 3.45 (t, J=4.8 Hz, 1H), 3.16 (s, 3H) , 0.99 (s, 9H). ESI-LCMS: m/z 589.5 [M+H] ⁺ .

Preparation of intermediate compound (6-6). BzCl (11.22 g, 79.56 mmol) was added dropwise to a solution of 6-5 (31.23 g, 53.04 mmol) in pyridine (300.00 mL) at 0°C. The mixture was stirred at room temperature. for 2 hours. Subsequently, the solution was cooled to 0°C and ammonium hydroxide (20 ml, 30%) was added and the mixture was allowed to warm to room temperature, after which the solvent was evaporated, 300 ml of H ₂ O and 600 ml of EA were added separately into solution, extracted the aqueous layer with EA, combined the organic layers and washed with brine, dried over anhydrous Na ₂ SO ₄ , removed the solvent and purified the residue on silica gel to give 6-6 (28.70 g, 41.42 mmol, yield 78.09%) as a white solid. ESI-LCMS: m/z 693.4 [M+H] ⁺ .

Preparation of intermediate compound (6-7). A solution of 6-6 (28.70 g, 41.42 mmol) in EA (150.00 ml) was added with Pd/C (3.00 g) and MeOH (150.00 ml) under H ₂ atmosphere. The mixture was stirred at room temperature. for 5 hours. Subsequently, the reaction mixture was filtered and the filtrate was concentrated to give 6-7 (25.49 g, 38.22 mmol, 92.27% yield) as a gray solid. ESI-LCMS: m/z 667.3 [M+H] ⁺ .

Preparation of intermediate compound (6-8). Collidine (13.89 g, 114.66 mmol) was added to a solution of 6-7 (25.49 g, 38.22 mmol) and AgNO ₃ (12.98 g, 76.44 mmol) in dCm (30.00 ml). ) and MMTrCl (19.43 g, 57.33 mmol) and the mixture was stirred at RT. for 2 hours. Subsequently, the reaction mixture was poured into ice water, the organic layer was extracted using DCM, washed with brine and dried with anhydrous Na ₂ SO ₄ , the solvent was removed and the residue was purified on silica gel to give 6-8 (32.79 g, 34.92 mmol, yield 91.36%) as a gray solid.

Preparation of intermediate compound (6-9). A solution of 6-8 (32.79 g, 34.92 mmol) in THF (300.00 mL) was added to TBAF (1 M, 35.00 mL) and the mixture was stirred at room temperature for 15 hours. Subsequently, the solvent was removed and purified residue on silica gel using EA to obtain 6-9

- 15 045540 (22.22 g, 31.71 mmol, 90.82% yield) as a white solid. 1H-NMR (400 MHz, CDCl ₃ ): δ=8.68 (s, 1H), 8.32 (s, 1H), 8.04 (d, J=7.2 Hz, 2H), 7.61 -7.57 (m, 1H), 7.53-7.48 (m, 6H), 7.40 (d, J=8.8 Hz, 2H), 7.217.12 (m, 6H), 6, 73 (d, J=8.8 Hz, 2H), 6.09 (d, J=2.4 Hz, 2H), 4.08-4.02 (m, 2H), 3.93-3.87 (m, 1H), 3.72 (s, 3H), 3.58-3.53 (m, 1H), 3.43-3.39 (m, 3H), 3.24-3.19 (m , 4H), 2.19 (br., 1H).

Receipt (6-10). To a solution of 6-9 (14.00 g, 19.98 mmol), DMAP (488.19 mg, 4.00 mmol) and DIPEA (6.46 g, 49.95 mmol, 8.73 ml) in dry DCM (100.00 mL) CEPCl (5.68 g, 23.98 mmol) was added dropwise under argon. The mixture was stirred at room temperature for 1 hour. Subsequently, the reaction mixture was washed with 10% NaHCO ₃ (aq) and brine, dried over Na ₂ SO ₄ , the solvent was removed and the residue was purified by column chromatography and then concentrated to obtain the crude product . The crude product (10 g, dissolved in 10 mL ACN) was purified by preparative flash HPLC to give 6-10 (12.60 g, 13.98 mmol, 69.99% yield) as a white solid. Subsequently, the product was dissolved in dry toluene (15 ml), concentrated three times and washed three times with dry ACN. 1H-NMR (400 MHz, CDCl ₃ ): δ=9.12 (d, J=46.8 Hz, 1H), δ=8.71 (d, J=11.6 Hz, 1H), 8.50 (s, 0.6H), 8.22 (s, 0.4H), 8.04 (t, J=7.2 Hz, 2H), 7.63-7.59 (m, 1H), 7, 55-7.46 (m, 6H), 7.40-7.37 (m, 2H), 7.19-7.06 (m, 6H), 6.69 (dd, J=8.8 Hz, 2H), 6.03 (d, J=3.2 Hz, 1H), 4.36-4.24 (m, 2H), 3.92-3.78 (m, 2H), 3.71 (d , J=11.6 Hz, 3H), 3.67-3.33 (m, 7H), 3.29 (d, J=11.2 Hz, 3H), 3.17-3.10 (m, 1H), 2.88 (dd, J=27.2 Hz, 1H), 2.65-2.50 (m, 2H), 2.38 (d, J=4.4 Hz, 0.4H), 1.80 (d, J=4.0 Hz, 0.6H), 1.23-1.15 (m, 12H). ³¹ iNMR (400 MHz, CDCl ₃ ): 148.86, 148.22. ESI-LCMS: m/z 901.3 [M+H]+.

Example 4.

NC

The guanosine-based synthesis of 2'-methoxyethoxyamidophosphite 7-13 is shown in Scheme 5. Stereoselective coupling of C-3-azidoribose 1 to the nucleotide base 2-amino-6-chloropurine was hypothesized to represent key steps in the preparation of various 3'-NH-MMTr-2' -Oalkylated amidophosphites based on guanosine. This is essential to obtain efficient 2'-O-alkylation and to prevent any alkylation of the base. For this purpose, C3'-azido-chloro-aminopurine nucleoside 7-2 b was synthesized from the nucleotide base by coupling 1 and 2-amino-6-chloropurine in ~70% actual yield. The 2-amino group of 7-2 was subsequently protected with MMTrCl to yield 7-3, which after deprotection of the 2'-O-acetyl and 5'-O-toluoyl groups with NH ₄ OH/0°C was converted to the 2'-hydroxyl intermediate connection 7-4.

Deprotection of the 5'-O-toluene group of intermediate 7-4 by treatment with a base followed by reprotection of the 5'-OH group with a trityl group resulted in the 2'-OH intermediate 9-6 for alkylation. This inventive deprotection and reprotection strategy was necessary for the efficient synthesis of 2'-Oalkylated intermediate 7-7 under alkaline conditions (NaHMDS/DMF).

- 16 045540

Scheme 5.

Subsequent simultaneous hydrolysis of the 6-methoxy group and detritylation under carefully controlled acidic conditions yielded 3'-azido-2'-methoxyguanosine intermediate 7-8, which was treated with iBuCl to yield bisisobutyrate 7-9 followed by reduction and protection with MMTr of the 3'amino group of the intermediate to give 3'-NH-MMTr -2'-methoxyethoxyguanosine nucleoside 7-11. By further selective deprotection of 5'-O-isobutyrate using alkaline conditions at low temperature, the precursor 7-12 2'methoxyethoxyguanosine amidite was obtained. Phosphytylation of 7-12 using standard protocols resulted in the desired 2'-O-methoxyethoxyguanosine amidophosphite monomer 7-13.

Preparation of intermediate compound (7-2). To a solution of compound 1 (60.00 g, 152.00 mmol) in dry ACN (1300.00 ml) was added 2-amino-6-chloropurine (54.0 g, 318.5 mmol) and BSA (136.0 g , 667.82 mmol). The resulting suspension was stirred at 50°C until a transparent state was achieved. Subsequently, the mixture was cooled to -20°C and TMSOTf (60.0 g, 271.00 mmol) was added dropwise. Subsequently, the mixture was stirred at 70°C for 18 hours, and quenched sat. NaHCO ₃ solution and extracted using DCM. The organic layer was dried over Na ₂ SO ₄ , purified on silica gel and recrystallized to give 7-2 (55.20 g, 113.38 mmol) as a white solid. 1H-NMR (400

MHz, CDCl ₃ ): δ ppm 7.87 (d, J=8.4 Hz, 2H), 7.82 (s, 1H), 7.25 (d, J=8.0 Hz, 2H ), 6.09 (dd, J=5.6 Hz, 1H), 5.97 (d, J=4.0 Hz, 1H), 5.17 (s, 2H), 4.84-4.80 (m, 2H), 4.60-4.56 (m, 1H), 4.44-4.41 (m, 1H), 2.43 (s, 3H), 2.22 (s, 3H). ESI-LCMS: m/z 487.1 [M+H]+.

Preparation of intermediate compound (7-3). To a solution of 7-2 (33.2 g, 68.20 mmol) dissolved in DCM (300.00 mL), DMAP (1.67 g, 13.60 mmol) and DIEA (22.04 g, 136.4 mmol), MMTrCl (42.0 g, 136.4 mmol) was added. And the mixture was stirred at room temperature. for 16 hours in an N2 atmosphere. Subsequently, the reaction mixture was quenched using sat. NaHCO ₃ solution and extracted using DCM. The organic layer was dried over Na ₂ SO ₄ and the residue was purified on silica gel to give 7-3 (51.00 g, 67.20 mmol, 98.51% yield) as a white solid. ESI-LCMS: m/z 759.4 [M+H] ⁺ .

Preparation of intermediate compound (7-4). To a solution of 7-3 (54.00 g, 212.06 mmol) in THF (400 L) was added NH ₄ OH (100.00 ml) at 0°C. The reaction mixture was stirred at room temperature. for 48 hours, and concentrated and purified on silica gel with 1-2% MeOH in DCM to give 7-4 (51.00 g, 178.48 mmol, 99.98% yield) as a yellow solid. ¹ H-NMR (400 MHz, DMSO): δ ppm 8.26 (d, J=4.4 Hz, 1H), 8.17 (s, 1H), 7.78-7.76 (m , 2H), 7.35-7.25 (m, 12H), 7.16 (t, J=7.2 Hz, 2H), 6.82 (dd, J=8.8 Hz, 2H), 6 .14 (s, 1H), 5.62 (s, 1H), 4.40-4.35 (m, 4H), 3.69 (s, 3H), 3.36 (s, 1H), 2. 39 (s, 3H). ESI-LCMS: m/z 717.4 [M+H] ⁺ .

Preparation of intermediate compound (7-5). To a solution of sodium methoxide in methanol (300.00 ml, 2 N) was added 7-4 (47.00 g, 65.54 mmol), the mixture was stirred at rt for 1 hour. The mixture was concentrated

- 17 045540 was collected in vacuo to give the crude product, which was purified by CC (DCM:methyl alcohol=50:1) to give 7-5 (36.50 g, 61.38 mmol, 93.66% yield) as white solid matter. 1H-NMR (400 MHz, DMSO): δ ppm 8.15 (s, 1H), 7.47 (s, 1H), 7.41-7.32 (m, 4H), 7.32- 7.23 (m, 6H), 7.18 (t, J=7.16 Hz, 2H), 6.85 (d, J=8.68 Hz, 2H), 6.19 (s, 1H), 5.77 (s, 1H), 5.28 (s, 1H), 4.81 (s, 1H), 4.204.06 (m, 1H), 3.90 (s, 1H), 3.72 (s , 3H), 3.65-3.50 (m, 2H). ESI-LCMS: m/z 595.4 [M+H]+.

Preparation of intermediate compound (7-6). To a solution of 7-5 (36.50 g, 61.38 mmol) in dry DCM (5.00 mL) was added DIPEA (15.87 g, 122.77 mmol, 21.44 mL), DMAP (1.50 g , 12.28 mmol) and TrtCl (20.48 g, 73.66 mmol). The resulting suspension was stirred at room temperature. for 20 hours under nitrogen and concentrated in vacuo. The product was purified with cc (PE:EA=10:1 ~ 3:1) to give 7-6 (46.00 g, 54.96 mmol, 89.55% yield) as a white solid. ¹ H-NMR (400 MHz, DMSO): δ ppm 8.08 (s, 1H), 7.47-7.35 (m, 7H), 7.34-7.10 (m, 20H) , 6.80 (d, J=7.76 Hz, 2H), 6.25 (s, 1H), 5.81 (s, 1H), 4.86 (s, 1H) 4.10-3.87 (m, 2H), 3.69 (s, 3H), 3.56-3.30 (m, 2H), 3.29-3.02 (m, 3H), 2.00 (s, 1H). ESI-LCMS: m/z 837.4 [M+H] ⁺ .

Preparation of intermediate compound (7-7). To a solution of 7-6 (32.00 g, 38.23 mmol) in dry DMF (300.00 ml) was added dropwise tetrabutylammonium iodide (3.82 g, 11.47 mmol), 1-bromo-2-methoxyethane ( 15.94 g, 114.69 mmol) and NaHMDS (21.03 g, 114.69 mmol) at -10°C. The resulting suspension was stirred at -10°C for 30 hours and stirred at rt for 2 hours. The product was quenched using sat. NH ₄ Cl solution and extracted using DCM. The organic layer was concentrated in vacuo to give the crude product, which was purified over a silica gel column to give 7-7 (31.46 g, 35.15 mmol, 91.94% yield) as a white solid. 1H NMR (400 MHz, DMSO): δ ppm 8.04 (s, 1H), 7.40-7.34 (m, 6H), 7.33-7.08 (m, 20H), 6. 78 (d, J=8.9 Hz, 2H), 5.83 (s, 1H), 4.04-3.98 (m, 1H), 3.68 (s, 3H), 3.67-3 .46 (m, 3H), 3.44-3.36 (m, 2H), 3.27-3.15 (m, 2H), 3.12 (s, 3H), 2.88 (s, 1H ). ESI-LCMS: m/z 895.4 [M+H] ⁺ .

Preparation of intermediate compound (7-8). To a solution of compound 7-7 (42.00 g, 46.93 mmol) in 1,4-dioxane (150.00 ml) was added 6 N. hydrochloric acid solution (46.93 mmol, 150.00 ml) at room temperature. The resulting suspension was stirred at room temperature. within 30 min. The mixture was extracted with petroleum ether. The aqueous layer was stirred at room temperature. for 24 hours. The mixture was concentrated in vacuo to give 7-8 (20.00 g, 37.12 mmol) as a white solid. ¹ H-NMR (400 MHz, DMSO): δ ppm 11.0810.82 (m, 1H), 8.41-8.13 (m, 1H), 7.35-6.99 (m, 1H), 6.85-6.62 (m, 2H), 5.90-5.78 (m, 1H), 4.71 (t, J=5.16 Hz, 1H), 4.46 (t , J=4.64 Hz, 1H), 4.02-3.97 (m, 1H), 3.78-3.70 (m, 2H), 3.70-3.62 (m, 1H), 3.57 (dd, J=12.12 Hz, 1H), 3.43 (t, J=4.60 Hz, 2H), 3.18 (s, 3H). ESI-LCMS: m/z 367.2 [M+H] ⁺ .

Preparation of intermediate compound (7-9). Isobutyl chloride (17.45 g, 163.80 mmol) was added dropwise to a solution of 7-8 (20.00 g, 54.60 mmol) in pyridine (100.00 mL) at room temperature. The resulting suspension was stirred at room temperature. for 1 hour. The mixture was extracted using DCM and washed with water. The organic layer was concentrated in vacuo. Dissolved in pyridine and added dropwise isobutyl chloride (17.45 g, 163.80 mmol) at room temperature. The resulting suspension was stirred at room temperature. for 1 hour, concentrated and purified on a silica gel column to give 7-9 (13.00 g, 25.67 mmol) as a white solid. ESI-LCMS: m/z 507.3 [M+H] ⁺ .

Preparation of intermediate compound (7-10). To a solution of 7-9 (13.00 g, 25.67 mmol) in THF (100.00 ml) was added 10% palladium on carbon (1.30 g), the mixture was stirred at room temperature. for 20 hours in an H2 atmosphere. The mixture was filtered and the filtrate was concentrated in vacuo to give 7-10 (11.70 g, 24.35 mmol) as a brown solid. ESI-LCMS: m/z 481.3 [M+H] ⁺ .

Preparation of intermediate compound (7-11). To a solution of 7-10 (11.70 g, 24.35 mmol) in DCM (150.00 mL) was added MMTrCl (11.25 g, 36.53 mmol) and 2,4,6-collidine (5.90 g , 48.70 mmol) at 0°C, the mixture was stirred at room temperature. for 30 min and AgNO ₃ (6.20 g, 36.53 mmol) was added at 0°C. The mixture was stirred at room temperature. for 2 hours under N2, filtered and the organic layer was washed with water and dried over Na ₂ SO ₄ , concentrated to give the crude product, which was purified on a silica gel column to give 7-11 (8.5 g, 11.22 mmol) in as a white solid. EPILCMS: m/z 753.5 [M+H] ⁺ .

Preparation of intermediate compound (7-12). To a solution of 7-11 (8.5 g, 11.29 mmol) in pyridine (50.00 ml) was added dropwise 2 N. NaOH (50.00 ml) at 0°C, the mixture was stirred at 0°C for 20 min. Subsequently, the reaction mixture was neutralized with a saturated solution of NH ₄ Cl (aq) to achieve pH=7-8, and 300 ml of H ₂ O and 800 ml of DCM were added separately to the solution, the aqueous layer was extracted with DCM, the combined organic layer was washed with brine, dried over anhydrous Na ₂ SO ₄ , the solvent was removed and the residue was purified on silica gel to give 7-12 (6.3 g, 9.23 mmol, 81.73% yield) as a white solid. 1H-NMR (400 MHz, DMSO): δ ppm 11.78 (br.s, 1H), 11.41 (s, 1H), 8.11 (s, 1H), 7.46 (t, J=7.28 Hz, 4H), 7.31-7.21 (m, 6H), 7.15 (t, J=7.32 Hz, 2H), 6.76 (d, J=8.92 Hz, 2H), 5.82 (s, 1H), 5.08 (t, J=4.08 Hz, 1H), 3.99-3.85 (m, 3H), 3.65 (s, 3H ), 3.62-3.55 (m, 1H), 3.423.38 (m, 2H), 3.29-3.19 (m, 1H), 3.10 (s, 3H), 3.06- 2.99 (m, 1H), 2.89-2.74 (m, 2H), 1.96 (d, J=4.08 Hz, 1H), 1.20-1.12 (m, 6H) . ESI-LCMS: m/z 883.4 [M+H] ⁺ .

Receipt (7-13). To a solution of 7-12 (6.3 g, 9.10 mmol) in DCM (60 ml) was added DMAP (222.00 mg) and

- 18 045540

DIPEA (5.9 ml). Subsequently, CEPCl (2.60 g) was added. The reaction mixture was stirred at room temperature. for 1 hour, the mixture was washed with saturated NaHCO ₃ solution and brine, dried over Na ₂ SO ₄ , purified using preparative flash HPLC. The result was 5.04 g of 7-13 as a white solid. ¹ H-NMR (400 MHz, CDCl ₃ ): δ ppm 11.96 (br. s, 1H), 8.57 (s, 1H), 7.98-7.67 (m, 1H), 7.55-7.46 (m, 4H), 7.42-7.34 (m, 2H), 7.25-7.18 (m, 4H), 7.18-7.08 (m, 2H ), 6.78-6.68 (m, 2H), 5.79-5.69 (m, 1H), 4.27-4.11 (m, 2H), 4.10-3.73 (m , 2H), 3.73-3.67 (m, 3H), 3.66-3.31 (m, 7H), 3.30-3.21 (m, 3H), 2.072.97 (m, 1H ), 2.94-2.78 (m, 1H), 2.70-2.50 (m, 3H), 2.30-1.68 (m, 1H), 1.30-1.07 (m , 18H). ³¹ P NMR (162 MHz, CDCl3): 149.09, 148.09. ESI-LCMS: m/z 883.4 [M+H]+.

Example 5.

Properly protected 2'-O-ethyl-3'-amino-5'-amidophosphite (Examples 8, 9, 10, 11) was obtained after the chemical transformations shown in Schemes 6-10.

First, for the synthesis of 3'-NH-MMtr-2'-O-ethyl thymine amidophosphites (Example 9), intermediate 2 was protected, for example, with methyl propiolate in the presence of dimethylaminopyridine (Scheme 6) to give intermediate 8-4 with the base N-3 protected to increase yield in 2'-O-alkylation. Further deacetylation of 8-4 to yield C-2'-hydroxy intermediate 8-5.

Scheme 6.

Additional alkylation using ethane iodine afforded 2'O-ethyl nucleoside 8-6. Intermediate 8-6 was converted to thymine 2'-O-ethyl-3'-amino-5'amidophosphite 8-11 via a chemical reaction similar to that for compound 4-10.

- 19 045540 shown in the previous diagram 1.

Preparation of intermediate compound (8-4). DMAP (1.2 g, 9.92 mmol) was added to a solution of 8-2 (22.0 g, 49.62 mmol) in MeCN (400 mL). 3 (5.8 g, 419.5 mmol) was subsequently added and the mixture was stirred at RT. under N2 for 2 hours. Concentrate and purify on a silica gel column using (PE:EA=6:1) to give 8-4 (22.0 g, 40.63 mmol, 81.9% yield) in in the form of yellow oil. ESI-LCMS: m/z 564 [M+Na]+.

Preparation of intermediate compound (8-5). To a solution of 8-4 (28.0 g, 51.71 mmol) in MeOH (400 ml) was added conc. aqueous solution of NH ₄ OH (28 ml) at 0°C. The reaction mixture was stirred at 0°C for 1.5 hours. The mixture was concentrated and purified by a silica gel column using (PE:EA=10:1~2:1) to obtain 8-5 (21.0 g, 42. 04 mmol, yield 81.3%) as a yellow oil. ESILCMS: m/z 522 [M+Na] ⁺ .

Preparation of intermediate compound (8-6). Ag ₂ O (18.6 g, 80.08 mmol) was added to a solution of 8-5 (20.0 g, 40.04 mmol) in iodine ethane (100 mL). The reaction mixture was stirred at 50°C for 5 hours, filtered through diatomaceous earth and concentrated to give 8-6 (16.0 g, 30.33 mmol, 75.7% yield) as a yellow oil, which was used directly in the next step . ESI-LCMS: m/z 528 [M+H] ⁺ .

Preparation of intermediate compound (8-7). Pyrrolidine (8.63 g, 121.32 mol, 12 mL) was added to a solution of 8-6 (16.0 g, 30.33 mmol) in MeCN (400 mL), and the reaction mixture was stirred at room temperature. during the night. Concentrate and purify on a silica gel column using (DCM:MeOH=100:1~50:1) to give 7 (12.0 g, 27.94 mmol, 92.1% yield) as a yellow oil. ESI-LCMS: m/z 430 [M+H] ⁺ .

Preparation of intermediate compound (8-8). Pd/C (1.2 g) was added to a solution of 8-7 (12.0 g, 27.94 mmol) in THF (200 ml), and the mixture was stirred at room temperature. in an H ₂ atmosphere overnight. According to LC-MS, 7 was completely consumed. Filter and wash with DCM (100 mLx3), subsequently concentrate to give 8-8 (11.0 g, 27.27 mmol, 97.6% yield) as a gray solid, which is used directly in the next step. ESI-LCMS: m/z 404 [M+H] ⁺ .

Preparation of intermediate compound (8-9). To a solution of 8-8 (10.0 g, 24.79 mmol) in DCM (80 mL) was added MMTrCl (11.4 g, 37.18 mmol), 2,4,6-collidine (2.0 g, 16 .61 mmol, 6.5 ml) and AgNO ₃ (6.3 g, 37.18 mmol), the mixture was stirred at room temperature. for 1.5 hours. The mixture was filtered and the organic layer was washed with water and dried over Na ₂ SO ₄ , subsequently concentrated and purified on a silica gel column using (PE:EA=5:1~1:1) to obtain 8-9 ( 16.0 g, 23.68 mmol, 95.5% yield) as a light yellow solid.

Preparation of intermediate compound (8-10). 8-9 (4.0 g, 5.92 mmol) was added to 1.0 N. NaOH solution (20 ml, MeOH/H ₂ O = 9:1). The reaction mixture was stirred at 40°C for 2 hours, concentrated and extracted using DCM (20 mlx2), the organic layer was dried over Na ₂ SO ₄ and concentrated, the residue was purified on a silica gel column using a mixture of (DCM:MeOH=200:1 ~50:1), yielding 8-10 (3.0 g, 53.8 mmol, 90.9 yield) as a white solid.

Receipt (8-11). To a solution of 8-10 (2.36 g, 4.23 mmol) in DCM (2.0 mL) was added DMAP (103 mg, 0.8 mmol) and DIPEA (2.2 g, 16.92 mmol, 2. 96 ml). CEPCl (1.0 g, 4.23 mmol) was subsequently added. The reaction mixture was stirred at room temperature. for 1 hour, washed with saturated NaHCO ₃ solution (5 ml), separated the organic layer and washed the aqueous layer with DCM (10 mlx2). The combined organic layer was washed with brine, dried over Na ₂ SO ₄ , concentrated and purified by preparative flash HPLC to give 8-11 (2.45 g, 3.23 mmol, 76.36% yield) as a white solid. ^1H NMR (400 MHz, CDCl ₃ ) δ 8.62 (s, 1H), 7.74 (dd, J=1.4 Hz, 0.5H), 7.60-7.50 (m, 4H) , 7.51-7.41 (m, 2H), 7.34-7.16 (m, 7H), 7.12 (d, J=1.4 Hz, 0.5H), 6.88-6 .76 (m, 2H), 5.66 (s, 1H), 4.37-4.23 (m, 1H), 4.16-4.05 (m, 1H), 4.05-3.94 (m, 0.5H), 3.88-3.74 (m, 4.5H), 3.72-3.35 (m, 3H), 3.22 (td, J=10.3, 4, 7 Hz, 0.5H), 3.03-2.89 (m, 1.5H), 2.80-2.69 (m, 1H), 2.61 (t, J=6.5 Hz, 1H ), 2.37 (td, J=6.6, 1.3 Hz, 1H), 1.97 (d, J=3.5 Hz, 0.5H), 1.91 (dd, J=11, 4, 1.2 Hz, 3H), 1.52 (d, J=4.7 Hz, 0.5H), 1.29-1.17 (m, 12H), 1.08 (td, J=7 ,0, 4.9 Hz, 3H). ³¹ P NMR (162 MHz, CDCh) δ 149.31, 147.14. ESI-LCMS: m/z 576 [M+H] ⁺ .

Example 6.

NC

MMTrHN'OEt 9-3

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2'-O-ethyl-NH-benzoyl-5-methylcytosine amidophosphite 9-3 was prepared by converting the thymidine intermediate 8-8 to the 3'-aminocytidine analogue 9-1 followed by phosphitylation using known protocols to yield the desired amidophosphite monomer 2' -Oethylcytidine 9-3, as shown in Scheme 7 below.

Scheme 7.

Preparation of intermediate compound (9-1). To a solution of 8-8 (8.2 g, 12.09 mmol) in dry MeCN (40 ml) was added TPSCl (2.5 g, 24.18 mmol) and DMAP (2.95 g, 24.18 mmol), TEA (2.5 g, 24.18 mmol, 3.4 mL) was subsequently added and the reaction mixture was stirred at rt under N2 for 12 h. Aqueous NH4OH (20 mL) was subsequently added and the resulting mixture was left to stir at rt. .T. for an additional 3 hours, then concentrated and purified on a silica gel column to give 9-1 (7.7 g, 11.35 mmol, 93.9% yield) as a light yellow oil.

Preparation of intermediate (9-2). To a solution of 9-1 (8.0 g, 11.86 mmol) in pyridine (80 ml) was added benzoyl chloride (2.0 g, 14.23 mmol, 1.6 ml) at 0°C, the reaction mixture was allowed to warm to room temperature and stirred for another 1 hour, 2.0 N was added. NaOH (80 ml, MeOH/H ₂ O = 9:1) at 0°C, subsequently the mixture was left to stir at 0°C for another 2 hours, then quenched with sat. NH ₄ Cl solution (20 ml), extracted with DCM (30 mlx2), dried over Na ₂ SO ₄ , filtered and concentrated. The residue was purified by silica gel chromatography (DCM:MeOH=200:1~50:1) to give 9-2 (5.4 g, 8.2 mmol, 66.3% yield) as a yellow solid. 1H NMR (400 MHz, CDCl ₃ ) δ 13.35 (s, 1H), 8.40-8.28 (m, 2H), 7.93 (d, J=1.3 Hz, 1H), 7, 61-7.51 (m, 5H), 7.51-7.43 (m, 4H), 7.33-7.22 (m, 6H), 6.89-6.72 (m, 2H), 5.66 (s, 1H), 4.26-4.19 (m, 2H), 4.08 (dt, J=10.4, 2.0 Hz, 1H), 3.80 (s, 3H) , 3.74-3.63 (m, 1H), 3.12 (td, J=10.3, 4.5 Hz, 1H), 2.95-2.84 (m, 2H), 2.05 (d, J=1.1 Hz, 3H), 1.91 (d, J=4.5 Hz, 1H), 1.29 (t, J=7.1 Hz, 1H), 1.13 (t , J=7.0 Hz, 3H).

Receipt (9-3). To a solution of 9-2 (4.2 g, 6.36 mmol) in DCM (4 ml) was added DMAP (776 mg, 6.36 mmol), DIPEA (821 mg, 6.36 mmol, 1.1 ml) and subsequently CEPCl (1.5 g, 6.36 mmol). The reaction mixture was stirred at room temperature. for 1 hour extinguished sat. NaHCO ₃ solution, extracted with DCM (30 mlx2), the organic layer was dried over Na ₂ SO ₄ and the solvent was evaporated, the residue was purified by preparative flash HPLC to give 9-3 (4.9 g, 5.69 mmol, yield 89.47%) as a yellow solid. 1H NMR (400 MHz, CDCl ₃ ) δ 13.31 (s, 1H), 8.40-8.31 (m, 2H), 8.03 (s, 0.5H), 7.60-7.52 (m, 5H), 7.52-7.43 (m, 4H), 7.40 (s, 0.5H), 7.31-7.18 (m, 7H), 6.86-6.78 (m, 2H), 5.67 (d, J=2.3 Hz, 1H), 4.42-4.26 (m, 1H), 4.24-4.11 (m, 1H), 4, 06-3.98 (m, 0.5H), 3.90-3.81 (m, 1.5H), 3.79 (d, J=3.0 Hz, 3H), 3.75-3, 56 (m, 2H), 3.55-3.37 (m, 1H), 3.22 (td, J=10.4, 4.5 Hz, 0.5H), 3.04-2.87 ( m, 1.5H), 2.86-2.73 (m, 1H), 2.63 (t, J=6.4 Hz, 1H), 2.40 (t, J=6.5 Hz, 1H ), 2.13 (dd, J=19.9, 1.1 Hz, 3H), 1.99 (d, J=3.8 Hz, 0.5H), 1.54 (d, J=4, 5 Hz, 0.5H), 1.31-1.21 (m, 12H), 1.10 (td, J=7.0, 4.8 Hz, 3H). ³¹ P NMR (162 MHz, CDCl ₃ ) δ 149.48, 147.08. ESI-LCMS: m/z 861 [M+H]+.

Example 7.

MMTrHN OE1

10-B

The synthesis of 2'-O-ethyladenosine amidophosphite 10-8 was performed as shown below in Scheme 8. To achieve 2'-O-alkylation in high yield, protection of the exocyclic amino group of 6-4 with a bulky protecting group, such as a 4-monomethoxytrityl group, is necessary. Subsequently, reaction of intermediate 6-4 with Ag ₂ O/EtI afforded 2'-O-ethyl intermediate 10-2 in 94% actual yield after deprotection of the MMTr- group. This protection strategy provided efficient synthesis of the key intermediate 3'-azido-2'-OEt 10-3 in high yield. This intermediate 10-3 was converted to 2'-O-ethyl adenosine amidophosphite 108 using chemical reactions similar to those described for compound 6-10 (Scheme 4).

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Scheme 8.

Preparation of intermediate compound (10-1). To a solution of compound 6-4 (23.6 g, 44 mmol) in DCM (400.00 ml), DMAP (543 mg, 4 mmol) and TEA (9.2 g, 9 mmol), MMTr-Cl (20 .6 g, 67 mmol), the mixture was stirred for 16 hours at 20°C under N ₂ atmosphere. According to LC-MS, 6-4 was consumed; the reaction mixture was quenched with sat. NaHCO ₃ solution and extracted using DCM. The organic layer was dried over Na ₂ SO ₄ and concentrated to give the crude product, which was purified on silica gel with 20-50% EtOAc in petroleum ether to give compound 10-1 (26.1 g, 32 mmol, 73.09% yield) as a yellowish solid. ¹ H-NMR (400 MHz, DMSO): δ=8.38 (s, 1H), 7.84 (s, 1H), 7.60-7.55 (m, 4H), 7.46-7, 20 (m, 19H), 6.84 (d, J=8.8 Hz, 2H), 6.35 (d, J=5.6 Hz, 1H), 5.95 (d, J=4.4 Hz, 1H), 5.14-5.12 (m, 1H), 4.46 (t, J=5.6 Hz, 2H), 4.11-4.05 (m, 1H), 3.90 -3.82 (m, 2H), 3.72 (s, 3H), 0.94 (s, 8H). ESI-LCMS: m/z 803.4 [M+H]+.

Preparation of intermediate compound (10-2). To a solution of compound 10-1 (26.1 g, 32 mmol) in CH ₃ CH ₂ I (200 ml) was added Ag ₂ O (9.7 g, 42 mmol). The resulting mixture was refluxed at 80°C for 1 hour. Subsequently, the mixture was filtered to obtain compound 10-2 (25.4 g, 30 mmol, 94.04% yield) as a yellow solid. ESI-LCMS: m/z 831.3 [M+H]+.

Preparation of intermediate compound (10-3). A solution of compound 10-2 (25.4 g, 30 mmol) was dissolved in DCM (200 ml), TCA (12 ml) was added and the mixture was stirred at 20°C for 6 hours. LC-MS indicated that 10-2 was consumed , after which the mixture was washed with saturated NaHCO ₃ solution, concentrated to obtain the crude product, which was purified on silica gel with 1-3% MeOH in DCM to obtain compound 10-3 (15.7 g, 28 mmol, 91.95% yield) as white solid. ¹ H-NMR (400 MHz, DMSO): δ=8.33 (s, 1H), 8.10 (s, 1H), 7.63-7.60 (m, 4H), 7.47-7, 37 (m, 8H), 6.09 (d, J=4.0 Hz, 1H), 4.95 (t, J=4.8 Hz, 1H), 4.67 (t, J=5.6 Hz, 1H), 4.17-4.13 (m, 1H), 3.97-3.93 (m, 1H), 3.86-3.82 (m, 1H), 3.683.63 (m, 2H), 1.15-1.11 (m, 3H), 0.97 (s, 9H). ESI-LCMS: m/z 589.2 [M+H] ⁺ .

Preparation of intermediate compound (10-4). To a solution of compound 10-3 (15.7 g, 28 mmol) in pyridine (150 ml) at 0°C, BzCl (4.7 g, 34 mmol) was added with a syringe over 5 min, the mixture was subsequently allowed to warm to 20° WITH. Subsequently, the mixture was stirred at rt under N ₂ for 1 hour. The solution was cooled to 0° C. and ammonium hydroxide (20 mL, 30%) was added and the mixture was allowed to warm to rt and stirred at rt. for 2 hours. The mixture was diluted with EA and water, extracted with EA, the combined organic layer was washed with brine, dried over anhydrous Na ₂ SO ₄ , concentrated to give the crude product, which was purified on silica gel to give compound 10-4 (10, 9 g, 16 mmol, yield 58.52%) as a white solid. ESI-LCMS: m/z 663.8 [M+H] ⁺ .

Preparation of intermediate compound (10-5). To a solution of compound 10-4 (10.9 g, 16 mmol) in

- 22 045540

THF (100 ml) was added Pd/C (1.9 g), the mixture was stirred at 20°C for 6 hours under H ₂ atmosphere, then filtered and the filtrate was concentrated to give compound 10-5 (9.8 g, 15 mmol , yield 93.95%) as a white solid. ESI-LCMS: m/z 637.4 [M+H]+.

Preparation of intermediate compound (10-6). To a solution of compound 10-5 (9.8 g, 15 mmol) in anhydrous DCM (100 ml), collidine (3.7 g, 31 mmol), AgNO ₃ (3.9 g, 31 mmol) and MMTr-Cl were added (5.7 g, 18 mmol), the mixture was stirred at 20°C for 1 hour under N2 atmosphere, then filtered, washed with _H2O and dried over _Na2SO4 , concentrated to obtain a residue, which was purified on _silica gel with 20-50% EA in petroleum ether to give compound 10-6 (10.5 g, 11 mmol, 75.19% yield) as a white solid.

Preparation of intermediate compound (10-7). To a solution of compound 10-6 (10.5 g, 11 mmol) in THF (100 ml) was added TBAF (1M) (11 mmol, 12 ml), the mixture was stirred at 20°C overnight, subsequently washed with saturated NaHCO ₃ solution , was concentrated to give a residue, which was purified on silica gel with 1-2% MeOH in DCM to give compound 10-7 (5.6 g, 8 mmol, 72.28% yield) as a white solid.

Receipt (10-8). To a solution of compound 10-7 (5.6 g, 8 mmol) in DCM (50 ml) was added DMAP (204 mg, 2 mmol) and DIPEA (3.2 g, 25 mmol, 4.3 ml) followed by CEPCl (2.3 g, 10 mmol). The reaction mixture was stirred at room temperature. TLC indicated that 10-7 was consumed, the mixture was washed with saturated NaHCO ₃ and brine, dried over Na ₂ SO ₄ , concentrated to give the crude product and purified by column chromatography and preparative flash HPLC to give 10-8 (5.30 g, 6.09 mmol, 72.88% yield) as a white solid. 1H NMR (400 MHz, CDCl ₃ ): δ=9.17 (s, 0.5H), 9.07 (s, 0.5H), 8.75 (s, 0.5H), 8.72 (s, 0.5H), 8.51 (s, 0.5H), 8.22 (s, 0.5H), 8.05 (t, J=7.2 Hz, 2H), 7.65-7.61 (m, 1H), 7.57-7.48 (m, 6H), 7.39 (dd, J=8.8 Hz, 2H), 7.21-7.10 (m, 6H), 6, 74-6.69 (m, 2H), 6.01 (d, J=3.9 Hz, 1H), 4.40-4.21 (m, 2H), 4.14-4.11 (m, 0.5H), 3.99-3.93 (m, 0.5H), 3.88-3.80 (m, 1H), 3.70-3.50 (m, 7H), 3.41- 3.32 (m, 1H), 2.93-2.80 (m, 2H), 2.68-2.50 (m, 2H), 2.27 (d, J=4.0 Hz, 0. 5H), 2.27 (d, J=4.0 Hz, 0.5H), 1.25-1.12 (m, 15H). ³¹ P NMR (162 MHz, CDQ3): 148.79, 148.08. ESI-LCMS: m/z 870.3 [M+H] ⁺ .

Example 8.

NC

12-9

The synthesis of guanosine amidophosphite 2'-O-ethyl 12-9 is shown below in Scheme 9, starting with the key 2'-hydroxyl intermediate 7-4. Intermediate 7-4 was 2'-alkylated using EtI/Ag ₂ O or Et//K ₂ CO ₃ ; The desired intermediate 2'OEt 7-5 could not be prepared in high yield, as shown below in Scheme 9.

Scheme 9.

Thus, an improved method was developed to improve on 2'-alkylation and create a more stable guanosine intermediate under acidic conditions, as shown below in Scheme 10. By reacting the 6-chloro intermediate 7-4 with NaH/BnOH/THF, simultaneous substitution of C-6-Cl with BnOH and hydrolysis of the 5'-O-toluene ester in one step to yield C-6-OBn intermediate 12-1.

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Scheme 10.

The 5'-Hydroxy group was selectively protected with DMTr to yield the 2'-OH nucleoside 12-2. The resulting intermediate 12-2 was reacted with NaH/EtI to give the 2'-O-ethyl intermediate 12-3, which was subjected to acidic conditions (TFA/DCM) to give the 5'-hydroxyl intermediate 12-4 without any depurinization. Subsequently, reaction of 12-4 with iBuCl afforded bis-isobutyrate intermediate 12-5, which upon hydrogenation using Pd/C converted to 2'-O-ethylguanosine intermediate 12-6. This deprotection and reprotection strategy was successfully applied for efficient 2′-O-alkylation under alkaline conditions (NaH/EtI/THF). The resulting 2'-Oethyl intermediate 12-6 was converted to the desired 2-0-ethylguanosine amidophosphite monomer 12-9 by following a procedure similar to that described for compound 7-13 in Example 12.

Preparation of intermediate compound (12-1). To a solution of compound 7-4 (48.0 g, 66 mmol) in 50 ml THF was added 90 ml sodium benzyl oxide in benzyl alcohol (freshly prepared by adding NaH (13 g) in 90 ml benzyl alcohol). The reaction mixture was stirred at room temperature for 1 hour and treated with 50 ml of saturated NH ₄ Cl solution. The mixture was extracted with CH ₂ Cl ₂ and the combined organic layers were dried over Na2SO4. The crude product was purified by column chromatography to give 12-1 (17.5 g, 26 mmol, 45.63%). ¹ H-NMR (400 MHz, DMSO): δ ppm 8.13 (s, 1H), 7.59 (s, 1H), 7.34-7.16 (m, 15H), 6.82 (d, J=8.8 Hz, 2H), 6.13 (s, 1H), 5.76 (s, 1H), 5.20-5.12 (m, 1H), 5.10-4, 78 (m, 2H), 3.88-3.70 (m, 2H), 3.60 (s, 3H), 3.58-3.51 (m, 1H), 3.34 (s, 1H) . ESI-LCMS: m/z 671.2 [M+H]+.

Preparation of intermediate compound (12-2). To a solution of compound 12-1 (17.5 g, 26 mmol) in pyridine (200 ml) was added DMTrCl (10.5 g, 31 mmol) at 0°C. Subsequently, the mixture was stirred at 35°C for 4 hours. The solvent was evaporated. The crude product was purified by column chromatography to give product 12-2 (21.8 g, 22 mmol, 85.87% yield) as a white solid. ESI-LCMS: m/z 974.6 [M+H] ⁺ .

Preparation of intermediate compound (12-3). To a solution of 12-2 (21.8 g, 22 mmol) in THF (300 ml) was added NaH (1.1 g, 44 mmol) at 0°C. Subsequently, EtI (10.4 g, 67 mmol) was added to the reaction mixture and the mixture was stirred at 35°C for 4 hours and then treated with 50 ml of saturated NH ₄ Cl solution. The mixture was extracted with CH ₂ Cl ₂ and the combined organic layers were dried over Na ₂ SO ₄ . The solvent was evaporated to give 12-3 (21.2 g, 21 mmol, 94.55% yield) as a yellow solid.

Preparation of intermediate compound (12-4). To a solution of 12-3 (21.2 g, 21 mmol) in DCM (200 ml) was added TFA (10 ml) at 0°C. The mixture was subsequently stirred at 35°C for 4 hours. The mixture was quenched with saturated NaHCO ₃ solution and washed with brine, dried over Na ₂ SO ₄ , purified by column chromatography to give 12-4 (8.7 g, 20 mmol, yield 96 .32%) as a white solid. 1H-NMR (400 MHz, DMSO): δ ppm 8.17 (s, 1H), 7.53-7.50 (m, 2H), 7.42-7.36 (m, 3H), 6.61 (d, J=8.8 Hz, 2H), 5.91-5.90 (m, 1H), 5.76 (s, 1H), 5.51-5.50 (m, 2H) , 4.76-4.75 (m, 1H), 4.55-4.54 (m, 1H),

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4.06-3.97 (m, 3H), 3.64-3.58 (m, 4H), 2.51 (s, 1H). ESI-LCMS: m/z 427.2 [M+H]+.

Preparation of intermediate compound (12-5). To a solution of 12-4 (8.7 g, 20 mmol) in pyridine (100 ml) iBuCl (5.4 g, 51 mmol) was added and the mixture was stirred at room temperature. for 1.5 hours. The organic layer was washed with water and dried over Na ₂ SO ₄ and purified by column chromatography to give 12-5 (8.5 g, 15 mmol, 73.54% yield) as a white solid. ESI-LCMS: m/z 567.4 [M+H] ⁺ .

Preparation of intermediate compound (12-6). To a solution of 12-5 (8.5 g, 15 mmol) in THF (100 ml) was added Pd/C (801 mg, 6 mmol) at 25°C. The mixture was subsequently stirred under H ₂ for 16 hours, filtered and washed with EA, concentrated to give crude product 12-6 (6.7 g, 14 mmol, 99.15% yield) as a yellow solid. ESI-LCMS: m/z 451.3 [M+H] ⁺ .

Preparation of intermediate compound (12-7). To a solution of compound 12-6 (6.7 g, 14 mmol) in anhydrous DCM (70 ml) was added collidine (3.7 g, 31 mmol), AgNO ₃ (3.0 g, 17 mmol) and MMTr-Cl ( 5.5 g, 17 mmol), the mixture was stirred at 20°C for 1 hour under N2, subsequently filtered, washed with H2O and dried over Na2SO4, concentrated to give a residue, which was purified by column chromatography to give compound 12-7 (8.4 g, 11 mmol, 75.19% yield) as a white solid.

Preparation of intermediate compound (12-8). Compound 12-7 (8.4 g, 11 mmol) was added to 80 ml of 1 N. a solution of NaOH in a mixture of MeOH/THF/H ₂ O (65/30/5) at 0°C. The suspension was stirred at 0°C for 2 hours. The reaction mixture was quenched by adding sat. NH ₄ Cl solution. The solution was extracted using DCM and the combined organic layers were washed with sat. NaHCO ₃ solution, saline, dried over Na2SO4, filtered and concentrated. The residue was purified by column chromatography to give product 12-8 (5.60 g, 8.35 mmol, 72.28% yield) as a white solid. ¹ H-NMR (400 MHz, DMSO): δ ppm 12.13 (br. s, 1H), 11.38 (s, 1H), 8.10 (s, 1H), 7.47-7 .44 (m, 4H), 7.30-7.13 (m, 8H), 6.75 (d, J=8.96 Hz, 2H), 5.79 (s, 1H), 5.10 ( t, J=4.28 Hz, 1H), 3.95-3.89 (m, 3H), 3.65 (s, 3H), 3.46-3.26 (m, 2H), 2.84 -2.80 (m, 2H), 2.79-2.72 (m, 1H), 1.85 (s, 1H), 1.18-1.13 (m, 6H). 1.08-0.98 (m, 3H).

Receipt (12-9). To a solution of 12-8 (5.60 g, 8.35 mmol) in DCM (50 ml) was added DMAP (220 mg) and DIPEA (3 ml). CEPCl (2.6 g) was subsequently added. The reaction mixture was stirred at room temperature. for 1 hour, washed with saturated NaHCO ₃ solution and brine, dried over Na ₂ SO ₄ , purified by column chromatography and preparative flash HPLC. The result was 5.4 g (70.04%) of 12-9 as a white solid. ¹ H-NMR (400 MHz, CDCl3): δ ppm 12.11 (br. s, 1H), 8.88 (s, 1H), 7.97-7.64 (m, 1H), 7 .51-7.49 (m, 4H), 7.39-7.34 (m, 2H), 7.23-7.09 (m, 6H), 6.75-6.69 (m, 2H) , 5.67-5.65 (m, 1H), 4.22-4.08 (m, 2H), 3.84-3.29 (m, JN), 2.87-2.51 (m, 5H), 2.08-1.61 (m, 1H), 1.24-1.07 (m, 18H), 1.01-0.98 (m, 3H). ³¹ iNMR (162 MHz, CDCl3): 149.03, 147.96. ESI-LCMS: m/z 853.4 [M+H] ⁺ .

Example 9.

- 25 045540

Scheme 11.

13-16

Preparation of intermediate compound (13-3). PMBCl (113.7 g, 908.2 mmol) was added to a solution of 13-2 (260.0 g, 605.5 mmol) and DBU (305.0 g, 1.21 mmol) in DMF (1.5 L) . The mixture was stirred at room temperature. for 2 hours, poured into cold water, extracted with EA, washed with brine and dried over anhydrous Na ₂ SO ₄ , concentrated to give crude product 13-3 (460.0 g, crude) as a yellow oil. ESI-LCMS: m/z 550 [M+H]+.

Preparation of intermediate compound (13-4). To a solution of 13-3 (460.0 g, crude) in a mixture of THF (1.5 L) and H ₂ O (500 ml) was added NaOH (167.4 g, 4.1 mol). The reaction mixture was stirred at room temperature. for 15 h and subsequently poured into cold water, extracted using EA. The combined organic layer was washed with brine and dried over anhydrous Na ₂ SO ₄ , concentrated to give the crude product, which was washed with PE:EA=5:1 to give product 13-4 (212.0 g, 544.4 mmol, yield 89, 92% in two stages) as a white solid. EPILCMS: m/z 390 [M+H] ⁺ .

Preparation of intermediate compound (13-5). To a solution of 13-4 (207.0 g, 531.6 mmol) and pyridine (210.2 g, 2.6 mol, 214 mL) in DCM (2 L) was added DMTrCl (197.6 g, 584.8 mmol ) in an N2 atmosphere. The mixture was stirred at room temperature for 1 hour, quenched with MeOH, concentrated to give a residue, which was purified by column chromatography to give 13-5 (330.0 g, 477.0 mmol, 89.7% yield) as yellow solid matter. 'H-NMR (400 MHz, DMSO-d6): δ=7.81 (d, J=8.0 Hz, 1H), 7.39 (d, J=7.6 Hz, 2H), 7.33 (t, J=7.6 Hz, 2H), 7.28-7.25 (m, 7H), 6.91 (d, J=7.6 Hz, 4H), 6.86 (d, J= 8.4 Hz, 2H), 6.33 (d, J=5.2 Hz, 1H, exchange with D2O), 5.49 (d, J=8.0 Hz, 1H), 4.91 (s, 2H), 4.51 (dd, J=5.2, 8.8 Hz, 1H), 4.21 (t, J=6.8 Hz, 1H), 4.08-4.05 (m, 1H ), 3.75 (s, 6H), 3.72 (s, 3H). ESI-LCMS: m/z 692 [M+H] ⁺ .

Preparation of intermediate compound (13-6). To a solution of 13-5 (150.0 g, 216.8 mmol) in dry THF (750 ml) was added NaH (10.4 g, 433.7 mmol) and methyl p-toluenesulfonate (66.1 g, 433. 7 mmol) at 0°C, the mixture was left to stir at room temperature. and TBAI (16.0 g, 43.3 mmol) was added. The reaction mixture was stirred at 80°C for 2 days and then cooled to saturation. aqueous NH ₄ Cl, extracted with EA, washed with brine, dried over Na ₂ SO ₄ , concentrated to give a residue which was purified by column chromatography to give 136 (55.0 g, 70.0 mmol, yield 32.28 %) as a yellow solid. ESI-LCMS: m/z 786 [M+H] ⁺ .

Preparation of intermediate compound (13-7). To a solution of 13-6 (52.0 g, 66.1 mmol) and K2CO3 (18.2 g, 132.3 mmol) in dry DMF (500 ml) was added methyl p-toluenesulfonate (18.4 g, 99. 2 mmol) in an argon atmosphere. The mixture was stirred at room temperature. for 15 hours, poured into cold water, extracted using EA, washed with brine and dried over anhydrous Na ₂ SO ₄ , concentrated to give a residue which was purified by column chromatography to give 7 (38.5 g, 48.1 mmol , yield 72.74%) as a white solid. 'H-NMR (400 MHz, DMSO-d ₆ ): δ=7.81 (d, J=8.4 Hz, 1H), 7.41-7.38 (m, 2H), 7.34-7 .31 (m, 2H), 7.28-7.23 (m, 7H), 6.92-6.90 (m, 4H), 6.84-6.81 (m, 2H), 5.98 (d, J=3.6 Hz, 1H), 5.59 (d, J=8.4 Hz, 1H), 5.40 (dd, J=3.6, 6.4 Hz, 1H), 4 .90 (dd, J=14.0, 32.0 Hz,

- 26 045540

2H), 4.67 (dd, J=6.4, 7.6 Hz, 1H), 4.09-4.05 (m, 1H), 3.74 (d, J=0.8 Hz, 6H ), 3.70 (s, 3H). ^-NMR (376 MHz, DMSO-d6): -75.31, -75.69, -76.69, -77.35. ESI-LCMS: m/z 800 [M+H]+.

Preparation of intermediate compound (13-8). To a solution of 13-7 (33.0 g, 41.2 mmol) in THF (600 ml) was added NaBH ₄ (2.3 g, 61.8 mmol) and EtOH (20 ml) at 0°C. The mixture was left to stir at rt for 4 hours and cooled to saturation. aqueous NH ₄ Cl, extracted using EA, washed with brine, dried over Na ₂ SO ₄ , concentrated to give a residue which was purified by column chromatography to give 13-8 (25.0 g, 32.3 mmol, yield 78 .51%) as a white solid. ¹ H-NMR (400 MHz, DMSO-d6): δ=7.78 (d, J=8.4 Hz, 1H), 7.41-7.38 (m, 2H), 7.347.30 (m, 2H), 7.28-7.21 (m, 7H), 6.92-6.89 (m, 4H), 6.84-6.82 (m, 2H), 5.98 (d, J= 4.4 Hz, 1H), 5.83 (t, J=6.8 Hz, 1H), 5.60 (d, J=8.0 Hz, 1H), 5.30 (t, J=5, 3 Hz, 1H), 4.90 (dd, J=14.0, 33.2 Hz, 2H), 4.61 (t, J=6.0 Hz, 1H), 4.084.05 (m, 1H) , 3.74 (d, J=0.8 Hz, 6H), 3.70 (s, 3H), 3.39 (dd, J=4.0, 14.8 Hz, 1H), 3.30 ( dd, J=2.8, 11.2 Hz, 1H). ¹⁹ F-NMR (376 MHz, DMSO-d6): -79.67, -80.04, -80.87, -81.25. ESI-LCMS: m/z 772 [M+H] ⁺ .

Preparation of intermediate compound (13-9). To a solution of 13-8 (26.0 g, 33.6 mmol) in THF (250 ml) was added NaH (808 mg, 33.6 mmol) and MeI (23.9 g, 168.4 mmol, 10.4 ml ) at 0°C. The mixture was left to stir at room temperature. for 15 hours and poured into cold water, extracted with EA, washed with brine, dried over Na ₂ SO ₄ , concentrated to give crude product 13-9 (29.0 g) as a yellow oil. ESI-LCMS: m/z 786 [M+H] ⁺ .

Preparation of intermediate compound (13-10). To a solution of 13-9 (29.0 g, 36.9 mmol) in DCM (200 mL) was added a solution of p-TsOH (10.0 g) in MeOH (20 mL). The mixture was stirred at room temperature. for 0.5 h and washed with saturated NaHCO ₃ solution until pH = 7, dried over Na ₂ SO ₄ , concentrated to obtain a residue, which was purified by column chromatography to obtain 13-10 (13.5 g, 27.9 mmol , yield 82.90% in two stages). ¹ H-NMR (400 MHz, DMSO-d ₆ ): 6=7.99 (d, J=4.4 Hz, 1H), 7.22 (d, J=8.4 Hz, 2H), 6, 85 (d, J=8.8 Hz, 2H), 6.01 (d, J=5.2 Hz, 1H), 5.89 (d, J=8.0 Hz, 1H), 5.46 ( t, J=4.8 Hz, 1H), 5.17 (t, J=6.4 Hz, 1H), 4.90 (dd, J=14.0, 32.4 Hz, 2H), 4, 43 (t, J=5.2 Hz, 1H), 4.00-3.97 (m, 1H), 3.74-3.67 (m, 6H), 3.64-3.60 (m, 1H), 3.33 (s, 3H). ESI-LCMS: m/z 484 [M+H] ⁺ .

Preparation of intermediate compound (13-11). To a solution of 13-10 (28.0 g, 57.9 mmol) and TEA (11.7 g, 115.8 mmol, 16 mL) in DCM (300 mL) was added BzCl (12.2 g, 86.8 mmol ) in an Ar atmosphere. The mixture was stirred at room temperature. for 2 hours and poured into cold water, extracted using EA, washed with brine, dried over Na ₂ SO ₄ , concentrated to give a residue which was purified by column chromatography to give 13-11 (31.0 g, 52.7 mmol, yield 91.10%) in the form of a clear oil. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ=8.00 (d, J=3.2 Hz, 1H), 7.77 (d, J=8.0 Hz, 1H), 7. 68 (t, J=7.6 Hz, 1H), 7.54 (t, J=7.6 Hz, 1H), 7.20 (d, J=8.8 Hz, 2H), 6.82 ( d, J=8.4 Hz, 2H), 5.97 (d, J=4.4 Hz, 1H), 5.78 (d, J=8.4 Hz, 1H), 5.38 (t, J=6.4 Hz, 1H), 4.87 (dd, J=14.0, 27.2 Hz, 2H), 4.65-4.60 (m, 2H), 4.56-4.51 (m, 1H), 4.28-4.24 (m, 1H), 3.76 (t, J=10.4 Hz, 2H), 3.69 (s, 3H), 3.35 (s, 3H). ESI-LCMS: m/z 588 [M+H] ⁺ .

Preparation of intermediate compound (13-12). To a solution of 13-11 (30.0 g, 51.0 mmol) in a mixture of ACN (300 ml) and H ₂ O (100 ml) was added CAN (83.9 g, 153.1 mmol). The mixture was then stirred at 45°C for 24 hours, poured into water, extracted using EA, washed with brine and dried over anhydrous Na2SO4, concentrated to give a residue, which was purified by column chromatography to give 13-12 (20.0 g, 42.7 mmol, yield 83.80%) as a white solid. ESI-LCMS: m/z 468 [M+H] ⁺ .

Preparation of intermediate compound (13-13). To a solution of 13-12 (13.0 g, 27.8 mmol) in THF (150 ml) was added TPP (10.9 g, 41.7 mmol) and H ₂ O (5.0 g, 278.1 mmol) . The mixture was stirred at 60°C for 5 hours, concentrated to give a residue, which was purified by column chromatography to give 13-13 (11.5 g, 26.0 mmol, 93.67% yield) as a white solid. ESI-LCMS: m/z 442 [M+H] ⁺ .

Preparation of intermediate compound (13-14). To a solution of 13-13 (10.5 g, 23.7 mmol) and DMAP (581 mg, 4.7 mmol) in DCM (100 ml) was added MMTrCl (11.0 g, 35.6 mmol) and TEA (4 .8 g, 47.5 mmol, 6.6 ml) under N2. The mixture was stirred at room temperature. for 1 hour, quenched with MeOH and concentrated to give a residue, which was purified by column chromatography to give 13-14 (15.7 g, 22.0 mmol, 92.47% yield) as a white solid. ESI-LCMS: m/z 714 [M+H] ⁺ .

Preparation of intermediate compound (13-15). A solution of 13-14 (4.0 g, 5.6 mmol) in 1 N was obtained. NaOH (MeOH:H2O=4:1) (80 ml), the mixture was stirred at room temperature for 1 hour and the reaction mixture was neutralized with a saturated aqueous solution of NH ₄ Cl to pH=7~8, extracted using EA, washed with saline, dried over anhydrous Na ₂ SO ₄ , concentrated to give a residue, which was purified by column chromatography to give 1315 (3.2 g, 5.2 mmol, 93.66% yield) as a white solid. ¹ H-NMR (400 MHz, DMSC-d6): 6=11.37 (d, J=2.4 Hz, 1H, exchange with D2O), 7.91 (d, J=8.0 Hz, 1H) , 7.50-7.48 (m, 4H), 7.39-7.36 (m, 2H), 7.29 (t, J=7.6 Hz, 4H), 7.22-7.18 (m, 2H), 6.86 (d, J=8.8 Hz, 2H), 6.19 (d, J=6.0 Hz, 1H), 5.67 (dd, J=2.0, 8.0 Hz, 1H), 5.16 (t, J=4.8 Hz, 1H), 4.28 (t, J=5.6 Hz, 1H), 3.78 (dd, J=7, 6, 12.8 Hz, 2H), 3.73 (s, 3H), 3.38 (s, 3H), 3.313.26 (m, 4H), 3.06-3.01 (m, 1H) . ¹⁹ F-NMR (376 MHz, DMSO-d6): -73.96, -74.34, -78.81, -79.20. ESI-LCMS: m/z 610 [M+H] ⁺ .

- 27 045540

Receipt (13-16). To a solution of 13-15 (1.5 g, 2.4 mmol) and DCI (145 mg, 1.2 mmol) in dry DCM (30 ml) was added CEP[N(iPr) ₂ ] ₂ (816 mg, 2. 7 mmol) in an Ar atmosphere. The mixture was stirred at room temperature. for 1 hour. The reaction mixture was washed with 10% aqueous NaHCO ₃ and brine, dried over Na ₂ SO ₄ and concentrated to give the crude product, which was purified by column chromatography. The result was 13-16 (1.5 g, 1.8 mmol, 75.28% yield) as a white solid. ¹ H-NMR (400 MHz, DMSO-d6): δ=11.41 (s, 1H), 7.64 (dd, J=8.0, 41.6 Hz, 1H), 7.51-7, 47 (m, 4H), 7.38-7.36 (m, 2H), 7.29 (t, J=7.6 Hz, 4H), 7.20 (t, J=7.2 Hz, 2H ), 6.86 (dd, J=3.6, 8.8 Hz, 2H), 6.14 (dd, J=6.8, 106.8 Hz, 1H), 5.69 (dd, J= 8.0, 66.4 Hz, 1H), 3.82-3.63 (m, 7H), 3.61-3.42 (m, 5H), 3.39-3.28 (m, 7H) , 3.20-2.93 (m, 1H), 2.75-2.72 (m, 1H), 2.68-2.64 (m, 1H), 1.10 (dd, J=4, 8, 6.8 Hz, 6H), 1.00 (dd, J=6.4, 36.4 Hz, 6H). ³¹ P-NMR (162 MHz, DMSC-44): 148.37, 147.75. ¹⁹ F-NMR (376 MHz, DMSO-d ₆ ): -73.87, -74.25, -74.62, 78.45, -78.83, -78.99, -79.37. ESI-LCMS: m/z 808 [M-H].

Example 10.

Scheme 12.

14-14 14-17 14-10

14-19 14-20

Preparation of intermediate compound (14-17). To a solution of 14-14 (7.1 g, 9.9 mmol), DMAP (2.4 mg, 19.9 mmol) and TEA (2.0 g, 19.9 mmol, 2.77 ml) in dry ACN (100 ml) TIPSCl (4.5 g, 14.9 mmol) was added. After stirring the mixture at room temperature. Conc. was added over 3 hours. NH ₄ OH (30 ml) and stirred the reaction mixture at room temperature. for 6 hours. After completion, the solvent was removed and the residue was dissolved in EA, washed with sat. aqueous NH ₄ Cl and brine, dried over anhydrous Na ₂ SO ₄ , concentrated to give a residue, which was purified by column chromatography to give 14-17 (6.1 g, 8.5 mmol, 86.03% yield) in as a white solid. ESI-LCMS: m/z 713 [M+H]+.

Preparation of intermediate compound (14-18). BzCl (1.5 g, 10.7 mmol) was added dropwise to a solution of 14-17 (5.1 g, 7.1 mmol) in pyridine (50 ml) at 0°C. After this, the mixture was stirred for 1 hour at room temperature, 200 ml of H ₂ O and 300 ml of EA were added to separate the solution, the aqueous layer was extracted with EA, the combined organic layer was washed with saturated saline, dried over Na ₂ SO ₄ , concentrated to give crude product 14-18 (6.2 g) as a yellow oil. ESI-LCMS: m/z 817 [M+H]+.

Preparation of intermediate compound (14-19). To a solution of crude product 14-18 (6.2 g) in pyridine (60 ml) was added dropwise 2 N. NaOH (in MeOH:H ₂ O = 4:1) (30 ml) at 0°C and stirred the mixture at 0°C for 15 minutes. The mixture was neutralized with sat. solution of NH ₄ Cl (aq.) until

- 28 045540 pH=7~8, and 300 ml H2O and 400 ml EA were added to separate the solution, the aqueous layer was extracted with EA, the combined organic layer was washed with brine, dried over Na ₂ SO ₄ , concentrated to obtain the crude product, which purified by medium pressure liquid chromatography (MPLC) to give 14-19 (4.1 g, 5.7 mmol, 80.3% yield over two steps) as a white solid. 1H-NMR (400 MHz, DMSO-d ₆ ): δ=11.30 (s, 1H, exchange with D2O), 8.47 (d, J=7.6 Hz, 1H), 8.02 (d, J=7.6 Hz, 2H), 7.63 (t, J=7.2 Hz, 1H), 7.54-7.45 (m, 6H), 7.35-7.25 (m, 7H ), 7.19 (t, J=7.2 Hz, 2H), 6.83 (d, J=8.8 Hz, 2H), 6.10 (d, J=3.6 Hz, 1H), 5.19 (t, J=4.0 Hz, 1H), 3.77-3.70 (m, 6H), 3.64-3.61 (m, 2H), 3.46-3.43 ( m, 1H), 3.35 (s, 3H), 3.31-3.26 (m, 1H), 3.03 (d, J=8.8 Hz, 1H, exchange with D2O). ¹⁹ F-NMR (376 MHz, DMSO-de): -73.47, -73.85, -78.26, -79.64. ESI-LCMS: m/z 713 [M+H]+.

Receipt (14-20). To a solution of 14-19 (1.7 g, 2.3 mmol) and DCI (170 mg, 1.1 mmol) in dry DCM (20 ml) was added CEP[N(iPr) ₂ ] ₂ (790 mg, 2. 6 mmol) in an Ar atmosphere. The mixture was stirred at room temperature. for 1 hour. The reaction mixture was washed with 10% aqueous NaHCO ₃ and brine, dried over Na ₂ SO ₄ and concentrated to give the crude product, which was purified by column chromatography. The result was 14-20 (1.8 g, 1.9 mmol, 82.7% yield) as a white solid. 'H-NMR (400 MHz, DMSO-d ₆ ): δ=11.35 (s, 1H), 8.20 (dd, J=8.0, 81.6 Hz, 1H), 8.03- 8.00 (m, 2H), 7.64 (t, J=7.6 Hz, 1H), 7.54-7.44 (m, 6H), 7.37-7.16 (m, 9H) , 6.82 (dd, J=9.2, 16.4 Hz, 2H), 6.14 (dd, J=4.8, 90.4 Hz, 1H), 4.15-4.02 (m , 1H), 3.90-3.40 (m, 11H), 3.36-3.24 (m, 6H), 3.12-2.98 (m, 1H), 2.77-2.73 (m, 2H), 1.13 (d, J=6.8 Hz, 6H), 1.05 (dd, J=6.8, 28.0 Hz, 6H). ³¹ P-NMR (162 MHz, DMSO-d6): 148.20, 147.96. ¹⁹ F-NMR (376 MHz, DMSO-d6): -73.36, -73.74, -73.98, -74.36, -77.89, -78.28, -78.55, -78 ,94. ESI-LCMS: m/z 913 [M+H] ⁺ .

Example 11.

Scheme 13.

Preparation of intermediate compound (15-21). To a solution of compound 15-1 (90 g, 238.5 mmol) in dry acetonitrile (900 ml) was added N-(5H-purin-6-yl)benzamide (85.6 g, 357.7 mmol) and BSA (155 .3 g, 763.2 mmol). The resulting suspension was stirred at 50°C for 30 minutes. Subsequently, the mixture was heated at -5°C and TMSOTf (79.5 g, 357.8 mmol) was added via syringe. Subsequently, the mixture was stirred at 80°C for 72 hours under N2 atmosphere. The mixture was cooled and concentrated. And subsequently water was added and extracted using EA. The extract was washed with brine, dried over Na ₂ SO ₄ and concentrated to give a residue, which was purified by column chromatography to give 15-21 (120 g, 215.62 mmol, 90.4% yield) as a yellow solid. 'H-NMR (400 MHz, DMSO-d6): δ=8.56 (s, 1H), 8.53 (s, 1H), 8.00 (t, J=8.0 Hz, 2H), 7 .76-7.74 (d, J=8.0 Hz, 2H), 7.64 ~ 7.60 (t, J=7.5 Hz, 1H), 7.54 ~ 7.50 (t, J =7.68 Hz, 2H), 7.27 ~ 7.25 (d, J=7.8 Hz, 2H,), 6.31 (d, J=3.3 Hz, 1H), 6.14 ~ 6.11 (m, 1H), 5.05 ~ 5.01 (t, J=8.0 Hz, 1H), 4.67 ~ 4.63 (m, 1H), 4.53-4.49 ( m, 1H), 4.37-4.34 (m, 1H), 2.31 (s, 3H), 2.12 (s, 3H). ESI-LCMS: m/z 556 [M+H] ⁺ .

- 29 045540

Preparation of intermediate compound (15-22). A solution of 15-21 (120 g, 215.6 mmol) and methylamine (267.9 g, 8.6 mol) in methanol (1300 ml) was stirred at room temperature. for 18 hours in N2 atmosphere. The mixture was concentrated and stirred at 25% EA in PET for 1 hour to give 15-22 (52 g, 1778.0 mmol, 82.5% yield) as a white solid. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ=8.36 (s, 1H), 8.15 (s, 1H), 7.40 (s, 2H), 6.24-6.22 (d, J=8.0Hz, 1H), 5.91~5.90 (d, J=4Hz, 1H), 5.63~5.60 (m, 1H), 5.04~5, 00 (m, 7H), 4.34~4.32 (m, 1H), 3.99~3.98 (d, J=4Hz, 1H), 3.72~3.55 (m, 2H) . ESI-LCMS: m/z 292 [M+H]+.

Preparation of intermediate compound (15-23). A solution of 15-22 (50 g, 171.1 mmol) and DMTrCl (121.7 g, 359.3 mmol) in pyridine (250 ml) was stirred for 2 h at RT. The mixture was quenched with water, extracted with EA, dried over Na ₂ SO ₄ , concentrated to give a residue which was purified by column chromatography to give 15-23 (117 g, 130.4 mmol, 76.2% yield) as a yellow solid substances. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ=8.29 (s, 1H), 7.76 (s, 1H), 7.20-7.12 (m, 19H), 6.74 (m, 8H), 5.87 (s, 1H), 5.08 (s, 1H), 4.39 (s, 1H), 3.62 (s, 12H), 3.32~3.10 ( m, 2H). ESI-LCMS: m/z 896 [M+H] ⁺ .

Preparation of intermediate compound (15-24). A solution of 15-23 (115 g, 128.2 mmol) and THF (800 ml) was stirred at 5°C under N2. NaH (4.0 g, 167.4 mmol) was added slowly at below 10°C and sodium chlorodifluoroacetate (25.5 g, 167.4 mmol) and TBAI (5.6 g, 16.7 mmol) were added at 5 °C. The reaction mixture was refluxed for 18 hours. After cooling to rt, the reaction mixture was quenched with sat. aqueous NH ₄ Cl and extracted with EA, washed with brine, dried over Na ₂ SO ₄ , concentrated to give a residue which was purified by column chromatography to give 15-24 (56 g, 56.6 mmol, yield 48.3 %) as a yellow crude solid. ESI-LCMS: m/z 990 [M+H] ⁺ .

Preparation of intermediate compound (15-25). A solution of 15-24 (56 g, 57.3 mmol), methyl ptoluenesulfonate (15.8 g, 84.8 mmol) and K2CO3 (15.6 g, 113.0 mmol) in dry DMF (560 ml) was stirred at rt under N2 for 18 hours. The mixture was quenched with water, extracted with EA, washed with brine and concentrated to give a residue which was purified by column chromatography to give 15-25 (33 g, 32.8 mmol, yield 58.1 %) as a white solid. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ=8.40 (s, 1H), δ=7.82 (s, 1H), 7.40 (s, 1H), 7.31-7 .16 (m, 18H), 6.826.79 (m, 8H), 6.24-6.23 (d, J=4.0 Hz, 1H), 6.00-5.99 (t, 1H), 4.88-4.85 (t, 1H), 4.17-4.14 (m, 1H), 3.79 (s, 3H), 3.68 (s, 12H), 3.31-3, 30 (m, 2H). ¹⁹ F-NMR (376 MHz, DMSO-d6): -75.75, -76.14, -76.89, -77.28. ESI-LCMS: m/z 1004 [M+H] ⁺ .

Preparation of intermediate compound (15-26). To a solution of 15-25 (30 mg, 29.9 mmol) in THF (300 ml) was added NaBH ₄ (1.7 g, 44.8 mmol) and EtOH (100 ml) at 5°C. The mixture was left to stir at room temperature. for 1.5 hours and extinguished sat. aqueous NH ₄ Cl, extracted using EA, washed with brine, dried over Na ₂ SO ₄ , concentrated to give a residue which was purified by column chromatography to give 15-26 (22 g, 22.5 mmol, yield 75.4 %) as a yellow solid. ¹ H-NMR (400 MHz, DMSO-d6): δ=8.35 (s, 1H), 7.79 (s, 1H), 7.26-7.13 (m, 21H), 6.77- 6.75 (m, 9H), 6.14-6.13 (d, J=4.0 Hz, 1H), 5.90 (t, 1H), 4.74 ~ 4.72 (t, 1H) , 4.11 (s, 1H), 3.63 (s, 12H), 3.28 ~ 3.25 (m, 2H). ¹⁹ F-NMR (376 MHz, DMSO-d6): -79.94, -80.31, -80.77, -81.15. ESI-LCMS: m/z 976 [M+H] ⁺ .

Preparation of intermediate compound (15-27). To a solution of 15-26 (20 g, 20.5 mmol) in THF (100 ml) was added NaH (491.7 mg, 20.5 mmol) and methyl p-toluenesulfonate (11.4 g, 61.5 mmol) at 5°C. The mixture was left to stir at room temperature. for 3 hours and poured into cold water, extracted with EA, washed with brine, dried over Na2SO4, concentrated to give a residue which was purified by column chromatography to give 15-27 (16.5 g, 16.7 mmol, yield 81 .3%) as a yellow solid. ¹ H-NMR (400 MHz, DMSO-d6): δ=8.40 (s, 1H), 7.86 (s, 1H), 7.377.30 (m, 4H), 7.28-7.26 ( m, 5H), 7.24-7.18 (m, 12H), 6.84-6.81 (m, 8H), 6.17-6.16 (d, J=4.0 Hz, 1H) , 5.92-5.89 (t, 1H), 4.79~4.76 (t, 1H), 4.15~4.11 (m, 1H), 3.70 (s, 12H), 3 .31 (s, 5H). ¹⁹ F-NMR (376 MHz, DMSO-d6): -76.83, -77.22, -78.05, -78.43. ESI-LCMS: m/z 990 [M+H] ⁺ .

Preparation of intermediate compound (15-28). To a solution of 15-27 (15.5 mg, 15.6 mmol) in DCM (155 ml) was added TsOH (6.2 mg, 36.0 mmol) in MeOH (30 ml). The mixture was stirred at room temperature. for 0.5 hours and cooled sat. solution of NaHCO ₃ to pH=7~8, extracted using EA, dried over Na ₂ SO ₄ , concentrated to obtain a residue, which was purified by column chromatography to obtain 15-28 (4.6 g, 11.4 mmol, yield 72 .8%) as a white solid. ESI-LCMS: m/z 386 [M+H] ⁺ .

Preparation of intermediate compound (15-29). To a solution of 15-28 (4.4 g, 11.3 mmol) in pyridine (40 ml) was added BzCl (8.0 g, 56.7 mmol) slowly at 5°C. The mixture was left to stir at room temperature. for 2 hours and quenched with a small amount of water, after which ammonium hydroxide was slowly added at 4°C until the intermediate product disappeared. The mixture was extracted using EA, washed with brine, dried over Na ₂ SO ₄ , concentrated to give the crude product, which was purified by column chromatography. The result was 15-29 (6.0 g, 10.1 mmol, 89.1% yield) as a white solid. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ=8.67-8.66 (t,

- 30 045540

1H), 8.47-8.44 (t, 1H), 8.02-7.97 (m, 4H), 7.69-7.62 (m, 2H), 7.56-7.52 ( m, 4H), 6.32-6.31 (d, J=4.0 Hz, 1H), 6.09-6.06 (m, 1H), 5.00-4.98 (m, 1H) , 4.71-4.67 (m, 1H), 4.58-4.54 (m, 1H), 4.43-4.41 (m, 1H), 3.77-3.72 (m, 2H), 3.31 (m, 3H). ¹⁹ B-NMR (376 MHz, DMSO-d ₆ ): -76.97, -77.35, -78.19, -78.57. ESI-LCMS: m/z 594 [M+H]+.

Preparation of intermediate compound (15-30). To a solution of 15-29 (6.0 g, 10.1 mmol) in THF (70 mL) was added PPh ₃ (4.0 g, 15.1 mmol) and water (1.8 g, 100.9 mmol) in atmosphere N2. The mixture was stirred at 45°C for 15 hours, poured into cold water, adjusted to pH=1-2 with 2N. aqueous HCl solution and extracted using EA. Subsequently, the aqueous phase was adjusted to pH=7-8 with saturated aqueous NaHCO ₃ solution, extracted using EA, washed with brine, dried over Na ₂ SO ₄ , concentrated to give 15-30 (5.5 g, 9.7 mmol, yield 95.9%) as a white solid. 1H-NMR (400 MHz, DMSO-d ₆ ): δ=8.67 (s, 2H), 8.03-8.01 (m, 2H), 7.88-7.86 (m, 2H), 7.65-7.61 (m, 2H), 7.56-6.46 (m, 5H), 6.30-6.29 (d, J=4.0 Hz, 1H), 5.42- 5.40 (m, 1H), 4.69-4.66 (m, 1H), 4.51-4.47 (m, 1H), 4.08-4.09 (m, 2H), 3. 87-3.80 (m, 2H), 3.46 (s, 5H). ¹⁹ F-NMR (376 MHz, DMSO-d6): -75.65, -76.03, -77.47, 77.86. ESI-LCMS: m/z 568 [M+H] ⁺ .

Preparation of intermediate compound (15-31). To a solution of 15-30 (5.5 g, 9.6 mmol) in DCM (55 mL) was added MMTrCl (4.5 g, 14.5 mmol) and TEA (2.0 g, 19.3 mmol) under atmosphere N2. The mixture was left to stir at room temperature. for 1.0 h and quenched with water, extracted with DCM, washed with brine, dried over Na ₂ SO ₄ , concentrated to give a residue which was purified by column chromatography to give 15-31 (7.3 g, 8.7 mmol , yield 89.8%) as a yellow solid. 1H-NMR (400 MHz, DMSO-d6): δ=8.46 (s, 1H), 8.41 (s, 1H), 8.00-7.98 (d, J=8.0 Hz, 2H ), 7.62-7.44 (m, 12H), 7.38-7.34 (m, 4H), 7.17-7.12 (m, 4H), 7.06-7.01 (m , 2H), 6.67 6.65 (d, J=8.0 Hz, 2H), 6.17 (s, 1H), 4.57-4.54 (d, J=12 Hz, 1H), 4.35-4.18 (m, 4H), 3.70-3.65 (m, 2H), 3.50 (s, 3H), 3.31 (s, 3H). ¹⁹ F-NMR (376 MHz, DMSO-d ₆ ): -73.61, -74.00, -77.94, -78.32. ESI-LCMS: m/z 840 [M+H] ⁺ .

Preparation of intermediate compound (15-32). To a solution of 15-31 (7.2 g, 8.5 mmol) in pyridine (70 ml) was slowly added 2 N. aqueous NaOH solution (510.0 mg, 10.3 mmol) at 5-10°C. The mixture was stirred at room temperature. for 10 min, neutralized with saturated aqueous NH4C1 to pH 7-8, extracted using EA, washed with brine, dried over Na2SO4, concentrated to give crude product, which was purified by column chromatography. The result was 15-32 (5.8 g, 7.9 mmol, 92.2% yield) as a white solid. 1H-NMR (400 MHz, DMSO-d6): δ=8.63 (s, 2H), 7.99-7.97 (d, J=8.0 Hz, 2H), 7.64-7.60 (m, 1H), 7.547.50 (m, 2H), 7.42-7.38 (m, 4H), 7.27-7.25 (d, J=8.0 Hz, 2H), 7, 21-7.16 (m, 4H), 7.12-7.09 (m, 2H), 6.72-6.70 (d, J=8.0 Hz, 2H), 6.28-6, 27 (d, J=8.0 Hz, 1H), 5.59 (m, 1H), 3.96 (s, 4H), 3.76-3.74 (m, 1H), 3.68-3 .63 (m, 2H), 3.60 (s, 3H), 3.47-3.41 (m, 2H), 3.27 (s, 3H). ¹⁹ F-NMR (376 MHz, DMSO-d ₆ ): -74.13, -74.51, -78.09, -78.47. ESI-LCMS: m/z 736 [M+H] ⁺ .

Receipt (15-33). To a solution of 15-32 (3 g, 4.1 mmol) and DCI (240.4 mg, 2.0 mmol) in anhydrous DCM (30 ml) was added CEP[N(iPr) ₂ ] ₂ (1.5 g, 4.9 mmol) in an N2 atmosphere. The mixture was left to stir at room temperature. for 1 hour, extinguished sat. NaHCO ₃ solution, washed with brine, dried over Na2SO4, concentrated to give the crude product, which was purified by column chromatography. The result was 15-33 (3.1 g, 3.3 mmol, 81.2% yield) as a white solid. 1H-NMR (400 MHz, DMSO-d ₆ ): δ=11.20 (s, 1H), 8.69-8.68 (d, J=4.0 Hz, 1H), 8.50-8, 48 (d, J=8.0 Hz, 1H), 8.05-8.03 (m, 2H), 7.66-7.62 (m, 1H), 7.57-7.45 (m, 6H), 7.40-7.33 (m, 2H), 7.30-7.11 (m, 6H), 6.85-6.76 (m, 2H), 6.45-6.30 ( m, 1H), 4.77-4.74 (m, 0.5H), 4.00-3.99 (m, 0.5H), 3.86-3.84 (m, 0.5H), 3.77-3.70 (m, 5H), 3.67-3.59 (m, 4H), 3.56-3.51 (m, 1H), 3.50-3.34 (m, 3H ), 3.32 (s, 3H), 3.08-3.06 (m, 0.5H), 2.77-2.65 (m, 2H), 1.08-0.99 (m, 12H ). ¹⁹ F-NMR (376 MHz, DMSO-d ₆ ): -73.92, -74.27, -74.30, -74.65, -77.64, -78.03, -78.55, 78 ,93. ESI-LCMS: m/z 936 [M+H] ⁺ .

Example 12.

MMTrHN

- 31 045540

Scheme 14.

Preparation of intermediate compound (16-34). To a solution of 16-1 (150.0 g, 397.5 mmol) and 6chloroguanine (101.1 g, 596.3 mmol) in dry ACN (1.5 L) was added BSA (259.5 g, 1.27 mol ) in an Ar atmosphere. The resulting suspension was stirred at 50°C for 1 hour. Subsequently, the mixture was cooled to 5°C and TMSOTf (590.5 g, 2.66 mol) was added via syringe. After this, the mixture was stirred at 60°C for 5 hours, concentrated to remove ACN, and the residue was dissolved in EA (2.0 L), washed with saturated aqueous NaHCO ₃ and saline, dried over Na ₂ SO ₄ , concentrated with yielding crude product 16-34 (265.0 g) as a yellow solid. ESI-LCMS: m/z 487 [M+H]+.

Preparation of intermediate compound (16-35). To a solution of 16-34 (crude, 265 g, 353.8 mmol) and TEA (107.4 g, 1.06 mol, 148 mL) in DCM (2.5 L) was added MMTrCl (164.0 g, 530 7 mmol) and DMAP (8.6 g, 70.8 mmol), the mixture was stirred at room temperature. for 5 hours, washed with water and brine, dried over Na ₂ SO ₄ , concentrated to give a residue, which was purified by column chromatography to give 16-35 (160.0 g, 210.8 mmol, 53.0% yield 2 stages) as a yellow solid. ESI-LCMS: m/z 759 [M+H] ⁺ .

Preparation of intermediate compound (16-36). To a solution of 35 (20.0 g, 26.39 mmol) in benzyl alcohol (200 ml) was added NaH (5.3 mg, 131.93 mmol) at 0°C. The resulting mixture was left to stir at room temperature. for 2 hours, cooled with cold water, extracted using EA, washed with water and brine, dried over Na ₂ SO ₄ , concentrated to give a residue which was purified by column chromatography to give 16-36 (11.2 g, 16.72 mmol, yield 63.1%) as a white solid. ESI-LCMS: m/z 671 [M+H] ⁺ .

Preparation of intermediate compound (16-37). To a solution of 16-36 (49.0 g, 73.1 mmol) and pyridine (28.9 g, 365.3 mmol, 29 mL) in DCM (500 L) was added DMTrCl (27.2 g, 80.4 mmol ) at k.t. The mixture was stirred at room temperature. for 3 hours, washed with water and brine, dried over Na ₂ SO ₄ , concentrated to give a residue which was purified by column chromatography to give 16-37 (58.9 g, 60.6 mmol, 82.9% yield) as a white solid. ESI-LCMS: m/z 973 [M+H] ⁺ .

Preparation of intermediate compound (16-38). To a solution of 16-37 (58.9 g, 60.6 mmol) in dry THF (300 ml) was added NaH (5.1 g, 127.8 mmol, 60% purity) and methyl p-toluenesulfonate (19.5 g, 127.8 mmol) at 0°C, the mixture was left to stir at room temperature. and TBAI (4.7 g, 12.8 mmol) was added. After this, the mixture was stirred at a temperature of 80°C for 24 hours and quenched with sat. aqueous NH4Cl, extracted with EA, washed with brine, dried over Na2SO4, concentrated to give a residue which was purified by column chromatography to give crude product 16-38 (30.9 g) as a colorless solid. ESI-LCMS: mz 1067 [M+H] ⁺ .

Preparation of intermediate compound (16-39). To a solution of crude 16-38 (30.9 g) and K2CO3 (8.0 g, 58.1 mmol) in DMF (200 ml) was added sodium chlorodifluoroacetate (5.4 g, 29.1 mmol) at room temperature. The mixture was stirred at rt for 15 hours, quenched with sat. aqueous NH ₄ Cl, extracted with EA, washed with brine, dried over Na2SO4, concentrated to give a residue which was purified by column chromatography to give 16-39 (9.8 g, 9.1 mmol,

- 32 045540 yield 15.0% in 2 stages) in the form of a yellow solid. ESI-LCMS: m/z 1081 [M+H]+.

Preparation of intermediate compound (16-40). To a solution of 16-39 (9.3 g, 8.6 mmol) in THF (180 ml) was added NaBH ₄ (488 mg, 12.9 mmol) and EtOH (15 ml) at 0°C. The mixture was stirred at 0°C for 1 hour, quenched with sat. aqueous NH4Cl, extracted using EA, washed with brine, dried over Na ₂ SO ₄ , concentrated to obtain a residue, which was purified by column chromatography. The result was 16-40 (8.5 g, 7.0 mmol, 81.63% yield, 87% purity) as a white solid. ESI-LCMS: m/z 1053 [M+H] ⁺ .

Preparation of intermediate compound (16-41). To a solution of 16-40 (8.3 g, 7.9 mmol) in THF (80 ml) was added NaH (472 mg, 11.8 mmol, 60% purity) and methyl p-toluenesulfonate (2.9 g, 15 .8 mmol) at 0°C. The mixture was left to stir at room temperature. for 15 hours and poured into cold water, extracted with EA, washed with brine, dried over Na ₂ SO ₄ , concentrated to give crude product 16-41 (9.1 g) as a yellow oil. ESI-LCMS: m/z 1067 [M+H] ⁺ .

Preparation of intermediate compound (16-42). To a solution of crude 16-41 (9.1 mg) in DCM (100 ml) was added a solution of p-TsOH (10 g) in MeOH (10 ml). The mixture was stirred at room temperature. for 0.5 h and neutralized with TEA until pH=7, concentrated to obtain a residue, which was purified by column chromatography. This gave 16-42 (3.2 g, 6.5 mmol, 82.3% yield over 2 steps) as a yellow solid. ¹ H-NMR (400 MHz, DMSO-d6): δ=8.14 (s, 1H), 7.52-7.50 (m, 2H), 7.42-7.33 (m, 3H), 6.62 (s, 2H, exchange with D2O), 6.01 (d, J=6.4 Hz, 1H), 5.62 (t, J=6.0 Hz, 1H), 5.49 (t , J=12.8 Hz, 2H), 5.39 (t, J=5.6 Hz, 1H, exchange with D2O), 4.64 (dd, J=3.6, 5.6 Hz, 1H) , 4.02 (dd, J=3.6, 7.2 Hz, 1H), 3.74-3.66 (m, 3H), 3.64-3.59 (m, 1H), 3.32 (s, 3H). ¹⁹ F-NMR (376 MHz, DMSO-d6): -77.52, -77.91, -78.17, 78.55). ESI-LCMS: m/z 493 [M+H] ⁺ .

Preparation of intermediate compound (16-43). To a solution of 16-42 (3.2 g, 6.5 mmol) in pyridine (30 ml) i-BuCl (2.1 g, 19.5 mmol) was added dropwise at 0°C. The mixture was stirred at 0°C for 1 hour and neutralized with MeOH and concentrated to give a residue, which was purified by column chromatography. The result was 16-43 (3.2 g, 5.5 mmol, 84.59% yield) as a yellow oil. ¹ H-NMR (400 MHz, DMSO-d6): δ=10.60 (s, 1H, exchange with D2O), 8.42 (s, 1H), 7.57-7.54 (m, 2H), 7.44-7.35 (m, 3H), 6.20 (d, J=2.8 Hz, 1H), 5.71 (dd, J=2.4, 5.6 Hz, 1H), 5 .68-5.64 (m, 3H), 4.41 (dd, J=3.6, 12.0 Hz, 1H), 4.24 (dd, J=5.6, 12.0 Hz, 1H ), 4.19-4.15 (m, 1H), 3.80 (t, J=10.4 Hz, 2H), 3.38 (s, 3H), 3.35 (s, 3H), 2 .45-2.38 (m, 1H), 1.12 (dd, J=2.4, 6.8 Hz, 6H), 0.97 (dd, J=6.8, 16.4 Hz, 6H ). ¹⁹ F-NMR (376 MHz, DMSO-d6): 76.48, -76.87, -77.62, -78.01). ESI-LCMS: m/z 633 [M+H] ⁺ .

Preparation of intermediate compound (16-44). Pd/C (200 mg) was added to a solution of 16-43 (1.6 g, 2.5 mmol) in THF (20 ml), and the mixture was stirred at room temperature. under H ₂ atmosphere for 5 hours. The reaction mixture was filtered and the filtrate was concentrated to give 16-44 (1.2 g, 2.3 mmol, 91.86% yield) as a white solid. 1H-NMR (400 MHz, DMSC-d ₆ ): δ=11.60 (s, 1H, exchange with D2O), 8.22 (s, 1H), 6.05 (d, J=2.8 Hz, 1H), 5.14 (dd, J=3.2, 5.6 Hz, 1H), 4.40 (dd, J=2.4, 12.0 Hz, 1H), 4.17 (dd, J =5.6, 12.0 Hz, 1H), 3.93-3.73 (m, 4H), 3.38 (s, 3H), 1.14 (d, J=7.2 Hz, 6H) , 1.04 (dd, J=7.2, 12.4 Hz, 6H). ¹⁹ F-NMR (376 MHz, DMSO-d6): -75.73, -76.12, -77.46, -77.84). ESI-LCMS: m/z 517 [M+H] ⁺ .

Preparation of intermediate compound (16-45). To a solution of 16-44 (3.0 g, 5.8 mmol) and collidine (1.8 g, 14.5 mmol) in DCM (40 ml) was added MMTrCl (2.7 g, 8.7 mmol) and AgNO ₃ (986 mg, 5.8 mmol), the mixture was stirred at room temperature. for 1 hour, filtered, and the filtrate was washed with water and dried over anhydrous Na2SO4, concentrated to give a residue, which was purified by column chromatography to give 16-45 (3.8 g, 4.8 mmol, 82.94% yield) in as a white solid. ¹ H-NMR (400 MHz, DMSO-d6): δ=12.16 (s, 1H, exchange with D2O), 11.48 (s, 1H, exchange with D2O), 7.86 (s, 1H), 7.50 (dd, J=5.2, 7.2 Hz, 4H), 7.37 (d, J=8.8 Hz, 2H), 7.28 (t, J=7.2 Hz, 4H ), 7.20 (t, J=7.2 Hz, 2H), 6.83 (d, J=9.2 Hz, 2H), 6.11 (d, J=3.2 Hz, 1H), 4.18 (dd, J=5.2, 8.8 Hz, 1H), 4.08 (t, J=4.4 Hz, 1H), 4.44-3.97 (m, 1H), 3 .74 (dd, J=7.6, 10.4 Hz, 2H), 3.70 (s, 3H), 3.46-3.41 (m, 1H), 3.16 (d, J=8 ,0 Hz, 1H, exchange with D2O), 2.86-2.79 (m, 1H), 2.33-2.26 (m, 1H), 1.13 (t, J=7.2 Hz, 6H), 0.89 (dd, J=7.2, 14.8 Hz, 6H). %-NMR (376 MHz, DMSO-d ₆ ): 74.01, -74.39, -78.00, -78.38). ESI-LCMS: m/z 789 [M+H] ⁺ .

Preparation of intermediate compound (16-46). To a solution of 16-45 (3.8 g, 4.8 mmol) in pyridine (40 ml) was added 2 N. NaOH (MeOH:H ₂ O=4:1) (15 ml) at 0°C, the mixture was stirred at 0°C for 1 hour. Subsequently, the reaction mixture was neutralized with a saturated aqueous solution of NH4Cl to pH=7~8, extracted using EA, washed with brine, dried over anhydrous Na2SO4, concentrated to obtain a residue, which was purified by column chromatography. The result was 16-46 (3.0 g, 4.2 mmol, 87.50% yield) as a white solid. ¹ H-NMR (400 MHz, DMSO-d6): δ=12.16 (s, 1H, exchange with D2O), 11.68 (s, 1H, exchange with D2O), 8.19 (s, 1H), 7.51 (dd, J=5.2, 7.2 Hz, 4H), 7.38 (d, J=8.8 Hz, 2H), 7.31 (t, J=7.2 Hz, 4H ), 7.21 (t, J=7.2 Hz, 2H), 6.87 (d, J=8.8 Hz, 2H), 6.26 (d, J=6.0 Hz, 1H), 5.15 (t, J=4.4 Hz, 1H), 4.76 (t, J=6.0 Hz, 1H, exchange with D2O), 3.75-3.70 (m, 5H), 3 .42 (d, J=2.4 Hz, 1H), 3.38-3.28 (m, 5H), 3.23 (d, J=6.4 Hz, 1H, exchange with D2O), 3. 16-3.10 (m, 1H), 2.88-2.81 (m, 1H), 1.15 (dd, J=6.4, 7.2 Hz, 6H). ¹⁹ F-NMR (376 MHz, DMSO-d6): -74.73, -75.12, -78.79, -79.17. ESI-LCMS: m/z 719 [M+H] ⁺ .

Receipt (16-47). To a solution of 16-46 (3.0 g, 4.2 mmol) and DCI (394 mg, 3.3 mmol) in dry DCM (50 ml)

- 33 045540 CEP[N(iPr)2]2 (1.5 g, 5.0 mmol) was added under Ar atmosphere. The mixture was stirred at room temperature. for 1 hour. The reaction mixture was washed with 10% aqueous NaHCO ₃ and water, dried over Na2SO4 and concentrated to give the crude product, which was purified by column chromatography. The result was 16-47 (3.0 g, 3.2 mmol, 77.81% yield) as a white solid. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ=12.13 (s, 1H), 11.65 (d, J=5.6 Hz, 1H), 8.11 (s, 0.35H ), 7.95 (s, 0.65H), 7.50 (t, J=7.2 Hz, 4H), 7.38-7.26 (m, 6H), 7.20 (dd, J= 8.4, 16.0 Hz, 2H), 6.88 (d, J=9.2 Hz, 0.7H), 6.82 (d, J=8.8 Hz, 1, 3H), 6, 31 (d, J=6.8 Hz, 0.35N), 6.14 (d, J=4.4 Hz, 0.65N), 4.97 (t, J=6.4 Hz, 0.35N ), 3.97 (t, J=4.4 Hz, 0.65H), 3.82-3.54 (m, 9H), 3.48-3.14 (m, 9H), 2.86- 2.69 (m, 2H), 1.24-0.92 (m, 18H). ³¹ P-NMR (162 MHz, DMSO-d6): 148.35, 148.24. ESI-LCMS: m/z 919 [M+H]+.

Example 13.

Scheme 15.

Preparation of intermediate compound (17-2). To a solution of 17-1 (120 g, 318 mmol) in ACN (1.2 ml) was added BSA (208 mg, 1016 mmol) and N ⁴ -benzoyl cytosine (102 g, 474 mmol). The mixture was stirred at 50°C for 1 hour until it became clear. Subsequently, the solution was cooled to -10°C. TMSOTf (84 g, 381 mmol) was slowly added to the mixture. Subsequently, the mixture was heated. The reaction mixture was stirred at 60°C for 12 hours. The mixture was cooled to 0°C and a saturated NaHCO ₃ solution was added until pH=7.5 was reached. The mixture was filtered and the filtrate was concentrated to obtain a residue. The residue was dissolved in EA and washed with water and brine, dried over Na ₂ SO ₄ The organic solution was concentrated to give crude 17-2 (160 g) as a yellow oil. EPILCMS: m/z 533 [M+H] ⁺ .

Preparation of intermediate compound (17-3). A solution of crude 17-2 (160 g) in pyridine (1.5 L) was added to 600 ml of 2N. a solution of NaOH in methanol and water at 0°C. The suspension was stirred at 0°C in

- 34 045540 within 30 minutes. The reaction mixture was quenched by adding sat. NH4Cl solution (1 l). The solution was added to water (15 L) to obtain a solid. The solid was filtered and washed (PE:EA=3:1) to give 17-3 (80 g, 214 mmol, 67% in two steps) as a white solid. ESI-LCMS: m/z 373 [M+H]+.

Preparation of intermediate compound (17-4). To a solution of 17-3 (80 g, 214 mmol) in pyridine (1 ml) was added DMTrCl (87 g, 257 mmol). The reaction mixture was stirred at room temperature. for 1 hour. MeOH (50 ml) was added to the mixture. The solution was concentrated to obtain the crude product. The crude product was dissolved in EA and washed with water and brine. The organic layer was dried over Na ₂ SO ₄ . The organic solution was concentrated to give crude 17-4 (160 g) as a yellow oil, which was used directly in the next step. ESI-LCMS: m/z 675 [M+H] ⁺ .

Preparation of intermediate compound (17-5). To a solution of crude 17-4 (160 g) in DCM (1 L) was added imidazole (22 g, 332 mmol) and TBSCl (46 g, 310.62 mmol). The reaction mixture was stirred at room temperature. for 12 hours. The mixture was diluted with DCM (1 L). Added water. The organic layer was washed with brine and concentrated to give crude 17-5 (200 g), which was used directly in the next step. ESI-LCMS: m/z 789 [M+H] ⁺ .

Preparation of intermediate compound (17-6). A solution of crude 17-5 (200 g) in methylamine (1 L) was stirred at room temperature. for 2 hours. The solution was concentrated and purified by column chromatography to give 17-6 (130 g, 204.42 mmol, 94.8% yield) as a white solid. ESI-LCMS: m/z 685 [M+H] ⁺ .

Preparation of intermediate compound (17-7). To a solution of 17-6 (142 g, 207.34 mmol) in DCM (1 L) was added collidine (50.25 g, 414.69 mmol) and MMTrCl (95.79 g, 311.01 mmol). Subsequently, AgNO ₃ (52.83 g, 311.01 mmol) was added to the solution. The reaction mixture was stirred at room temperature. for 1 hour. The mixture was filtered. The organic solution was washed with water. The organic layer was concentrated to give crude 17-7 (190 g, 198.50 mmol, 95.73% yield) as a yellow solid, which was used directly in the next step. ESI-LCMS: m/z 957.4 [M+H] ⁺ .

Preparation of intermediate compound (17-8). To a solution of 17-7 (190 g, 198.50 mmol) in THF (1 L) was added TBAF (77.85 g, 297.74 mmol). The mixture was stirred at room temperature. for 12 hours. Water was added. The product was extracted with EA. The organic layer was washed with brine and dried over Na ₂ SO ₄ . The organic solution was concentrated and purified by column chromatography to give 17-8 (160 g, 189.81 mmol, 95.6% yield) as a white solid. EPILCMS: m/z 843.4 [M+H] ⁺ .

Preparation of intermediate compound (17-9). To a solution of 17-8 (154 g, 182.70 mmol) in DMF (1 L) was added Ag ₂ O (84.68 g, 365.39 mmol, 11.86 ml), (2-bromoethoxy)-tert-butyldimethylsilane (78.67 g, 328.85 mmol) and NaI (41.08 g, 274.04 mmol). The reaction mixture was stirred at 45°C for 12 hours. Water was added. The mixture was filtered and the product was extracted using EA. The organic layer was concentrated and purified by column chromatography to give 17-9 (90 g, 89.89 mmol, 49.2% yield) as a black solid. ESI-LCMS: m/z 1001.4 [M+H] ⁺ .

Preparation of intermediate compound (17-10). To a solution of 17-9 (90 g, 89.89 mmol) in THF (500 mL) was added TBAF (35.25 g, 134.83 mmol). The reaction mixture was stirred at room temperature. for 12 hours. Water was added to the mixture. The product was extracted with EA. The organic layer was concentrated and purified by column chromatography to give 17-10 (75 g, 84.56 mmol, 94.1% yield) as a yellow solid. ESI-LCMS: m/z 887.2 [M+H] ⁺ .

Preparation of intermediate compound (17-11). To a solution of 17-10 (54 g, 60.88 mmol) in THF (400 mL) was added NaH (1.75 g, 73.06 mmol) at 0°C. After stirring at rt for 30 min. the reaction mixture was cooled to 0°C, CS2 (9.50 g, 121.76 mmol) was added to the mixture. After 10 min at this temperature, MeI (15.45 g, 109.58 mmol) was added to the mixture. And subsequently the mixture was stirred at room temperature. for 12 hours. Water was added. The product was extracted with EA. The organic layer was washed with brine and dried over Na ₂ SO ₄ . The organic solution was concentrated and purified by column chromatography to give 17-11 (50 g, 51.17 mmol, 84.1% yield) as a white solid. ESI-LCMS: m/z 977.2 [M+H] ⁺ .

Preparation of intermediate compound (17-12). To a solution of 17-11 (17 g, 17.40 mmol) in DCM (100 mL) was added PTSA (5.99 g, 34.79 mmol) in methanol (10 mL). The mixture was stirred at room temperature. for 1 hour. Cooled to 0°C. Added conc. NH4OH to obtain pH=7.5. Water was added and the product was extracted with DCM. The organic layer was concentrated and purified by column chromatography to give 17-12 (10 g, 14.82 mmol, 85.2% yield) as a white solid. ESI-LCMS: m/z 675.1 [M+H] ⁺ .

Preparation of intermediate compound (17-13). To a solution of 17-12 (40 g, 59.28 mmol) in DCM (800 ml) was added TFA (150 ml). The mixture was stirred at room temperature. for 1 hour. Cooled to 0°C. Added conc. NH4OH to the solution until pH = 7.5. Water was added and the product was extracted with DCM. The organic layer solution was dried over Na ₂ SO ₄ and concentrated to give the crude product, which was purified by column chromatography to give 17-13 (21 g,

- 35 045540

52.18 mmol, 88.0% yield) as a white solid. ESI-LCMS: m/z 403.0 [M+H]+.

Preparation of intermediate compound (17-14). To a solution of 17-13 (26 g, 64.60 mmol) in pyridine (500 ml) was added Ac ₂ O (19.79 g, 193.81 mmol). The reaction mixture was stirred at 40°C for 1 hour. Water was added. The solution was concentrated to obtain the crude product. The crude product was purified by column chromatography and MPLC to give product 17-14 (21 g, 43.16 mmol, 66.8% yield) as a white solid. ESI-LCMS: m/z 487.0 [M+H] ⁺ .

Preparation of intermediate compound (17-15). To a solution of DBH (4.19 g, 14.64 mmol) in DCM (75 ml) was added HF/pyridine (10 ml) at -60°C. Subsequently, SM-1 (2.5 g, 5.14 mmol) dissolved in DCM was slowly added to the mixture. The reaction mixture was stirred at -40°C for 1 hour. DCM was added to the mixture. Added sat. NaHCO ₃ solution slowly to the mixture until pH = 7.0 is reached. The solution was concentrated to obtain the crude product. The crude product was purified by column chromatography and MPLC to give product 17-15 (1.1 g, 2.37 mmol, 46.1% yield) as a white solid. ESI-LCMS: m/z 465.1 [M+H] ⁺ . ¹⁹ F NMR (376 MHz, chloroform-d) δ -55.49.

Preparation of intermediate compound (17-16). A solution of 17-15 (4.6 g, 9.91 mmol) in methylamine (30 ml) was stirred at room temperature. for 1 hour. The solution was concentrated and purified by column chromatography to give 17-16 (3.7 g, 9.73 mmol, 98.2% yield) as a white solid. ESI-LCMS: m/z 381.1 [M+H] ⁺ .

Preparation of intermediate compound (17-17). NaNO2 (30.40 g, 447.04 mmol) was added to a solution of 17-16 (1.7 g, 4.47 mmol) in AcOH (5 mL) and water (5 mL). The mixture was stirred at room temperature. within 3 hours, LCMS showed 70% conversion of the substance. EA and water were added. The organic layer was concentrated and purified by MPLC to give 17-17 (1.5 g, 3.93 mmol, 88.0% yield) as a white solid. ESI-LCMS: m/z 382.2 [M+H] ⁺ .

Preparation of intermediate compound (17-18). To a solution of 17-17 (2.23 mg, 5.85 mmol) in DCM (30 mL) was added imidazole (796.36 mg, 11.70 mmol) and TBSCl (1.32 mg, 8.77 mmol). The reaction mixture was stirred at room temperature. for 12 hours. Water was added. The organic layer was washed with brine and dried over Na ₂ SO ₄ . The organic layer was concentrated to give crude 17-18 (2.8 g, 5.65 mmol, 96.6% yield) as a white solid. ESI-LCMS: m/z 496.4 [M+H] ⁺ .

Preparation of intermediate compound (17-19). To a solution of 17-18 (2.8 g, 5.65 mmol) in THF (30 ml) was added PPh ₃ (1.78 g, 6.78 mmol) and water (203.59 g, 11.30 mmol). The mixture was stirred at 50°C for 2 hours under N ₂ atmosphere. The mixture was concentrated and purified by column chromatography to give 17-19 (2.5 g, 5.32 mmol, 94.2% yield) as a white solid. ESI-LCMS: m/z 470.3 [M+H] ⁺ .

Preparation of intermediate compound (17-20). To a solution of 17-19 (2.8 g, 5.96 mmol) in DCM (40 mL) was added TEA (1.21 g, 11.93 mmol, 1.66 mL) and MMTrCl (2.76 g, 8. 95 mmol). The mixture was stirred at room temperature. for 1 hour. Water was added. The organic layer was dried over Na2SO4 and concentrated, the residue was purified by column chromatography to give 17-20 (3.1 g, 4.18 mmol, 70.1% yield) as a white solid. 1H NMR (400 MHz, DMSO-d ₆ ) δ 11.36 (d, J=2.2 Hz, 1H), 7.67 (d, J=8.0 Hz, 1H), 7.46 (td, J=8.6, 1.3 Hz, 4H), 7.37-7.30 (m, 2H), 7.25 (t, J=7.5 Hz, 4H), 7.22-7.14 (m, 2H), 6.846.77 (m, 2H), 5.54 (s, 1H), 5.44 (dd, J=8.0, 2.2 Hz, 1H), 4.17-4, 01 (m, 4H), 3.90 (dd, J=9.8, 2.4 Hz, 1H), 3.70 (s, 4H), 3.07 (td, J=10.1, 4, 5 Hz, 1H), 3.03-2.93 (m, 1H), 2.82 (d, J=10.3 Hz, 1H), 1.51 (d, J=4.5 Hz, 1H) , 0.84 (s, 9H), 0.12 (s, 3H), -0.03 (s, 3H). ¹⁹ F NMR (376 MHz, DMSO-d6) δ -58.72.

Preparation of intermediate compound (17-21). To a solution of 17-20 (3.1 g, 4.18 mmol) in THF (30 mL) was added TBAF (1.64 g, 6.27 mmol). The mixture was stirred at room temperature. for 15 hours. The mixture was concentrated and purified by column chromatography to give 17-21 (2.2 g, 3.51 mmol, 83.9% yield) as a white solid. 1H NMR (400 MHz, DMSO-d ₆ ) δ 11.30 (d, J=2.0 Hz, 1H), 7.98 (d, J=8.1 Hz, 1H), 7.52-7, 42 (m, 4H), 7.37-7.30 (m, 2H), 7.30-7.23 (m, 4H), 7.23-7.14 (m, 2H), 6.87- 6.79 (m, 2H), 5.52 (d, J=2.0 Hz, 1H), 5.50 (d, J=1.5 Hz, 1H), 5.17 (t, J=3 .8 Hz, 1H), 4.13 (t, J=4.2 Hz, 2H), 4.05 (dd, J=4.2, 2.1 Hz, 2H), 3.90 (dt, J =10.0, 2.2 Hz, 1H), 3.72 (s, 3H), 3.67 (dt, J=11.4, 3.7 Hz, 1H), 3.39 (dt, J= 6.4, 5.1 Hz, 1H), 3.10 (td, J=10.3, 4.3 Hz, 1H), 2.97 (dt, J=11.8, 4.7 Hz, 1H ), 2.68 (d, J=10.6 Hz, 1H), 1.44 (d, J=4.3 Hz, 1H). ¹⁹ F NMR (376 MHz, DMSO-d ₆ ) δ -58.82.

Receipt (17-22). To a solution of 17-21 (1.4 g, 2.23 mmol) in DCM (10 ml) was added DCI (318.88 mg, 2.23 mmol) and CEP[N(iPr) ₂ ] ₂ (872.87 mg , 2.90 mmol). The mixture was stirred at room temperature. for 1 hour. The solution was diluted with DCM. The organic layer was washed with sat. NaHCO3 solution, water and saline solution. The organic layer was dried over Na2SO4 and purified by column chromatography to obtain 1.3 g of crude product. The product was dried in vacuum at 40°C overnight. The result was 17-22 (1.15 g, 1.39 mmol, 62.28% yield) as a white solid. MS m/z [M-N] ^- (IER): 826.4; 1H NMR (400 MHz, DMSO46) δ 11.37 (s, 1H), 7.78 (d, J=8.1 Hz, 0.6 H), 7.59-7.44 (m, 4.4 H), 7.41-7.33 (m, 2H), 7.27 (m, 4H), 7.24-7.14 (m, 2H), 6.88-6.79 (m, 2H) , 5.57-5.48 (m, 1.4H), 5.44 (d, J=8.0 Hz, 0.6H), 4.25-4.13 (m, 2H), 4.13 -3.90 (m, 3H), 3.79-3.38 (m, 8H), 3.03 (m, 2H), 2.86 (dd, J=26.4, 10.4 Hz, 1H ), 2.79-2.67 (m, 2H), 1.67 (d, J=4.5 Hz, 0.4 N), 1.26 (d, J=4.5 Hz, 0.6 H), 1.18 (d, J=6.7 Hz, 3H), 1.15-1.02 (m, 9H). ³¹ P NMR (162 MHz, DMSO-d6) δ 148.03, 146.68.

- 36 045540

Example 14.

Scheme 16.

Preparation of intermediate compound (18-23). To a solution of 18-16 (1.2 g, 3.16 mmol) in pyridine (10 ml) was added BzCl (971.92 mg, 6.94 mmol) at 0°C. The mixture was stirred at room temperature. for 1 hour. Water was added. The product was extracted with EA. The organic layer was washed with brine and dried over Na ₂ SO ₄ . The organic layer was concentrated and purified by column chromatography to provide 18-23 (1.58 g, 2.68 mmol, 85.1% yield) as a white solid. ESI-LCMS: m/z 589.1 [M+H]+.

Preparation of intermediate compound (18-24). To a solution of 18-23 (1.47 g, 2.50 mmol) in THF (20 ml) was added water (90.00 mg, 5.00 mmol) and PPh ₃ (786.20 mg, 3.00 mmol). The mixture was stirred at 50°C under N2 atmosphere. The mixture was concentrated and purified by column chromatography to give 18-24 (1.35 g, 2.40 mmol, 96.1% yield) as a white solid. ESI-LCMS: m/z 563.2 [M+H]+.

Preparation of intermediate compound (18-25). To a solution of 18-24 (1.4 g, 2.49 mmol) in DCM (40 ml) was added TEA (503.71 mg, 4.98 mmol, 694.29 μl) and MMTrCl (1.15 g, 3. 73 mmol). The mixture was stirred at room temperature. for 1 hour. Water was added. The organic layer was dried over Na ₂ SO ₄ and concentrated, the residue was purified by column chromatography to give 18-25 (2 g, 2.40 mmol, 96.3% yield) as a white solid. ¹⁹ F NMR (376 MHz, DMSO-d ₆ ) δ -58.72.

Preparation of intermediate compound (18-26). To a solution of 18-25 (2 g, 2.40 mmol) in pyridine (30 ml) was added NaOH (287.46 mg, 7.19 mmol) in methanol (12 ml) and water (3 ml) at 0°C. The mixture was stirred at room temperature. for 0.5 hours. Added sat. NH ₄ Cl solution until pH=8 is reached. The product was extracted with EA. The organic layer was concentrated and purified by MPLC to give 18-26 (1.5 g, 2.05 mmol, 85.69% yield) as a white solid. 1H NMR (400 MHz, DMSO-dg) δ 11.27 (s, 1H), 8.55 (d, J=7.5 Hz, 1H), 8.11-7.94 (m, 2H), 7 .77-7.57 (m, 1H), 7.57-7.40 (m, 6H), 7.40-7.10 (m, 9H), 6.97-6.76 (m, 2H) , 5.56 (s, 1H), 5.36-5.18 (m, 1H), 4.15 (dd, J=9.7, 5.4 Hz, 4H), 4.01 (d, J =10.2 Hz, 1H), 3.84 (d, J=14.7 Hz, 1H), 3.72 (s, 3H), 3.18-2.97 (m, 2H), 2.66 (d, J=10.9 Hz, 1H), 1.48 (d, J=4.l Hz, 1H). ¹⁹ F NMR (376 MHz, DMSO-d ₆ ) δ -58.75.

Receipt (18-27). To a solution of 18-26 (1.4 g, 1.92 mmol) in DCM (30 ml) was added DCI (226.08 mg, 1.92 mmol) and CEP[N(iPr) ₂ ] ₂ (749.69 mg , 2.49 mmol). The mixture was stirred at room temperature. for 1 hour. The solution was diluted with DCM. The organic layer was washed with water and brine. The organic layer was dried over Na ₂ SO ₄ and concentrated to obtain the crude product. The crude product was purified by HPLC. The product was dried in vacuum at 40°C overnight. The result was 18-27 (1.65 g, 1.77 mmol, 92.51% yield) as a white solid. ESI-LCMS: m/z 877 [M+H] ⁺ . 1H NMR (400 MHz, DMSO-d6) δ 11.33 (s, 1H), 8.34 (d, J=7.4 Hz, 0.6H), 8.20 (d, J=7.4 Hz , 0.6H), 8.02 (dt, J=8.3, 1.2 Hz, 2H), 7.73-7.57 (m, 1H), 7.57-7.40 (m, 6H ), 7.40-7.30 (m, 3H), 7.30-7.10 (m, 6H), 6.866.71 (m, 2H), 5.60 (d, J=1.1 Hz, 1H), 4.41-4.03 (m, 5H), 4.00-3.39 (m, 8H), 3.05 (m, 2H), 2.92-2.70 (m, 3H) , 1.53

- 37 045540

Scheme 17.

(d, J=4.2 Hz, 0.4H), 1.29 (d, J=4.2 Hz, 0.6H), 1.26-1.00 (m, 12H). ³¹ P NMR (162 MHz, DMSO-d6) δ 148.41, 146.88.

Example 15.

Preparation of intermediate compound (19-2). To a solution of 19-1 (48.0 g, 138.2 mmol) in anhydrous pyridine (500 ml) was added TIPDSCl (52.2 g, 165.8 mmol) at 0°C, the mixture was left to stir at room temperature. for 5 hours. The mixture was then poured into cold water, extracted using EA, washed with brine, dried over anhydrous Na ₂ SO ₄ and concentrated to give a residue, which was purified by column chromatography to give 19-2 (67 g, 113, 6 mmol, yield 82.2%) as a white solid. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ=11.29 (s, 1H, exchange with D2O), 8.21 (d, J=7.2 Hz, 1H), 8.02-8 .00 (m, 2H), 7.65-7.61 (m, 1H), 7.54-7.50 (m, 2H), 7.38 (d, J=7.2 Hz, 1H), 5.83 (d, J=3.6 Hz, 1H, exchange with D ₂ O), 5.64 (s, 1H), 4.24 (d, J=13.2 Hz, 1H), 4.11 (s, 3H), 3.95 (d, J=12.8 Hz, 1H), 1.09-0.96 (m, 28H). ESI-LCMS: m/z 590 [M+H]+.

Preparation of intermediate compound (19-3). To a solution of 19-2 (54 g, 91.5 mmol) and EtI (285.6 g, 1.8 mol, 146 ml) in toluene (500 ml) was added Ag ₂ O (63.6 g, 274.6 mmol ), the mixture was stirred at 50°C for 4 hours, filtered and the filtrate was concentrated to give a residue, which was purified by column chromatography to give 19-3 (41.2 g, 66.4 mmol, 72.5% yield) as white solid. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ=11.34 (s, 1H, exchange with D2O), 8.19 (d, J=7.2 Hz, 1H), 7.97 (d , J=7.6 Hz, 2H), 7.63 (t, J=7.2 Hz, 1H), 7.51 (t, J=7.6 Hz, 2H), 7.38 (d, J =7.2 Hz, 1H), 5.67 (s, 1H), 4.22 (d, J=13.6 Hz, 1H), 4.14 (dd, J=4.0, 9.6 Hz , 1H), 4.08 (d, J=9.6 Hz, 1H), 3.95-3.91 (m, 2H), 3.82 (dd, J=6.8, 13.6 Hz, 2H), 1.17 (t, J=6.8 Hz, 3H), 1.04-0.94 (m, 28H). ESI-LCMS: m/z 618 [M+H] ⁺ .

Preparation of intermediate compound (19-4). To a solution of 19-3 (41.2 g, 66.4 mmol) in THF (400 ml) was added TBAF (1M in THF) (132.7 mmol, 133 ml), the mixture was stirred at room temperature. for 1 hour, poured into cold water, extracted with EA, washed with brine, dried over anhydrous Na ₂ SO ₄ and concentrated to a solid residue, which was washed with water (PE:EA=1:1) to give 19-4 (19.1 g, 50.6 mmol, 76.3% yield) as a white solid. 1HNMR (400 MHz, DMSO-d ₆ ): δ=11.29 (s, 1H, exchange with D2O), 8.57 (d, J=7.6 Hz, 1H), 8.01 (d, J= 7.6 Hz, 2H), 7.63 (t, J=7.6 Hz, 1H), 7.52 (t, J=8.0 Hz, 2H), 7.36 (d, J=7, 6 Hz, 1H), 5.87 (d, J=2.8 Hz, 1H), 5.26 (t, J=5.2 Hz, 1H, exchange with D2O), 5.05 (d, J= 7.2 Hz, 1H, exchange with D2O), 4.11-4.06 (m, 1H), 3.94-3.91 (m W), 3.86-3.74 (m, 3H), 3.71-3.61 (m, 2H), 1.17 (t, J=7.6 Hz, 3H). ESI-LCMS: m/z 376 [M+H] ⁺ .

Preparation of intermediate compound (19-5). To a solution of 19-4 (19.1 g, 50.6 mmol) and imidazole (5.2 g, 75.9 mmol) in pyridine (200 ml) was added TBDPSCl (7.1 g, 60.7 mmol), a mixture stirred at room temperature. for 1 hour, poured into cold water, extracted using EA, washed with brine, dried over anhydrous Na ₂ SO ₄ and concentrated to give a solid, which was purified by column chromatography to give 19-5 (20.2 g, 32 .6 mmol, yield 64.4%) as a white solid. ESI-LCMS: m/z 614 [M+H]+.

Preparation of intermediate compound (19-6). To a solution of 19-5 (14.0 g, 22.8 mmol) and collidine (6.9 g, 57.0 mmol) in DCM (150 ml) was added DMTrCl (11.6 g, 34.2 mmol) and AgNO ₃ (3.9 g, 22.8 mmol) under Ar atmosphere. The mixture was stirred at room temperature. for 15 hours, filtered, and the filtrate was washed with water, dried over anhydrous Na ₂ SO ₄ and concentrated to give crude product 19-6 (26.2 g) as a yellow oil. ESI-LCMS: m/z 916 [M+H] ⁺ .

- 38 045540

Preparation of intermediate compound (19-7). To a solution of 19-6 (26.2 g, (crude)) in THF (200 ml) was added TBAF (1 M in THF) (23 ml), the mixture was stirred at 50°C for 3 hours, poured into cold water, extracted using EA, washed with brine, dried over anhydrous Na ₂ SO ₄ and concentrated to give a residue which was purified by column chromatography to give 19-7 (11.2 g, 16.5 mmol, 72.37% yield in two stage) as a white solid. ¹ H-NMR (400 MHz, DMSO-d6): δ=11.30 (s, 1H, exchange with D ₂ O), 8.40 (d, J=7.6 Hz, 1H), 8.02 ( d, J=7.6 Hz, 2H), 7.65-7.61 (m, 1H), 7.54-7.49 (m, 4H), 7.35-7.30 (m, 6H) , 7.26-7.23 (m, 1H), 6.89-6.86 (m, 4H), 5.96 (d, J=3.2 Hz, 1H), 5.08 (t, J =4.4 Hz, 1H, exchange with D2O), 4.01 (t, J=5.2 Hz, 1H), 3.79-3.78 (m, 1H), 3.73 (d, J= 1.2 Hz, 6H), 3.66-3.55 (m 2H), 3.40-3.38 (m, 1H), 3.25-3.17 (m, 1H), 2.97- 2.95 (m, 1H), 1.17 (t, J=6.8 Hz, 3H). ESI-LCMS: m/z 678 [M+H]+.

Receipt (19-8). To a solution of 19-7 (5.0 g, 7.4 mmol) and DCI (2.2 mg, 7.4 mmol) in dry DCM (50 ml) was added CEP[N(iPr) ₂ ] ₂ (2.7 g, 8.8 mmol) in an Ar atmosphere. The mixture was stirred at room temperature. for 1 hour. The reaction mixture was washed with 10% aqueous NaHCO ₃ and water, dried over Na ₂ SO ₄ and concentrated to give the crude product, which was purified by preparative flash HPLC. The result was 19-8 (5.5 g, 6.26 mmol, 84.91% yield) as a white solid. ¹ H-NMR (400 MHz, DMSO-d6): δ=11.34 (s, 1 H), 8.16 (dd, J=7.6, 39.2 Hz, 1H), 8.03-8 .00 (m, 2H), 7.65-7.61 (m, 2H), 7.54-7.45 (m, 4H), 7.35-7.24 (m, 8H), 6.88 -6.82 (m, 4H), 5.89 (dd, J=3.2, 88.8 Hz, 1H), 4.253.86 (m, 3H), 3.75-3.66 (m, 9H ), 3.62-3.38 (m, 4H), 3.26-3.05 (m, 1H), 2.78 (dd, J=5.6, 10.4 Hz, 2H), 1, 20-1.11 (m, 9H), 1.04 (dd, J=6.4, 27.6 Hz, 6H). 31P-NMR (162 MHz, DMSO-d6): 148.09, 147.84. ESI-LCMS: m/z 878 [M+H] ⁺ .

Preparation of intermediate compound (20-2). To a solution of thymine (8.15 g, 64.68 mmol, 1.5 eq.) in acetonitrile (300 ml) under an inert nitrogen atmosphere at room temperature was added N,O-Bis(trimethylsilyl)acetamide (39 g, 194.2 mmol, 4.5 eq.). The resulting solution was stirred for 2 hours at 80°C. The reaction mixture was cooled to 0°C and 20-1 (30 g, 43.16 mmol, 1.00 eq.) was added. Subsequently, trimethylsilyl trifluoromethanesulfonate (57.48 g, 258.95 mmol, 6.0 eq.) was added dropwise at

- 39 045540 stirring at 0°C. The resulting solution was left for another 1 hour for the reaction to proceed with stirring at a temperature of 80°C. The reaction mixture was cooled to 0°C and quenched by adding a saturated sodium bicarbonate solution. The resulting solution was extracted with ethyl acetate and the organic layers were combined. The organic layer was washed with water and saturated sodium chloride solution, respectively. The residue was purified by column chromatography. The result was 18 g (55%) of 20-2 as a brown solid. MS m/z [M+H]+ (ESI): 762.

Preparation of intermediate compound (20-3). To a solution of 20-2 (18 g, 23.9 mmol, 1.00 eq.) in tetrahydrofuran (180 ml) under an inert nitrogen atmosphere was added sodium hydroxide (47 ml, 2 N in water, 4.0 eq.) at 0°C. The resulting solution was stirred for 4 hours at 0°C. The resulting mixture was extracted with ethyl acetate and the organic layers were combined. The organic layer was dried over anhydrous sodium sulfate and concentrated. The crude product was purified by preparative flash HPLC. The result was 11.4 g (88%) of 20-3 as a white solid. MS m/z [M+H]+ (ESI): 548.

Preparation of intermediate compound (20-4). To a solution of 20-3 (11.4 g, 20.84 mmol, 1.00 eq.) in tetrahydrofuran (100 ml) was added 10% palladium on activated carbon (1.4 g). The flask was evacuated and purged with hydrogen five times. The resulting solution was stirred for 8 hours at room temperature. The solids were filtered. The resulting mixture was concentrated under reduced pressure. The result was 10 g (92%) of 20-4 as a white solid. MS m/z [M+H]+ (ESI): 522.

Preparation of intermediate compound (20-5). To a solution of 20-4 (10 g, 19.19 mmol, 1.00 eq.) in pyridine (50 ml) under an inert nitrogen atmosphere at room temperature was added 1(chlorodiphenylmethyl)-4-methoxybenzene (8.8 g, 28. 79 mmol, 1.5 eq.). The resulting solution was stirred for 12 hours at room temperature. Subsequently, the reaction was quenched by the addition of methanol (20 ml). The crude product was purified by preparative flash HPLC. Fractions (500 ml) were diluted with dichloromethane and dried over anhydrous sodium sulfate. Solids were filtered off. The filtrate was concentrated under reduced pressure. The result was 12.2 g (80%) of 20-5 as a white solid. MS m/z [Μ-H]’ (IER): 792.

Preparation of intermediate compound (20-6). To a solution of 20-5 (4.2 g, 5.29 mmol, 1.00 eq.) in dichloromethane (40 ml) under an inert nitrogen atmosphere at room temperature was added tetrabutylammonium fluoride (1M in THF, 13.0 ml, 2. 5 eq.).

The resulting solution was stirred for 10 hours at room temperature. The crude product was purified by preparative flash HPLC. The fractions were diluted with dichloromethane and dried over anhydrous sodium sulfate. Solids were filtered off. The filtrate was concentrated under reduced pressure. The result was 2.5 g (85%) of 20-6 as a white solid. MS m/z [M—H]'(ESI): 554. 1H NMR: (DMSO-d ₆ , 300 Hz, ppm): δ 11.25 (s, 1H), 7.95 (s, 1H), 7.46 (m, 4H), 7.317.11 (m, 8H), 6.76 (m, 2H), 5.61 (s, 1H), 5.22 (t, J=3.0 Hz, 1H), 3.95 (m, 1H), 3.85-3.61 (m, 6H), 2.85 (m, 1H), 2.45 (d, J=9.0 Hz, 1H ), 2.23-2.06 (m, 1H), 1.61 (m, 4H), 1.34 (d, J=12.0 Hz, 1H).

Receipt (20-7). To a solution of 20-6 (2.5 g, 3.78 mmol, 1.00 eq.) in dichloromethane (20 ml) under an inert nitrogen atmosphere at room temperature was added bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.7 g, 5.67 mmol, 1.5 eq.). 4,5-dicyanoimidazole (440 mg, 4.15 mmol, 1.10 eq.) was added to this mixture at room temperature. The resulting solution was stirred for 3 hours at room temperature and diluted with dichloromethane and washed with water. The crude product was purified by preparative flash HPLC. The fractions were diluted with dichloromethane and dried over anhydrous sodium sulfate. Solids were filtered off. The filtrate was concentrated under reduced pressure. The result was 2.3 g (67%) of 20-7 as a white solid. MS m/z [M-H]' (ESI): 754 NMR: (DMSO-d ₆ , 300 Hz, ppm): δ 11.31 (s, 1H), 7.76-7.41 ( m, 5H), 7.37-7.10 (m, 8H), 6.87-6.68 (m, 2H), 5.66 (m, 1H), 4.04 (m, 1H), 3 .83 (m, 2H), 3.70 (m, 3H), 3.69-3.45 (m, 4H), 3.30-3.00 (m, 1H), 2.88-2.54 (m, 3H), 2.452.12 (m, 2H), 1.87-1.64 (m, 4H), 1.51 (m, 1H), 1.17 (m, 12H). P NMR (DMSO-d6, 300 Hz, ppm): 152.69, 150.53.

Example 17.

- 40 045540

Scheme 19.

MsCl(l.2 eq.)

21-39

BUT . 10%

O Pd/C (10%, v/v)

21-40

CEOPlN(iPr) ₂ | ₂

DC1, DCM, ct

Preparation of intermediate compound (21-21). To a solution of (3aR,5S,6S,6aR)-5-(2,2-dimethyl-1,3dioxolan-4-yl)-2,2-dimethyltetrahydro-2H-furo[2,3-d][1,3 ]dioxol-6-ol (500 g, 1.92 mol, 1.00 eq.) in N,N-dimethylformamide (5000 ml) under an inert nitrogen atmosphere at room temperature was added (bromomethyl)benzene (345 g, 2.02 mol, 1.05 eq.). Sodium hydride (123 g, 3.08 mol, 1.60 eq. 60%) was added for several batches at 0°C. The resulting solution was stirred for 2 hours at 25°C. Subsequently, the reaction mixture was quenched by adding an aqueous solution of ammonium chloride. The resulting solution was extracted with ethyl acetate, and the organic layers were combined and dried over anhydrous sodium sulfate and concentrated under reduced pressure. The result was 670 g (crude) 2121 as a yellow oil. MS m/z [M+H]+ (ESI): 351.

Preparation of intermediate compound (21-22). 21-21 (500 g, 1.43 mol, 1.00 eq.) was dissolved in 80% acetic acid (5000 ml). The resulting solution was stirred overnight at room temperature. The resulting mixture was concentrated under reduced pressure. The resulting solution was diluted with ethyl acetate. The resulting mixture was washed with an aqueous solution of sodium bicarbonate. The mixture was dried over anhydrous sodium sulfate and concentrated under reduced pressure. This resulted in 440 g (crude) of 21-22 as a yellow oil. MS m/z [M+Na]+ (ESI): 333.

Preparation of intermediate compound (21-23). To a solution of 21-22 (500 g, 1.61 mol, 1.00 eq.) in 1,4dioxane (5000 ml) was added dropwise a solution of sodium periodate (345 g, 1.61 mol, 1.00 eq.) in water (5000 ml) with stirring at 0°C. The resulting solution was stirred for 2 hours at room temperature. The resulting solution was diluted with ethyl acetate. The resulting mixture was washed with water. The mixture was dried over anhydrous sodium sulfate and concentrated under reduced pressure. This resulted in 400 g of (crude) 21-23 as a yellow oil. MS m/z [M+H]+ (ESI): 279.

- 41 045540

Preparation of intermediate compound (21-24). To a solution of 21-23 (250 g, 898.3 mmol, 1.00 eq) in tetrahydrofuran/water (1250/1250 ml) was added a solution of formaldehyde (37%, 600 ml) at room temperature. 2 N was added dropwise to the resulting mixture. sodium hydroxide solution (1500 ml, 3.12 eq.) with stirring at 0°C. The resulting solution was stirred overnight at room temperature. The resulting solution was extracted with ethyl acetate and the organic layers were combined. The resulting mixture was washed with water. The mixture was dried over anhydrous sodium sulfate and concentrated under reduced pressure. This resulted in 191 g (crude) of 21-24 as a yellow oil. MS m/z [M+Na]+ (ESI): 333.

Preparation of intermediate compound (21-25). To a solution of 21-24 (300 g, 966.7 mmol, 1.00 eq) in pyridine (3000 ml) under an inert nitrogen atmosphere was added benzoyl chloride (409.4 g, 2.91 mol, 3.01 eq) dropwise .) at 0°C, and subsequently stirred for 2 hours at room temperature. The resulting solution was diluted with ethyl acetate. The resulting mixture was washed with an aqueous solution of sodium bicarbonate. The mixture was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography. The result was 410 g (82%) of 21-25 as a yellow oil.

Preparation of intermediate compound (21-26). To a solution of 21-25 (250 g, 482.11 mmol, 1.00 eq.) in methanol (2500 ml) was added 10% palladium on carbon (125 g) and acetic acid (29 g, 482.11 mmol 1.00 eq.). H ₂ (gaseous) was subsequently introduced. The resulting solution was stirred for 72 hours at 50°C. The solids were filtered. The resulting mixture was concentrated under reduced pressure. The resulting solution was diluted with dichloromethane and washed with saturated aqueous sodium bicarbonate. The mixture was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The result was 185 g (90%) of 21-26 as a light yellow oil.

Preparation of intermediate compound (21-27). Pyridine (300 mL) was added to a solution of 21-26 (128 g, 299.06 mmol, 1.00 eq.) in dichloromethane (900 mL) under an inert nitrogen atmosphere at room temperature. Trifluoromethanesulfonic anhydride (109.4 g, 387.9 mmol, 1.30 eq.) was added dropwise to the resulting mixture at -20°C. The resulting solution was stirred for 2 hours at -20°C. The resulting solution was diluted with ethyl acetate. The resulting mixture was washed with water and saline. The mixture was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The result was 158 g (crude) of 21-27 as a yellow oil.

Preparation of intermediate compound (21-28). To a solution of 21-27 (158 g, 281.89 mmol, 1.00 eq.) in N,N-dimethylformamide (1500 mL) under an inert nitrogen atmosphere was added sodium azide (55 g, 846.02 mmol, 3.00 eq. .) and tetrabutylammonium iodide (10.4 g, 28.18 mmol, 0.10 eq.) at room temperature. The resulting solution was stirred overnight at 100°C. Subsequently, the reaction was quenched by adding ice water. The resulting solution was extracted with ethyl acetate and the organic layers were combined. The resulting mixture was washed with water and saline. The mixture was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography. The result was 58 g (45%) of 21-28 as a yellow oil.

Preparation of intermediate compound (21-29). To a solution of 21-28 (70 g, 154.4 mmol, 1.00 eq.) in methanol/water (850/150 ml) was added potassium carbonate (6.4 g, 46.0 mmol, 0.30 eq. ). The resulting solution was stirred for 5 hours at 0°C. The pH of the solution was adjusted to 7 using a 10% hydrochloric acid solution. The resulting mixture was concentrated under reduced pressure. The resulting solution was diluted with ethyl acetate and washed with water. The mixture was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography. The result was 26.7 g (71%) of 21-29 as a white solid.

Preparation of intermediate compound (21-30). To a solution of 21-29 (12 g, 48.93 mmol, 1.00 eq.) in dichloromethane (360 mL) under an inert nitrogen atmosphere at room temperature was subsequently added triethylamine (14.85 g, 146.75 mmol, 3.00 eq. eq.). Tert-butyl(chloro)diphenylsilane (13.5 g, 49.12 mmol, 1.00 eq.) was added dropwise to the resulting mixture at 0°C. The resulting solution was stirred overnight at room temperature. The resulting solution was diluted with dichloromethane and washed with water. The mixture was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography. The result was 12.8 g (54%) of 21-30 as a white solid. MS m/z [M+Na] ⁺ (ESI): 506.

Preparation of intermediate compound (21-31). To a solution of oxalyl chloride (5.1 mL, 1.50 eq.) in dichloromethane (300 mL) under an inert nitrogen atmosphere, dimethyl sulfoxide (8.1 mL, 3.00 eq.) was added dropwise with stirring at -78°C. The resulting solution was stirred at -78°C for 30 minutes. To this was added a solution of 21-30 (26 g, 43 mmol, 1.00 eq.) in dichloromethane (100 ml) with stirring at -78°C, stirring for an additional 45 minutes at -78°C. Subsequently, triethylamine (23.5 ml, 4.50 eq.) was added and the reaction was allowed to proceed with stirring for an additional 1 hour at 25°C. The resulting solution was diluted with dichloromethane and washed with hydrochloric acid, saturated sodium bicarbonate solution and saturated sodium chloride solution, respectively. The mixture was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The result was 20 g (80%) of 21-31 as a colorless oil.

- 42 045540

Preparation of intermediate compound (21-32). To a solution of 21-31 (20 g, 41.53 mmol, 1.00 eq.) in ether (200 ml) under an inert nitrogen atmosphere was added methylmagnesium bromide (3 M) (28 ml, 62.30 mmol, 1 .50 eq.) with stirring at -100°C. The resulting solution was stirred for 2 hours at -100°C. Subsequently, the reaction was quenched by adding saturated aqueous ammonium chloride (50 ml) and diluted with ethyl acetate. The resulting mixture was washed with water and saturated sodium chloride solution, respectively. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The result was 14.5 g (70%) of 21-32 as a colorless oil. MS m/z [M+Na]+ (ESI): 520.

Preparation of intermediate compound (21-33). To a solution of PH21-32 (15 g, 30.14 mmol, 1.00 eq.) in pyridine (150 ml) under an inert nitrogen atmosphere was added dropwise methanesulfonyl chloride (4.5 g, 39.28 mmol, 1.30 eq. .) with stirring at 0-5°C. The resulting solution was stirred for 3 hours at 0-5°C. Subsequently, the reaction was quenched by adding methanol (20 ml) and diluted with ethyl acetate. The resulting mixture was washed with water and saturated sodium chloride solution. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The result was 13.8 g (70%) of 21-33 as a brown solid. MS m/z [M+Na] ⁺ (ESI): 598.

Preparation of intermediate compound (21-34). To a solution of 21-33 (10 g, 17.37 mmol, 1.00 eq.) in acetic acid (150 mL) under an inert nitrogen atmosphere was added acetyl acetate (19.5 g, 191.01 mmol, 11.00 eq. ) and sulfuric acid (170 mg, 1.73 mmol, 0.10 eq.). The resulting solution was stirred at room temperature for 1 hour. The resulting solution was diluted with ethyl acetate. The resulting mixture was washed with water and sat. sodium chloride solution. The mixture was dried over anhydrous sodium sulfate and concentrated. The crude product was purified by preparative flash HPLC. The result was 6.5 g (60%) of 21-34 as a yellow solid. MS m/z [M+Na] ⁺ (ESI): 642.

Preparation of intermediate compound (21-35). To a solution of 5-methyl-1,2,3,4-tetrahydropyrimidine2,4-dione (1.5 g, 11.90 mmol, 1.50 eq.) in acetonitrile (125 ml) under an inert nitrogen atmosphere was added N,O -bis(trimethylsilyl)acetamide (5.75 g, 28.19 mmol, 3.50 eq.) at room temperature. The resulting solution was stirred for 2 hours at 80°C. To the resulting mixture was added 21-34 (5 g, 8.07 mmol, 1.00 eq.) at 0°C, and subsequently trimethylsilyl trifluoromethanesulfonate (2.34 g, 10.53 mmol, 1.30 eq.) was added dropwise. ) with stirring at 0°C. The resulting solution was left for another 6 hours for the reaction to proceed with stirring at a temperature of 80°C. The reaction mixture was cooled to 0°C and quenched by adding a saturated ammonium chloride solution. The resulting solution was extracted with ethyl acetate, and the organic layers were combined and dried over anhydrous sodium sulfate and concentrated. The residue was purified by column chromatography. The result was 4.5 g (81%) of 21-35 as a white solid. MS m/z [M+H] ⁺ (ESI): 686.

Preparation of intermediate compound (21-36). Potassium carbonate (5.4 g, 38.79 mmol, 3.00 eq) was added to a solution of 21-35 (9 g, 13.12 mmol, 1.00 eq) in methanol (90 mL) under an inert nitrogen atmosphere. . The resulting solution was stirred for 2 hours at 65°C and diluted with ethyl acetate. The resulting mixture was washed with water and sat. sodium chloride solution. The organic layer was dried over anhydrous sodium sulfate and concentrated. The crude product was purified by preparative flash HPLC. The result was 5 g (70%) of 21-36 as a white solid. MS m/z [M+H] ⁺ (ESI): 548.

Preparation of intermediate compound (21-37). To a solution of 21-36 (6 g, 10.96 mmol, 1.00 eq.) in tetrahydrofuran (60 ml) was added 10% palladium on activated carbon (2 g). The flask was evacuated and purged with hydrogen five times. The resulting solution was stirred at room temperature for 8 hours. The solids were filtered. The resulting mixture was concentrated under reduced pressure. The result was 5.3 g (93%) of 21-37R and 21-37S as a white solid. Subsequently, the mixture was separated by preparative supercritical fluid chromatography (SFC). The result was 3.5 g of product as a white solid. MS m/z [M+H] ⁺ (ESI): 522.

Preparation of intermediate compound (21-38). 1(Chlorodiphenylmethyl)-4-methoxybenzene (2.7 g, 8.74 mmol, 1.30 eq.). The resulting solution was stirred at room temperature for 2 hours. Subsequently, the reaction was quenched by adding methanol (20 ml). The resulting solution was diluted with ethyl acetate. The resulting mixture was washed with saturated sodium bicarbonate solution and saline. The organic layer was dried over anhydrous sodium sulfate and concentrated. The residue was purified by column chromatography. The result was 4 g (75%) of 21-38 as a white solid. MS m/z [MH] ^- (IER): 792.

Preparation of intermediate compound (21-39). Triethylamine (1 g, 9.9 mmol, 2.0 eq) was added to a solution of 21-38 (4 g, 5.04 mmol, 1.00 eq) in dichloromethane (40 mL) under an inert nitrogen atmosphere at room temperature. . Triethylamine trihydrofluoride (0.97 g, 7.56 mmol, 1.50 eq.) was added to this mixture at room temperature.

The resulting solution was stirred at room temperature for 12 hours and diluted with dichloromethane. The resulting mixture was washed with water. The mixture was dried over anhydrous sulfate for

- 43 045540 sodium and concentrated. The crude product was purified by preparative flash HPLC. The result was 2.3 g (80%) of 21-39 as a white solid. 1H NMR (DMSO- _d6 , 300 Hz, ppm): δ 11.33 (s, 1H), 7.45 (m, 5H), 7.20 (m, 8H), 6.80 (m, 2H ), 5.35 (t, J=5.l Hz, 1H), 5.15 (s, 1H), 4.23 (m, 1H), 4.05 (m, 1H), 3.81 (m , 1H), 3.73 (s, 3H), 2.92 (d, J=9.7 Hz, 1H), 2.25 (d, J=9.8 Hz, 1H), 2.05 (s , 1H), 1.54 (s, 3H), 1.12 (d, J=6.1 Hz, 3H).

Receipt (21-40). To a solution of 21-39 in dichloromethane (23 mL) under an inert nitrogen atmosphere at room temperature was added bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.62 g, 5.38 mmol, 1.30 eq.). 4,5-dicyanoimidazole (490 mg, 4.15 mmol, 1.10 eq.) was added to this mixture at room temperature. The resulting solution was stirred at room temperature for 1 hour and diluted with dichloromethane. Subsequently, the mixture was washed with water. The organic layer was dried over anhydrous sodium sulfate and concentrated. The crude product was purified by preparative flash HPLC. The result was 2.3 g (70%) of 21-40 as a white solid. MS m/z [MH] ^- (ESI): 754. 1H NMR (DMSO-d6, 400 Hz, ppm) δ 11.37 (s, 1H), 7.45 (m, 4H), 7. 28 (m, 9H), 6.80 (m, 2H), 5.18 (d, J=6.3 Hz, 1H), 4.35 (m, 1H), 4.21 (m, 1H), 4, 01 (m, 1H), 3.7 (m, 6H), 3.6 (m, 1H), 2.90 (m, 3H), 2.33 (m, 1H), 2.13 (m, 1H ), 1.59 (d, J=21.6 Hz, 3H), 1.20 (m, 15H). P NMR (DMSO-d _fo 400 Hz, ppm): 147.45, 147.08.

Example 18.

Scheme 20.

PH-ALB-ON14-038-11

PH-ALB-ON14-038-21

Preparation of intermediate compound (22-2). To a solution of triphenylphosphane (10.5 g, 40.03 mmol, 2.00 eq.) in toluene (100 ml) under an inert nitrogen atmosphere at room temperature was added diisopropyl azodicarboxylate (8.10 g, 40.03 mmol, 2.00 eq. eq.). To the resulting mixture was added

- 44 045540 nitrobenzoic acid (6.70 g, 40.03 mmol, 2.00 eq.) at 0°C. The resulting solution was stirred for 2 hours at room temperature. 22-1 (10 g, 20.09 mmol, 1.00 eq.) was added to the resulting mixture at room temperature. The resulting solution was left to react for an additional 8 hours at 60°C. The reaction mixture was diluted using ethyl acetate. The resulting mixture was washed with water and saturated sodium chloride solution, respectively. The mixture was dried over anhydrous sodium sulfate and concentrated. The crude product was purified by preparative flash HPLC. The result was 5 g (38%) of 22-2 as a yellow solid.

Preparation of intermediate compound (22-3). To a solution of 22-2 (27.5 g, 42.52 mmol, 1.00 eq.) in methanol (300 ml) under an inert nitrogen atmosphere at 0°C was added sodium hydroxide (4.6 g, 85 mmol, 2. 0 eq.). The resulting solution was stirred for 2 hours at 25°C. The pH of the solution was adjusted to 8 with acetic acid. The resulting mixture was concentrated and subsequently diluted with ethyl acetate.

The resulting mixture was washed with water and saturated sodium chloride solution. The organic layer was dried over anhydrous sodium sulfate and concentrated. The crude product was purified by preparative flash HPLC. The result was 16.5 g (78%) of 22-3 as a yellow oil.

Preparation of intermediate compound (22-4). To a solution of 22-3 (16.5 g, 33.15 mmol, 1.00 eq.) in pyridine (160 ml) under an inert nitrogen atmosphere was added methanesulfonyl chloride (4.95 g, 43.23 mmol, 1. 30 eq.) with stirring at 0-5°C. The resulting solution was stirred for 3 hours at 0-5°C. Subsequently, the reaction was quenched by adding methanol (30 ml) and diluted with ethyl acetate. The resulting mixture was washed with water and sat. sodium chloride solution. The mixture was dried over anhydrous calcium chloride and concentrated under reduced pressure. The result was 15.3 g (70%) of 22-4 as a brown solid. MS m/z [M+Na] ⁺ (ESI): 598.

Preparation of intermediate compound (22-5). To a solution of 22-4 (15.3 g, 26.57 mmol, 1.00 eq.) in acetic acid (150 mL) under an inert nitrogen atmosphere was added acetyl acetate (30 g, 292.2 mmol, 11.00 eq. ) and sulfuric acid (260 mg, 2.65 mmol, 0.10 eq.). The resulting solution was stirred at room temperature for 1 hour and diluted with ethyl acetate. The resulting mixture was washed with water and sat. sodium chloride solution. The organic layer was dried over anhydrous sodium sulfate and concentrated. The crude product was purified by preparative flash HPLC. The result was 10 g (60%) of 22-5 as a yellow solid. MS m/z [M+Na] ⁺ (ESI): 642.

Preparation of intermediate compound (22-6). To a solution of 5-methyl-1,2,3,4-tetrahydropyrimidine2,4-dione (3.0 g, 23.8 mmol, 1.50 eq.) in acetonitrile (250 ml) under an inert nitrogen atmosphere was added N,O -bis(trimethylsilyl)acetamide (11.5 g, 56.1 mmol, 3.50 eq.) at room temperature. The resulting solution was stirred for 2 hours at 80°C. To the resulting mixture was added 22-5 (10 g, 16.14 mmol, 1.00 eq.) at 0°C, and subsequently trimethylsilyl trifluoromethanesulfonate (4.68 g, 21.06 mmol, 1.30 eq.) was added dropwise. ) with stirring at 0°C. The resulting solution was left for another 6 hours for the reaction to proceed with stirring at a temperature of 80°C. The reaction mixture was cooled to 0°C and quenched by adding a saturated ammonium chloride solution. The resulting solution was extracted with ethyl acetate, and the organic layers were combined and dried over anhydrous sodium sulfate and concentrated. The residue was purified by column chromatography. The result was 9 g (81%) of 22-6 as a white solid. MS m/z [M+H] ⁺ (ESI): 686.

Preparation of intermediate compound (22-7). To a solution of 22-6 (9 g, 13.12 mmol, 1.00 eq) in methanol (90 mL) under an inert nitrogen atmosphere was added potassium carbonate (5.4 g, 38.79 mmol, 3.00 eq) at room temperature. The resulting solution was stirred for 2 hours at 65°C. The resulting solution was diluted with ethyl acetate. The resulting mixture was washed with water and sat. sodium chloride solution.

The organic layer was dried over anhydrous sodium sulfate and concentrated. The crude product was purified by preparative flash HPLC. The result was 5 g (70%) of 22-7 as a white solid. MS m/z [M+H] ⁺ (ESI): 548.

Preparation of intermediate compound (22-8). To a solution of 22-7 (5 g, 9.13 mmol, 1.00 eq.) in tetrahydrofuran (50 ml) was added 10% palladium on activated carbon (2 g). The flask was evacuated and purged with hydrogen five times. The resulting solution was stirred at room temperature for 8 hours. The solids were filtered. The resulting mixture was concentrated under reduced pressure. The result was 4.4 g (93%) of 22-8R and 22-8 as a white solid. Subsequently, the mixture was separated by preparative supercritical fluid chromatography (SFC). The result was 1.9 g of 22-8 as a white solid. MS m/z [M+H] ⁺ (ESI): 522.

Preparation of intermediate compound (22-9). 1(Chlorodiphenylmethyl)-4-methoxybenzene (1.23 g, 4.01 mmol, 1.10 eq.). The resulting solution was stirred at room temperature for 2 hours. Subsequently, the reaction was quenched by adding methanol (10 ml). The resulting solution was diluted with ethyl acetate. The resulting mixture was washed with saturated sodium bicarbonate solution and saturated sodium chloride solution. The organic layer was dried over anhydrous sodium sulfate and concentrated. The residue was purified by column chromatography. The result was 2.3 g (80%) of 22-9 as a white solid. MS m/z [MH] ^- (IER): 792.

- 45 045540

Preparation of intermediate compound (22-10). To a solution of 22-9 (2.3 g, 2.90 mmol, 1.00 eq.) in dichloromethane (20 mL) under an inert nitrogen atmosphere at room temperature was added triethylamine (723 mg, 7.25 mmol, 2.50 eq. .). To this mixture was added triethylamine trihydrofluoride (2.8 g, 17.4 mmol, 6.00 eq.) at room temperature and the resulting solution was stirred at 25°C for 2 hours. The resulting solution was diluted with dichloromethane, washed with water and saturated chloride solution sodium respectively. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by preparative flash HPLC. The result was 1.7 g (80%) of 22-10 as a white solid. ^1H NMR (DMSO- _d6 , 400 Hz, ppm) δ 11.33 (s, 1H), 7.41 (m, 4H), 7.20 (m, 7H), 7.19 (m , 2H), 6.80 (m, 2H), 5.25 (t, J=5.2 Hz, 1H), 5.15 (s, 1H), 4.15 (m, 2H), 3.91 (kv, J=6.7 Hz, 1H), 3.73 (s, 3H), 2.73 (d, J=9.5 Hz, 1H), 2.25 (m, 1H), 2.05 (m, 1H), 1.55 (s, 3H), 1.38 (d, J=6.8 Hz, 3H).

Receipt (22-11). Bis(diisopropylamino)(2cyanoethoxy)phosphine (1.35 g, 4.3 mmol, 1.4 eq.). 4,5-dicyanoimidazole (451 mg, 3.6 mmol, 1.20 eq.) was added to this mixture at room temperature. The resulting solution was stirred at 25°C for 2 hours and diluted with dichloromethane. The resulting mixture was washed with water and saturated sodium chloride solution, respectively. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by preparative flash HPLC. The result was 1.82 g (76%) of 22-11 as a white solid. MS m/z [M-H] ^- (ESI): 754. ¹ H NMR (DMSO-d6, 400 Hz, ppm) δ 11.4 (d, J=9.8 Hz, 1H), 7 .40 (m, 4H), 7.28 (m, 8H), 7.07 (m, 1H), 6.79 (m, 2H), 5.22 (d, J=4.0 Hz, 1H) , 4.38 (m, 1H), 4.27 (m, 1H), 4.01 (kv, J=6.7 Hz, 1H), 3.7 (m, 7H), 2.82 (m, 1H), 2.70 (m, 1H), 2.13 (m, 1H), 2.32 (d, J=60.2 Hz, 1H), 2.13 (dd, J=28.6 Hz and 6.0 Hz, 1H), 1.60 (d, J=18.0 Hz, 3H),

1.45 (t, J=6.9 Hz, 3H), 1.20 (m, 12H). P NMR (DMSO-d6, 400 Hz, ppm): δ 148.03, 147.53. Example 19.

Preparation of intermediate compound (23-2). To a solution of 23-1 (7.2 g, 9.07 mmol, 1.0 eq.) in acetonitrile (70 mL) under an inert nitrogen atmosphere was added triethylamine (2.75 g, 27.24 mmol, 3.0 eq. ), 4(dimethylamino)pyridine (3.4 g, 27.24 mmol, 3.0 eq.) and 2,4,6-triisopropylbenzenesulfonyl chloride (8.226 g, 27.24 mmol, 3.0 eq.). The resulting solution was stirred for 12 hours at room temperature. Subsequently, ammonium hydroxide (20 ml) was added and stirred for 1 hour at room temperature. The resulting solution was extracted with dichloromethane. The organic layers were combined and washed with water, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by preparative flash HPLC. The result was 5.7 g (79%) of 23-2 as a yellow solid. MS m/z [M-N] ^- (IER): 791.

Preparation of intermediate compound (23-3). To a solution of 23-2 (5.7 g, 7.20 mmol, 1.00 eq.) in pi

- 46 045540 to ridine (70 ml), benzoyl chloride (1.14 g, 1.2 eq.) was added dropwise under an inert nitrogen atmosphere at 0°C. The resulting solution was stirred for 12 hours at room temperature. The reaction was subsequently quenched by the addition of methanol. The resulting mixture was concentrated. The residue was purified by column chromatography. The result was 5 g (76%) of 23-3 as a light yellow solid. MS m/z [M-N]· (IER): 895.

Preparation of intermediate compound (23-4). To a solution of 23-3 (5 g, 5.58 mmol, 1.00 eq) in tetrahydrofuran (50 ml) was added tetrabutylammonium fluoride (1M In tetrahydrofuran, 11 ml, 2.0 eq). The resulting solution was stirred for 4 hours at room temperature. The resulting mixture was concentrated. The crude product was purified by preparative flash HPLC. The result was 2.5 g (68%) of 23-4 as a white solid. MS m/z [M-H] ^- (ESI): 657. 1H NMR (DMSOd6, 300 Hz, ppm): δ 13.08 (s, 1H), 8.32-8.18 (m, 2H), 7.70-7.35 (m, 8H), 7.31-7.08 (m, 8H), 6.75 (m, 2H), 5.68 (s, 1H), 5.33 (s, 1H), 4.01-3.91 (m, 1H), 3.79 (m, 3H), 3.69 (s, 3H), 2.85 (m, 1H), 2.46- 2.05 (m, 2H), 1.88 (s, 3H), 1.70 (s, 1H), 1.36 (m, 1H).

Receipt (23-5). To a solution of 23-4 (2.5 g, 3.80 mmol, 1.00 eq.) in dichloromethane (25 ml) under an inert nitrogen atmosphere at room temperature was added bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.7 g, 5.70 mmol, 1.50 eq.). 4,5-dicyanoimidazole (490 mg, 4.17 mmol, 1.10 eq.) was added to this mixture at room temperature. The resulting solution was stirred at room temperature for 3 hours and diluted with dichloromethane. The resulting mixture was washed with water. The organic layer was dried over anhydrous sodium sulfate and concentrated. The crude product was purified by preparative flash HPLC. The result was 2.9710 g (91%) of 23-5 as a white solid. MS m/z [M+H]+ (ESI): 859. ¹ H NMR (DMSO-d6, 300 Hz, ppm): δ 13.07 (s, 1H), 8.15 (m, 2H ), 7.70-7.42 (m, 8H), 7.23 (m, 8H), 6.75 (m, 2H), 5.81-5.68 (m, 1H), 4.17- 3.97 (m, 1H), 3.96-3.55 (m, 9H), 3.10-2.55 (m, 4H), 2.38-2.19 (m, 1H), 1. 98 (m, 4H), 1.81-1.38 (m, 2H), 1.20 (m, 12H). P NMR (DMSO-d ₆ , 300 Hz, ppm): 148.11, 145.59.

Example 20.

- 47 045540

Scheme 22.

24*6

OAS

TBDPSO

10% Pd/C (50%, 06./06.), H ₂

AcOH, MeOH, 50 °C, 2 days

during the night (SOS1) ₂ . DM SO

TEA, CH2CI2 (1Рт ₂ P(JG H ₂ G Н N

DC1, DCM, CT, 1 h

CH ₂ O, NaOH

THF/water, ct, overnight 24-5 (50%, ???), N

OH, MeON, 5 4 days

BBN, THF, 35 12h

2. NaOH(2N), Н2О2, 0 - rt, 1 h

6-M-benzoyladenine, BSA

TMSOTf, CH3CN, reflux, (30%, v/v), H ₂

THR ct, 2 and

24-24

Preparation of intermediate compound (24-2). To a solution of (3aR,5S,6S,6aR)-5-(2,2-dimethyl-1,3dioxolan-4-yl)-2,2-dimethyltetrahydro-2H-furo[2,3-d][1,3 ]dioxol-6-ol (500 g, 1.92 mol, 1.00 eq.) in N,N-dimethylformamide (5000 ml) under an inert nitrogen atmosphere at room temperature was added (bromomethyl)benzene (345 g, 2.02 mol, 1.05 eq.). Sodium hydride (123 g, 3.0 mol, 1.60 eq) was added to several batches at 0°C. The resulting solution was stirred for 2 hours at 25°C. Subsequently, the reaction was quenched by adding saturated ammonium chloride solution (2000 ml). The resulting solution was extracted with ethyl acetate, and the organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated. The result was 670 g (crude) 24-2 as a yellow oil. This crude product was used in the next step without further purification. MS m/z [M+H]+ (ESI): 351.

Preparation of intermediate compound (24-3). 24-2 (300 g, 856.14 mmol, 1.00 eq.) was dissolved in acetic acid (80% in water, 3000 ml). The resulting solution was stirred overnight at room temperature. The resulting mixture was concentrated and diluted with ethyl acetate. The resulting mixture was washed with saturated sodium bicarbonate solution accordingly. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. The result was 237 g (not

- 48 045540 purified) 24-3 as a yellow oil. This crude product was used in the next step without further purification. MS m/z [M+Na]+ (ESI): 333.

Preparation of intermediate compound (24-4). To a solution of 24-3 (200 g, 644.44 mol, 1.00 eq.) in 1,4dioxane (2000 ml) was added dropwise a solution of sodium periodate (138 g, 644.44 mol, 1.00 eq.) in water (2000 ml) with stirring at 0°C. The resulting solution was stirred for 2 hours at room temperature. The resulting solution was diluted with ethyl acetate and washed with water. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. This gave 160 g of (crude) 24-4 as a yellow oil. This crude product was used in the next step without further purification. MS m/z [M+H] ⁺ (IER): 279.

Preparation of intermediate compound (24-5). To a solution of 24-4 (200 g, 718.64 mmol, 1.00 eq) in tetrahydrofuran/water (1000 ml/1000 ml) was added a solution of formaldehyde (37%, 480 ml) at room temperature. 2 N was added dropwise to the resulting mixture. sodium hydroxide solution (1200 ml, 3.12 eq.) with stirring at 0°C. The resulting solution was stirred overnight at room temperature. The reaction mixture was extracted with ethyl acetate and the organic layers were combined. The organic layer was washed with water. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. The result was 152.8 g (crude) 24-5 as a yellow oil. This crude product was used in the next step without further purification. MS m/z [M+Na] ⁺ (ESI): 333.

Preparation of intermediate compound (24-6). To a solution of 24-5 (500 g, 1.61 mmol, 1.00 eq.) in methanol/acetic acid (5000/500 ml) in a 20 L pressure vessel reactor under an inert nitrogen atmosphere was added 10% palladium on activated coal (250 g). The pressure reservoir was evacuated and purged with hydrogen five times. The resulting solution was stirred for 4 days at 50°C (8 atm). The solids were filtered. The resulting mixture was concentrated. The residue was purified by column chromatography. The result was 280 g (79%) of 24-6 as a white solid.

Preparation of intermediate compound (24-7). To a solution of 24-6 (200 g, 908.2 mmol, 1.00 eq.) in dichloromethane (6000 ml) under an inert nitrogen atmosphere at 0°C, benzaldehyde (436.0 g, 2722.9 mmol) was added dropwise with stirring , 3.00 eq.). Subsequently, camphorsulfonic acid (2 g) was added at 0°C. The resulting solution was stirred overnight at room temperature. Subsequently, the reaction was quenched by adding 2000 ml of saturated sodium bicarbonate solution. The resulting solution was extracted with dichloromethane and the organic layers were combined. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography. The result was 13.5 g (48%) of 24-7 as a white solid. MS m/z [M+Na] ⁺ (ESI): 331 ¹ H NMR (DMSO-d ₆ , 400 Hz, ppm): δ 7.44-7.30 (m, 6H), 5.97 (d, J=4.l Hz, 1H), 5.46 (s, 1H), 5.06 (t, J=5.4 Hz, 1H), 4.64-4.52 (m, 2H) , 4.14 (d, J=13.0 Hz, 1H), 4.08-3.93 (m, 1H), 3.74-3.65 (m, 1H), 3.38 (m, 1H ), 1.46 (s, 3H), 1.23 (s, 3H).

Preparation of intermediate compound (24-8). To a solution of oxalyl chloride (30.9 g, 243.31 mmol, 1.50 eq.) in dichloromethane (1000 ml) under an inert nitrogen atmosphere at -78°C, dimethyl sulfoxide (37.9 g, 485 .09 mmol, 3.00 eq.). The resulting solution was stirred for 30 minutes at -78°C. A solution of 24-7 (50 g, 162.17 mmol, 1.00 eq) in dichloromethane (200 ml) was added dropwise to the resulting mixture with stirring at -78°C. The resulting solution was left to react with stirring for another 1.5 hours at -78°C. Subsequently, triethylamine (73.9 g, 730.31 mmol, 4.50 eq.) was added dropwise with stirring at -78°C. The resulting solution was left to react for another 2 hours with stirring at a temperature of -78°C. The resulting solution was diluted with dichloromethane and washed with 5% hydrochloric acid, saturated sodium bicarbonate solution and saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. The result was 49 g (crude) 24-8 as a white solid. This crude product was used in the next step without further purification. MS m/z [M+H] ⁺ (ESI): 307.

Preparation of intermediate compound (24-9). To a solution of methyltriphenylphosphonium bromide (139.8 g, 2.00 eq.) in tetrahydrofuran (600 ml) under an inert nitrogen atmosphere was added potassium tert-butoxide (43.8 g, 390.33 mmol, 2.00 eq.). The resulting solution was stirred for 30 minutes at room temperature. A solution of 24-8 (60 g, 195.87 mmol, 1.00 eq) in tetrahydrofuran (150 ml) was added dropwise to the resulting solution with stirring at 0°C. The resulting solution was left to react for an additional 2 hours with stirring at room temperature. Subsequently, the reaction was quenched by adding saturated ammonium chloride solution (400 ml). The resulting solution was extracted with ethyl acetate and the organic layers were combined. The organic layer was washed with water and saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography. The result was 38.7 g (65%) of 24-9 as a white solid. MS m/z [M+H] ⁺ (ESI): 305.

Preparation of intermediate compound (24-10). To a solution of 24-9 (60 g, 197.16 mmol, 1.00 eq.) in

- 49 045540 9-borabicyclo[3.3.1]nonane (0.5 M in tetrahydrofuran, 789 ml, 2.00 eq.) was added to tetrahydrofuran (600 ml) under an inert nitrogen atmosphere. The resulting solution was stirred overnight at 35°C. Subsequently, sodium hydroxide (2N in water, 591 mL, 6.00 eq.) was added at room temperature. Hydrogen peroxide (30%, 120 mL, 6.00 eq.) was added dropwise to the resulting mixture while stirring at room temperature. The resulting solution was left for the reaction to proceed. The resulting solution was extracted with dichloromethane and the organic layers were combined. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography. The result was 43.5 g (68%) of 24-10 as a white solid. MS m/z [M+Na]+ (ESI): 345.

Preparation of intermediate compound (24-11). To a solution of 24-10 (100 g, 310.2 mmol, 1.00 eq.) in pyridine (1000 ml) under an inert atmosphere of nitrogen, benzoyl chloride (130 g, 924.8 mmol, 3.00 eq.) was added dropwise. with stirring at 0°C. The resulting solution was stirred for 3 hours at room temperature. Subsequently, the reaction was quenched by the addition of methanol (200 ml). The resulting mixture was concentrated. The solid was diluted with ethyl acetate. The resulting mixture was washed with water and saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography. The result was 110 g (83%) of 24-11 as a white solid. MS m/z [M+Na] ⁺ (ESI): 449.

Preparation of intermediate compound (24-12). To a solution of 24-11 (200 g, 469 mmol, 1.00 eq.) in methanol/tetrahydrofuran (500/100 ml) was added 10% palladium on activated carbon (100 g). The flask was evacuated and purged with hydrogen five times. The resulting solution was stirred for 48 hours at 40°C. The solids were filtered. The resulting mixture was concentrated. The residue was purified by column chromatography. The result was 35 g (88%) of 24-12 as a colorless oil. MS m/z [M+Na] ⁺ (ESI): 361.

Preparation of intermediate compound (24-13). To a solution of 24-12 (50 g, 147.77 mmol, 1.00 eq.) and imidazole (19.5 g, 286.43 mmol, 2.00 eq.) in N,N-dimethylformamide (500 ml) in Under an inert nitrogen atmosphere, tert-butyldiphenylsilane (44.5 g, 1.10 eq.) was added dropwise at 0°C. The resulting solution was stirred for 2 hours at room temperature. Subsequently, the reaction was quenched by adding methanol (100 ml). The resulting mixture was concentrated. The residue was dissolved in dichloromethane. The resulting mixture was washed with water and saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography. The result was 60 g (70%) of 24-13 as a solid. MS m/z [M+Na] ⁺ (ESI): 599. ¹ H NMR (DMSO-d6, 300 Hz, ppm): 7.90-7.80 (m, 2H), 7.72- 7.54 (m, 5H), 7.49-7.30 (m, 8H), 5.85 (d, J=4.4 Hz, 1H), 5.59 (d, J=4.9 Hz , 1H), 4.54 (m, 1H), 4.37 (t, J=7.0 Hz, 2H), 4.09 (m, 1H), 3.29 (s, 2H), 2.22 (m, 2H), 1.42 (s, 3H), 1.21 (s, 3H), 0.96 (s, 9H).

Preparation of intermediate compound (24-14). To a solution of 24-13 (64 g, 110.97 mmol, 1.00 eq.) in dichloromethane/pyridine (448/134 ml) under an inert nitrogen atmosphere was added trifluoromethanesulfonic anhydride (46.9 g, 166.23 mmol) dropwise , 1.50 eq.) with stirring at 0°C. The resulting solution was stirred for 2 hours at 0°C. The resulting solution was diluted with dichloromethane. The resulting mixture was washed with water and saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. The result was 78 g (crude) 24-14 as a solid. This crude product was used in the next step without further purification. MS m/z [M+Na] ⁺ (ESI): 731.

Preparation of intermediate compound (24-15). To a solution of 24-14 (78 g, 110.04 mmol, 1.00 eq.) and tetrabutylammonium iodide (4 g, 10.83 mmol, 0.10 eq.) in N,N-dimethylformamide (800 ml) in an inert Sodium azide (21.5 g, 330.72 mmol, 3.00 eq.) was added under nitrogen atmosphere. The resulting solution was stirred overnight at 100°C. Subsequently, the reaction was quenched by adding ice water (200 ml). The resulting solution was diluted with ethyl acetate. The resulting mixture was washed with water and saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography. This resulted in 45 g (68%) of 24-15 as a brown oil. MS m/z [M+Na] ⁺ (ESI): 624.

Preparation of intermediate compound (24-16). To a solution of 24-15 (91 g, 151.22 mmol, 1.00 eq.) in methanol (900 mL) under an inert nitrogen atmosphere was added sodium methoxide (30% in methanol, 50 mL, 2.00 eq.). The resulting solution was stirred for 2 hours at room temperature. The resulting solution was extracted with ethyl acetate and the organic layers were combined. The organic layer was washed with water and saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography. The result was 55 g (73%) of 24-16 as a white solid. MS m/z [M+Na] ⁺ (ESI): 520.

Preparation of intermediate compound (24-17). To a solution of 24-16 (56 g, 112.53 mmol, 1.00 eq.) and triethylamine (67.2 g, 664.10 mmol, 6.00 eq.) in dichloromethane (825 ml) under an inert atmosphere of nitrogen at 4-dimethylaminopyridine (1 g, 8.19 mmol, 0.31 eq.) and 4-toluenesulfonyl chloride (31.5 g, 165.22 mmol, 1.50 eq.) were added at room temperature. The resulting solution was stirred during

- 50 045540 2 hours at room temperature. The resulting solution was diluted with dichloromethane. The resulting mixture was washed with water and saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography. The result was 55 g (75%) of 24-17 as a colorless oil. MS m/z [M+Na]+ (ESI): 674.

Preparation of intermediate compound (24-18). To a solution of 24-17 (100 g, 153.4 mmol, 1.00 eq.) in acetic acid (1000 mL) under an inert nitrogen atmosphere was added acetyl acetate (17.2 g, 168.48 mmol, 11.00 eq. ) and sulfuric acid (300 mg, 3.08 mmol, 0.20 eq.). The resulting solution was stirred for 2 hours at room temperature. Subsequently, the reaction was quenched by adding ice water (600 ml). The resulting solution was extracted with ethyl acetate. The resulting mixture was washed with water and saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography. The result was 52 g (49%) of 24-18 as a brown solid. MS m/z [M+Na] ⁺ (ESI): 718.

Preparation of intermediate compound (24-19). To a solution of 6-N-benzoyladenine (6.7 g, 1.50 eq.) in acetonitrile (100 ml) under an inert nitrogen atmosphere was added N,O-Bis(trimethylsilyl)acetamide (18.9 g, 92.91 mmol, 5.00 eq.). The resulting solution was stirred for 1 hour at 85°C. 2418 (13 g, 18.68 mmol, 1.00 eq) in acetonitrile (100 ml) was then added at 0°C. Trifluoromethanesulfonic acid trimethylsilyl ester (20.7 g, 92.91 mmol, 5.00 eq.) was added dropwise to the resulting mixture at 0°C. The resulting solution was left to react for another 4 hours with stirring at a temperature of 85°C. The reaction was subsequently quenched by adding saturated sodium bicarbonate solution (100 ml). The resulting solution was extracted with ethyl acetate and the organic layers were combined. The organic layer was washed with water and saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by preparative flash HPLC. The result was 8 g (49%) of 24-19 as a white solid. MS m/z [M+H] ⁺ (ESI): 875.

Preparation of intermediate compound (24-20). To a solution of 24-19 (7.4 g, 8.46 mmol, 1.00 eq.) in tetrahydrofuran (55 ml) under an inert nitrogen atmosphere at 0°C was added a solution of lithium hydroxide (1.01 g, 42.17 mmol , 5.00 eq.) in water (25 ml). The resulting solution was stirred for 4 hours at room temperature. The resulting solution was diluted with ethyl acetate. The resulting mixture was washed with water and saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography. The result was 4.3 g (77%) of 24-20 as a white solid. MS m/z [M+H] ⁺ (ESI): 661.

Preparation of intermediate compound (24-21). To a solution of 24-20 (4 g, 6.05 mmol, 1.00 eq.) in tetrahydrofuran (40 ml) was added 10% palladium on activated carbon (1.2 g). The flask was evacuated and purged with hydrogen five times. The resulting solution was stirred for 3 hours at 25°C. The solids were filtered. The resulting mixture was concentrated. The result was 3.8 g (99%) of 24-21 as a white solid. MS m/z [M+H] ⁺ (ESI): 635.

Preparation of intermediate compound (24-22). To a solution of 24-21 (3.8 g, 5.99 mmol, 1.00 eq.) in pyridine (38 ml) under an inert nitrogen atmosphere was added 4-methoxytriphenylmethyl chloride (2.0 g, 6.59 mmol, 1. 10 equiv.) at 0°C. The resulting solution was stirred for 3 hours at 25°C. Subsequently, the reaction was quenched by the addition of methanol (5 ml). The resulting mixture was concentrated. The residue was dissolved in dichloromethane. The resulting mixture was washed with water and saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by preparative flash HPLC. The result was 2.7 g (50%) of 2422 as a white solid. MS m/z [M+H] ⁺ (IER): 908.

Preparation of intermediate compound (24-23). To a solution of 24-22 (2.7 g, 2.98 mmol, 1.00 eq) in dichloromethane (27 mL) under an inert nitrogen atmosphere, triethylamine (750 mg, 7.41 mmol, 2.50 eq) was added sequentially. and triethylamine trihydrofluoride (2.87 g, 17.80 mmol, 6.00 eq.). The resulting solution was stirred for 2 hours at 25°C. The resulting solution was diluted with dichloromethane. The resulting mixture was washed with water and saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by preparative flash HPLC. The result was 1.8 g (90%) of 24-23 as a white solid. MS m/z [M+H] ⁺ (ESI): 669. ¹ H NMR (DMSO-d6, 300 Hz, ppm): δ 11.18 (s, 1H), 8.63 (d, J =13.7 Hz, 2H), 8.13-8.03 (m, 2H), 7.71-7.49 (m, 3H), 7.42-7.29 (m, 4H), 7. 23-6.92 (m, 8H), 6.62-6.51 (m, 2H), 6.25 (s, 1H), 5.27 (t, J=4.3 Hz, 1H), 4 .04 (m, J=12.0 Hz, 4.6 Hz, 1H), 3.88 (t, J=5.5 Hz, 3H), 3.57 (s, 3H), 3.04 (d , J=10.6 Hz, 1H), 2.41 (d, J=10.8 Hz, 1H), 2.20 (m, J=12.2 Hz, 7.7 Hz, 1H), 1, 57 (d, J=2.5 Hz, 1H), 1.44 (d, J=11.7 Hz, 1H).

Receipt (24-24). Bis(diisopropylamino)(2cyanoethoxy)phosphine (1.26 g, 1.40 eq.) and 4,5-dicyanoimidazole (422 mg, 1.20 eq.). The resulting solution was stirred for 1 hour at 25°C. The resulting solution was diluted with dichloromethane. The resulting mixture was washed with saturated sodium bicarbonate solution and saturated chloride solution for

- 51 045540 triya. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by preparative flash HPLC. The result was 2.1 g (77%) of 24-24 as a white solid. MS m/z [M+H]+ (ESI): 869. ¹ H NMR (DMSO-d ₆ , 300 Hz, ppm): δ 11.20 (s, 1H), 8.62 (d, J=17.2 Hz, 1H), 8.40 (m, 1H), 8.13-8.03 (m, 2H), 7.65 (m, 1H), 7.56 (m, 2H), 7.47-7.33 (m, 4H), 7.21 (t, J=8.3 Hz, 2H), 7.15-6.92 (m, 6H), 6.59 (d, J= 8.5 Hz, 2H), 6.31 (d, J=3.6 Hz, 1H), 4.22-4.02 (m, 1H), 4.00-3.37 (m, 9H), 3.04 (d, J=10.6 Hz, 1H), 2.92-2.67 (m, 2H), 2.55 (d, J=11.3 Hz, 1H), 2.31 (m , 1H), 1.73-1.47 (m, 2H), 1.27-0.99 (m, 13H), 0.93-0.76 (m, 1H). P-NMR (DMSO-d6, 300 Hz, ppm): δ 148.21, 147.04.

Example 21.

Scheme 23.

10% Pd/C, n ₂ but

(iPr ₂ N) ₂ POCH ₂ CH ₂ CN,

DC1, DCM, CT, 2 h

TBDPSOt _about

2-M-isobutyrylguavine, BSA <\TMSOTf, DCE, ^reflux , 8 hours

25-1

25-6

Preparation of intermediate compound (25-2). To a solution of N-2-acetylguanine (12.4 g, 56.36 mmol, 3.00 eq.) in 1,2-dichloroethane (200 ml) under an inert nitrogen atmosphere at room temperature was added N,O-bis(trimethylsilyl) acetamide (19 g, 93.6 mmol, 5.00 eq.). The resulting solution was stirred for 3 hours at 85°C. To the resulting mixture was added 25-1 (13 g, 18.68 mmol, 1.00 eq.) at 0°C, and subsequently trimethylsilyl trifluoromethanesulfonate (20.8 g, 93.58 mmol, 5.00 eq.) was added dropwise. ) with stirring at 0°C. The resulting solution was left for another 2 hours for the reaction to proceed with stirring at 80°C. The reaction mixture was cooled to 0°C and quenched by adding a saturated ammonium chloride solution (80 ml). The resulting solution was extracted with ethyl acetate, and the organic layers were combined and dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography. The result was 5.6 g (35%) of 25-2 as a white solid. MS m/z [M+H]+ (ESI): 857.

Preparation of intermediate compound (25-3). To a solution of 25-2 (5.6 g, 6.53 mmol, 1.00 eq.) in tetrahydrofuran (56 ml) under an inert nitrogen atmosphere was added 1 M sodium hydroxide (56 ml) at 0°C. The resulting solution was stirred for 1 hour at 0°C. The pH of the solution was adjusted to 7 with acetic acid. The resulting solution was diluted with ethyl acetate. The resulting mixture was washed with water and saturated sodium chloride solution, respectively. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by preparative flash HPLC. The result was 2.9 g (69%) of 25-3 as a white solid.

Preparation of intermediate compound (25-4). To a solution of 25-3 (2.9 g, 4.51 mmol, 1.00 eq.) in tet

- 52 045540 ragidrofuran (30 ml) was added 10% palladium on activated carbon (1.2 g). The flask was evacuated and purged with hydrogen five times. The resulting solution was stirred at room temperature for 5 hours. The solids were filtered. The resulting mixture was concentrated under reduced pressure. The result was 2.5 g (90%) of 25-4 as a white solid.

Preparation of intermediate compound (25-5). To a solution of 25-4 (2.5 g, 4.05 mmol, 1.00 eq.) in pyridine (25 ml) under an inert nitrogen atmosphere at room temperature, 1(chlorodiphenylmethyl)-4-methoxybenzene (1.9 g , 6.15 mmol, 1.50 eq.) and 4-dimethylaminopyridine (150 mg, 1.23 mmol, 0.30 eq.). The resulting mixture was stirred for 2 hours at room temperature and subsequently quenched by adding methanol (10 ml). The resulting solution was diluted with ethyl acetate. The resulting mixture was washed with water and saturated sodium chloride solution, respectively. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by preparative flash HPLC. The result was 2.7 g (75%) of 25-5 as a white solid.

Preparation of intermediate compound (25-6). To a solution of 25-5 (2.7 g, 3.04 mmol, 1.00 eq.) in dichloromethane (30 mL) under an inert nitrogen atmosphere at room temperature was added triethylamine (1 g, 9.88 mmol, 3.00 eq. .). Triethylamine trihydrofluoride (1.5 g, 9.32 mmol, 3.0 eq.) was added to this mixture at room temperature. The resulting solution was stirred at 25°C for 2 hours and diluted with dichloromethane. The resulting mixture was washed with water and saturated sodium chloride solution, respectively. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by preparative flash HPLC. The result was 1.7 g (86%) of 25-6 as a white solid. MS m/z [M+H]+ (ESI): 651. 1H NMR (DMSO-d ₆ , 400 Hz, ppm) δ 12.16 (s, 1H), 11.77 (s, 1H) , 8.15 (s, 1H), 7.42 (m, 4H), 7.15 (m, 8H), 6.56 (d, J=8.4 Hz, 2H), 5.97 (s, 1H), 5.22 (t, J=4.3 Hz, 1H), 4.01 (m, 1H), 3.85 (m, 2H), 3.73 (m, 1H), 3.60 ( s, 3H), 3.01 (m, 1H), 2.82 (m, 1H), 2.39 (d, J=10.9 Hz, 1H), 2.17 (m, 1H), 1, 58 (d, J=2.6 Hz, 1H), 1.41 (m, 1H), 1.18 (d, J=6.8 Hz, 3H), 1.12 (d, J=6.8 Hz, 3H).

Receipt (25-7). Bis(diisopropylamino)(2-cyanoethoxy)phosphine (950 mg, 3.16 mmol, 1.30 eq.). 4,5-dicyanoimidazole (340 mg, 2.91 mmol, 1.10 eq.) was added to this mixture at room temperature. The resulting solution was stirred at 25°C for 2 hours and diluted with dichloromethane. The resulting mixture was washed with water and saturated sodium chloride solution, respectively. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by preparative flash HPLC. The result was 1.8 g (81%) of 25-7 as a white solid. MS m/z [M+H]+ (ESI): 851. H NMR (DMSO-d ₆ , 400 Hz, ppm) δ 12.18 (s, 1H), 11.80 (s, 1H) , 7.98 (d, J=64.4 Hz, 1H), 7.47 (m, 4H), 7.15 (m, 8H), 6.56 (m, 2H), 6.01 (d, J=10.0 Hz, 1H), 3.95 (m, 3H), 3.77 (m, 2H), 3.66 (m, 1H), 3.59 (d, J=9.8 Hz, 3H), 3.38 (m, 2H), 2.91 (m, 4H), 2.30 (m, 1H), 1.55 (m, 2H), 1.15 (m, 14H), 1, 04 (d, J=6.7 Hz, 4H). P NMR (DMSO-d6, 400 Hz, ppm): 148.62, 147.09.

Example 22.

The synthesis of 2'-O-vinyladenosine analogue 26-8 was performed as shown in Scheme 26. Intermediate 6-2 was treated with KI ₃ /MeOH to give compound 26-1. To prevent side reactions of the vinyl group with the azide, the C3'-azide was reduced with a reducing agent such as H ₂ , Pd/C to give intermediate 26-2, the free 3'-NH ₂ group was protected by the MMTr protecting group education 26-3. After deprotection of the N6 position of the base to give 26-4, alkylation was performed using allyl bromide to give 26-5. In this method, reduction of the C3'-azide was performed prior to alkylation to avoid side reactions of the vinyl group with the azide. Reprotection of the N6 position with benzyl chloride afforded 26-6, which was deprotected at the 5' position with Py/NaOH/MeOH/H ₂ O to give 26-7. To obtain the target compound 26-8, standard phosphitylation conditions were used.

- 53 045540

Scheme 24.

Preparation of intermediate 26-1. To a solution of 6-2 (32.6 g, 58.6 mmol) in methanol (500 L) was added NH ₃ H ₂ O (50 ml) at 0°C. The reaction mixture was stirred at 0°C for 2 hours and concentrated, the residue was dissolved in EA, washed with brine, dried over anhydrous Na ₂ SO ₄ , the solvent was removed and the residue was purified on silica gel to give 26-1 (28.5 g, 55.4 mmol, yield 94.62%) as a yellow solid. ESI-LCMS: m/z 515 [M+H]+.

Preparation of intermediate 26-2. Pd/C (3.0 mg) was added to a solution of 26-1 (28.5 g, 55.4 mmol) in THF (400.00 mL), and the mixture was stirred at rt for 6 hours under Hg. The mixture was filtered and the filtrate was concentrated to give 26-2 (21.5 g, 44.1 mmol, 79.60% yield) as a gray solid. ESI-LCMS: m/z 489 [M+H] ⁺ .

Preparation of intermediate 26-3. To a solution of 26-2 (21.5 g, 44.1 mmol) in pyridine (300 ml) was added MMTrCl (20.4 g, 66.1 mmol). The mixture was stirred at room temperature for 1 hour. The reaction mixture was quenched with MeOH and concentrated to give a residue, which was purified on silica gel to give 26-3 (22.0 g, 28.9 mmol, 65.53% yield) as a white solid substances. ESI-LCMS: m/z 761 [M+H] ⁺ .

Preparation of intermediate 26-4. To a solution of 26-3 (22.0 g, 28.9 mmol) in a mixture of pyridine (200 ml) and AcOH (50 ml) was added hydrazine hydrate (80%) (173.5 mmol, 10 ml) and the reaction mixture was stirred at room temperature for 15 hours. The reaction mixture was poured into ice water, extracted using EA, washed with brine and dried over anhydrous Na ₂ SO ₄ , concentrated to give the crude product, which was purified on silica gel to give 26-4 (18.0 g, 27.4 mmol, yield 94.79%) as a white solid. ESI-LCMS: m/z 657 [M+H] ⁺ .

Preparation of intermediate 26-5. Ag ₂ O (9.5 g, 41.1 mmol, 1.33 ml) and NaI (8.2 g, 54.8 mmol) were added to solution 26-4 (18 g, 27.4 mmol) and bromide was added vinyl (6.6 g, 54.8 mmol) in dry DMF (250 ml). The mixture was stirred at room temperature for 1.5 hours. The reaction mixture was poured into ice water, extracted using EA, washed with brine and dried over anhydrous Na ₂ SO ₄ , concentrated to give the crude product, which was purified by recrystallization (PE:EA =1:1) to give 26-5 (12.1 g, 17.3 mmol, 63.36% yield) as a white solid. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ=7.99 (d, J=2.0 Hz, 1H), 7.537.48 (m, 6H), 7.40-7.37 (m , 2H), 7.28 (br. s, 2H, exchange with D2O), 7.21-7.14 (m, 6H), 7.09-7.04 (m, 2H), 6.68 (d , J=9.2 Hz, 2H), 5.93 (s, 1H), 5.87-5.77 (m, 1H), 5.22-5.16 (m, 1H), 5.10- 5.07 (m, 1H), 4.81-4.78 (m, 1H), 4.68 (dd, J=3.2, 12.4 Hz, 1H), 4.05-3.99 ( m, 1H), 3.94-3.89 (m, 1H), 3.53 (s, 3H), 3.22-3.18 (m, 1H), 2.92 (d, J=10, 8 Hz, 1H, exchange with D2O), 2.49 (d, J=4.8 Hz, 1H), 2.37 (s, 3H). ESI-LCMS: m/z 697 [M+H] ⁺ .

Preparation of intermediate 26-6. BzCl (4.8 g, 34.4 mmol) was added dropwise to a solution of 26-5 (12 g, 17.2 mmol) in pyridine (120 ml) at 0°C. After stirring the mixture for 1 hour at rt, 300 ml of H2O and 500 ml of EA were added to separate the solution, the aqueous phase was extracted using EA. The combined organic layers were washed with brine, dried over Na ₂ SO ₄ , concentrated to give crude product 26-6 (16.2 g) as a yellow oil. ESI-LCMS: m/z 801 [M+H] ⁺ .

Preparation of intermediate 26-7. To a solution of crude 26-6 (16.2 g) in pyridine

- 54 045540 (200 ml) 2 N was added dropwise. NaOH (MeOH:H ₂ O=4:1) (50 ml) at 0°C. The mixture was stirred at 0°C for 0.5 h and then neutralized with saturated aqueous NH ₄ Cl to obtain pH=7~8 and 500 ml of H ₂ O. 800 ml of EA was added to separate the solution, the aqueous phase was extracted using EA , the combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give the crude product, which was purified by MPLC to give 26-7 (9.3 g, 13.6 mmol, 79.07% yield over two steps) as a white solid. ¹ H-NMR (400 MHz, DMSO46): δ=11.15 (s, 1H, exchange with D2O), 8.68 (s, 1H), 8.65 (s, 1H), 8.07-8, 05 (m, 2H), 7.67-7.63 (m, 1H), 7.57-7.53 (m, 2H), 7.44-7.41 (m, 4H), 7.27 ( d, J=9.2 Hz, 2H), 7.20-7.16 (m, 4H), 7.13-7.09 (m, 2H), 6.72 (d, J=9.2 Hz , 2H), 6.08 (s, 1H), 5.93-5.83 (m, 1H), 5.27-5.22 (m, 1H), 5.18 (t, J=4.0 Hz, 1H, exchange with D2O), 5.135.10 (m, 1H), 4.11-3.99 (m, 4H), 3.64 (s, 3H), 3.46-3.42 (m, 1H), 3.38-3.32 (m, 1H), 2.71 (d, J=10.8 Hz, 1H, exchange with D ₂ O), 1.91 (d, J=4.4 Hz , 1H). ESI-LCMS: m/z 683 [M+H]+.

Getting 26-8. Phosphytylation reagent (3.4 g, 11.4 mmol) was added to a solution of 26-7 (6.5 g, 9.5 mmol) and DCI (1.1 g, 9.5 mmol) in dry DCM (70 mL). in an atmosphere of Ar. The mixture was stirred at rt for 1 hour. The reaction mixture was subsequently washed with 10% aqueous NaHCO ₃ and brine, dried over Na2SO4 and concentrated to give the crude product, which was purified by preparative flash HPLC. The result was 26-8 (7.1 g, 8.0 mmol, 84.46%) as a white solid. ¹ H-NMR (400 MHz, CDCl3): δ=9.02 (br. s, 1 H), 8.74 (d, J=10.4 Hz, 1H), 8.37 (d, J=108 .4 Hz, 1H), 8.04 (t, J=7.2 Hz, 2H), 7.65-7.61 (m, 1H), 7.57-7.53 (m, 2H), 7 .51-7.48 (m, 4H), 7.41-7.38 (m, 2H), 7.21-7.10 (m, 6H), 6.73-6.70 (m, 2H) , 6.03 (d, J=2.4 Hz, 1H), 5.91-5.79 (m, 1H), 5.31-5.21 (m, 2H), 5.17-5.13 (m, 1H), 4.40-4.25 (m, 2H), 4.16-4.08 (m, 1H), 4.05-3.94 (m, 1H), 3.89-3 .80 (m, 1H), 3.72 (dd, J=0.8, 10.8 Hz, 3H), 3.70-3.47 (m, 4H), 3.42-3.35 (m , 1H), 2.86 (dd, J=10.8, 23.2 Hz, 1H), 2.68-2.51 (m, 2H), 1.62 (dd, J=4.4, 210 .8 Hz, 1H), 1.26-1.18 (m, 12H). ³¹ P NMR (162 MHz, CDCl3): 148.85, 148.24. ESI-LCMS: m/z 883 [M+H] ⁺ .

Example 23.

The synthesis of guanosine 2'-vinyl phosphoramidate 27-9 was performed as shown in Scheme 27. The C3'-azide of compound 7-2 was reduced to the corresponding amine to give 27-1. Protection of the 3'amine with N-Boc afforded intermediate 27-2. 2'-O-allylation was performed using allyl bromide in the presence of Ag ₂ O and NaI to form intermediate 27-3. It was found that the introduction of a Boc protecting group at the 3'-NH position provides a higher alkylation yield compared to MMTr in this particular case. Cleavage of the exocyclic amino acid by MMTr to produce 27-4 followed by deprotection with iBuCl afforded 27-5. Subsequently, deprotection of the 3'-amine yielded 27-6, which upon re-protection with MMTrCl was converted to 27-7. As a result of cleavage of the 5'-thiol group to form 27-8 and final phosphitylation, the target compound 27-9 was obtained.

- 55 045540

Scheme 25.

Preparation of intermediate (27-1). To a solution of 7-2 (50.5 mg, 69.7 mmol) in THF (500 ml) was added PPh ₃ (27.4 g, 104.5 mmol) and H2O (1.3 g, 69.7 mmol). The mixture was stirred at 60°C for 15 hours. The reaction mixture was concentrated and the residue was purified on silica gel to give 27-1 (41.1 g, 59.3 mmol, 85.08% yield) as a white solid. ESI-LCMS: m/z 691 [M+H]+.

Preparation of intermediate compound (27-2). To a solution of 27-1 (41.1 g, 59.3 mmol) in a mixture of THF (300 ml) and sat. aqueous NaHCO ₃ solution (200 ml) was added Boc ₂ O (15.5 g, 71.2 mmol). The mixture was stirred at room temperature for 2 hours, extracted with EA, washed with brine and dried over anhydrous Na ₂ SO _4. It was finally concentrated to give the crude product, which was purified on silica gel to give 27-2 (46.2 g , 58.2 mmol, yield 98.00%) as a white solid. ESI-LCMS: m/z 791 [M+H] ⁺ .

Preparation of intermediate compound (27-3). To vinyl bromide (45 g, 56.8 mmol) and 27-2 (10.3 g, 85.3 mmol) in dry DMF (400 ml) was added Ag ₂ O (19.7 g, 85.3 mmol) and NaI (12.8 g, 85.3 mmol), the mixture was stirred at room temperature for 0.5 h, poured into ice water, extracted with EA, washed with brine and dried over anhydrous Na ₂ SO ₄ , concentrated to give the crude product, which was purified on silica gel to give 27-3 (35.3 g, 42.4 mmol, 74.66% yield) as a white solid. ESI-LCMS: m/z 831 [M+H] ⁺ .

Preparation of intermediate compound (27-4). TCA (100 g, 42.1 mmol) was added to a solution of 27-3 (35 g, 42.1 mmol) in DCM (400 ml) and the mixture was stirred at room temperature for 4 hours. At the end of this time, the reaction mixture was neutralized with saturated an aqueous solution of NaHCO ₃ to obtain pH=8~9. EA was added and washed with saline. The organic layers were dried over Na ₂ SO ₄ and concentrated to give the crude product, which was purified on silica gel to give 27-4 (18.5 g, 33.1 mmol, 78.61% yield) as a white solid. ESI-LCMS: m/z 559 [M+Na] ⁺ .

Preparation of intermediate compound (27-5). iBuCl (5.2 g, 48.3 mmol) was added dropwise to a solution of 27-4 (18 g, 32.2 mmol) in pyridine (200 ml) at 0°C. After this, the mixture was stirred at 0°C for 0.5 h, 500 ml of H ₂ O and 800 ml of EA were added to separate the solution. The aqueous phase was extracted using EA, the combined organic layer was washed with brine, dried over Na ₂ SO ₄ and finally concentrated to give the crude product, which was purified on silica gel to give 27-5 (18.1 g, 28.77 mmol, yield 89.35%) as a white solid. ESI-LCMS: m/z 629 [M+H] ⁺ .

Preparation of intermediate compound (27-6). 27-5 (18 mg, 28.6 mmol) was dissolved in a mixture of TFA (180 ml) and H2O (60 ml). The reaction mixture was stirred at room temperature for 15 hours and neutralized with saturated aqueous NaHCO ₃ solution. The crude product was extracted with

- 56 045540 using EA, washed with saline solution and dried over anhydrous Na2SO4. Concentration by purification on silica gel gave 27-6 (12.5 g, 24.5 mmol, 85.57% yield) as a white solid. ESI-LCMS: m/z 511 [M+H]+.

Preparation of intermediate compound (27-7). To a solution of 27-6 (12.5 g, 24.5 mmol) in anhydrous DCM (150 ml) was added collidine (5.9 g, 49.0 mmol), AgNO3 (6.2 g, 36.7 mmol) and MMTr-Cl (11.3 g, 36.7 mmol) and the mixture was stirred at rt for 1 hour under N2. After completion of the reaction, the crude reaction mixture was filtered, washed with H2O, dried over Na ₂ SO ₄ and concentrated to give a residue which was purified on silica gel to give crude product 27-7 (18.5 g, 23.6 mmol, 96.56% yield ) as a white solid. ESI-LCMS: m/z 783 [M+H] ⁺ .

Preparation of intermediate compound (27-8). To a solution of crude 27-7 (18.5 g, 23.6 mmol) in pyridine (200 ml) was added 2 N. NaOH (in MeOH:H ₂ O = 4:1) (50 ml) at 0°C, the mixture was stirred at 0°C for 15 minutes. The mixture was neutralized with a saturated aqueous solution of NH4Cl to obtain pH=7~8 and 500 ml of H ₂ O and 800 ml of EA were added. The aqueous phase was extracted using EA, the combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give the crude product, which was purified by MPLC to give 27-8 (12.1 g, 18.2 mmol, 77.21 %) as a white solid. 1H-NMR (400 MHz, CDCl ₃ ): δ=12.13 (s, 1H, exchange with D2O), 11.34 (s, 1H, exchange with D2O), 8.11 (s, 1H), 7, 47-7.43 (m, 4H), 7.28-7.21 (m, 6H), 7.17-7.13 (m, 2H), 6.77-6.75 (m, 2H), 5.89-5.80 (m, 2H), 5.25-5.20 (m, 1H), 5.13-5.09 (m, 1H), 4.03-3.92 (m, 4H ), 3.65 (s, 3H), 3.46-3.41 (m, 1H), 3.31-3.24 (m, 1H), 2.87-2.80 (m, 1H), 2.73 (d, J=10.0 Hz, 1H, exchange with D2O), 1.91 (d, J=4.8 Hz, 1H), 1.16 (dd, J=6.8, 11, 2 Hz, 6H). ESI-LCMS: m/z 665 [M+H] ⁺ .

Receipt (27-9). Phosphytylation reagent (4.4 g, 14.7 mmol) was added to a solution of 27-8 (7.5 g, 11.3 mmol) and DCI (1.5 g, 12.4 mmol) in dry DCM (80 mL). in an Ar atmosphere. The mixture was stirred at rt for 1 hour, washed with 10% aqueous NaHCO ₃ and brine, dried over Na ₂ SO ₄ and concentrated to give the crude product, which was purified by preparative flash HPLC. The result was 27-9 (7.4 g, 8.5 mmol, 75.79% yield) as a white solid. ¹ H-NMR (400 MHz, CDCl3): δ=7.90 (d, J=119.2 Hz, 1H), 7.54-7.51 (m, 4H), 7.42-7.37 ( m, 2H), 7.287.12 (m, 6H), 6.78-6.72 (m, 2H), 5.79-5.65 (m, 2H), 5.18-5.04 (m, 2H), 4.30-4.09 (m, 3H), 3.93-3.33 (m, JN), 2.86 (dd, J=10.0, 28.8 Hz, 1H), 2 .72-2.52 (m, 3H), 2.10 (dd, J=4.8, 211.6 Hz, 1H), 1.28-1.11 (m, 18H). ³¹ P NMR (162 MHz, CDCl3): 149.14, 148.15. ESI-LCMS: m/z 865 [M+H] ⁺ .

Example 24.

The synthesis of uridine 2'-O-allyl 28-5 was performed as shown below in Scheme 28. Briefly, MMTrCl-mediated protection of the 3'-NI G group of compound 8-5 afforded compound 28-4, which was alkylated with bromide allyl to give 28-2. The propiolate group was deprotected using pyrrolidine, resulting in the formation of 28-3. Subsequently, cleavage of the 5'-ester provided intermediate 28-4, which was phosphotylated to yield the target phosphoramidite 28-5.

Scheme 26.

Preparation of intermediate compound (28-1). MMTrCl (15.3 g, 49.6 mmol) was added under Ar atmosphere to a solution of 8-5 (19.0 g, 41.3 mmol) in pyridine (300 ml), the mixture was stirred at room temperature for 1 hour. The mixture quenched with MeOH and concentrated to give a residue, which was purified

- 57 045540 silica gel to give 7 (24.5 g, 33.4 mmol, 80.97% yield) as a white solid. 1H-NMR (400 MHz, DMSO-d ₆ ): δ=8.08 (d, J=14.8 Hz, 1H), 7.59-7.48 (m, 7H), 7.35-7, 28 (m, 4H), 7.23-7.16 (m, 4H), 7.12-7.03 (m, 2H), 6.87 (d, J=14.8 Hz, 1H), 6 .73 (d, J=4.8 Hz, 1H), 5.93 (d, J=4.4 Hz, 1H, exchange with D ₂ O), 5.425.40 (m, 2H), 4.73 ( d, J=12.4 Hz, 1H), 4.61 (dd, J=2.8, 12.8 Hz, 1H), 4.28-4.19 (m, 3H), 3.58 (s , 3H), 3.13-3.04 (m, 2H), 2.38 (s, 3H), 2.15 (t, J=4.0 Hz, 1H), 1.27 (t, J =7.2 Hz, 3H). ESI-LCMS: m/z 732 [M+H]+.

Preparation of intermediate compound (28-2). NaI (9.2 g, 61.5 mmol) was added to a solution of 28-1 (22.5 g, 30.7 mmol) and allyl bromide (7.4 g, 61.5 mmol) in dry DMF (250 mL). and Ag ₂ O (10.7 g, 46.1 mmol) under Ar, the mixture was stirred at room temperature for 0.5 h. The reaction mixture was poured into ice water, extracted with EA, washed with brine and dried over anhydrous Na ₂ SO ₄ was concentrated to give the crude product, which was purified on silica gel to give 28-2 (18.8 g, 24.3 mmol, 79.22% yield) as a white solid. ^! HNMR (400 MHz, DMSO-d ₆ ): δ=8.06 (d, J=14.8 Hz, 1H), 7.59 (t, J=8.0 Hz, 3H), 7.50 (t , J=8.4 Hz, 4H), 7.33 (d, J=8.4 Hz, 2H), 7.27 (d, J=8.0 Hz, 2H), 7.22-7.15 (m, 4H), 7.10 (t, J=7.2 Hz, 1H), 7.04 (t, J=7.6 Hz, 1H), 6.87 (dd, J=2.4, 22.8 Hz, 1H), 6.72 (d, J=8.8 Hz, 2H), 5.88-5.78 (m, 1H), 5.38 (d, J=8.0 Hz, 1H), 5.22 (dd, J=1.6, 17.2 Hz, 1H), 5.11-5.08 (m, 1H), 4.87-4.75 (m, 2H), 4 .34 (d, J=10.4 Hz, 1H), 4.20 (dd, J=7.2, 14.4 Hz, 2H), 4.06-3.99 (m, 1H), 3, 55 (s, 3H), 3.32-3.30 (m, 2H), 2.79 (d, J=10.4 Hz, 1H, exchange with D2O), 2.37 (s, 3H), 1 .84 (d, J=4.4 Hz, 1H), 1.26 (t, J=7.2 Hz, 3H). ESI-LCMS: m/z 772 [M+H] ⁺ .

Preparation of intermediate compound (28-3). To a solution of pyrrolidine (1.8 g, 25.9 mmol) was added a solution of 28-2 (20.0 g, 25.9 mmol) in acetonitrile (200 ml). The mixture was stirred at room temperature for 15 hours. The solvent was evaporated and the residue was purified on silica gel to give 28-3 (15.5 g, 23.0 mmol, 88.81% yield) as a yellowish solid. ESI-LCMS: m/z 696 [M+Na]+.

Preparation of intermediate compound (28-4). A mixture of 28-3 (6.0 g, 8.9 mmol) and 2 N. NaOH (in MeOH:H ₂ O = 4:1) (60 ml) was stirred at rt for 0.5 h. After TLC showed that 28-3 was completely consumed, 28-4 was extracted using DCM and washed with saline solution, dried over Na2SO4 and concentrated to give the crude product, which was purified by MPLC to give 28-4 (4.3 g, 7.7 mmol, 87.05%) as a white solid. ¹ NMR (400 MHz, DMSO-d ₆ ): δ=11.27 (s, 1H, exchange with D2O), 7.97 (d, J=8.0 Hz, 1H), 7.47-7.44 (m, 4H), 7.34 (d, J=8.8 Hz, 2H), 7.27 (t, J=7.2 Hz, 4H), 7.21-7.17 (m, 2H) , 6.84 (d, J=8.8 Hz, 2H), 5.86-5.76 (m, 1H), 5.25-5.49 (m, 2H), 5.20-5.15 (m, 1H), 5.08-5.05 (m, 1H), 4.06-4.01 (m, 2H), 3.96-3.90 (m, 2H), 3.72 (s , 3H), 3.30-3.25 (m, 1H), 3.16-3.09 (m, 1H), 2.69 (d, J=10.4 Hz, 1H, exchange with D2O), 1.57 (d, J=4.4 Hz, 1H). ESI-LCMS: m/z 556 [M+H] ⁺ .

Receipt (28-5). To a solution of 28-4 (4.3 g, 7.7 mmol) and DCI (999 mg, 8.5 mmol) in dry DCM (40 ml) was added 28-4 (3.0 g, 10.0 mmol) in atmosphere Ar. The mixture was stirred at rt for 1 hour. The reaction mixture was washed with 10% aqueous NaHCO ₃ and brine, dried over Na ₂ SO ₄ and concentrated to give the crude product, which was purified by preparative HPLC. 28-5 (4.4 g, 5.8 mmol, 75.68% yield) was obtained as a white solid. 1H-NMR (400 MHz, CDCl ₃ ): δ=7.80 (dd, J=8.0, 182.4 Hz, 1H), 7.55-7.43 (m, 6H), 7.30- 7.18 (m, 6H), 6.82-6.78 (m, 2H), 5.84-5.74 (m, 1H), 5.66 (d, J=4.8 Hz, 1H) , 5.60 (d, J=8.0 Hz, 1H), 5.22-5.17 (m, 1H), 5.11-5.08 (m, 1H), 4.324.29 (m, 1H ), 4.19-4.01 (m, 3H), 3.89-3.81 (m, 1H), 3.78 (s, 3H), 3.66-3.46 (m, 3H), 3.39-3.35 (m, 1H), 3.22-3.01 (m, 1H), 2.94-2.88 (m, 1H), 2.64-2.44 (m, 1H ), 1.52 (dd, J=4.8, 140.0 Hz, 1H), 1.25-1.15 (m, 12H). ³¹ P-NMR (162 MHz, CDC13): 149.16, 148.20. ESI-LCMS: m/z 756 [M+H] ⁺ .

Example 25.

MMTrHN b

- 58 045540

Scheme 27.

Preparation of intermediate compound (29-1). DMAP (3.6 g, 29.7 mmol), TEA (3.0 g, 29.6 mmol, 4 mL) and TPSCl (6.7 g, 22.2 mmol) were added under Ar atmosphere to solution 28-3 (10.0 g, 14.8 mmol) in dry acetonitrile (100 ml). After stirring the mixture at rt for 1 hour, conc. NH3H2O (25 ml) and the reaction mixture was stirred at rt for another 15 hours. After completion of the reaction, the solvent was removed and the residue was dissolved in EA, washed with sat. aqueous NH ₄ Cl and brine, dried over Na ₂ SO ₄ and concentrated to give the crude product, which was purified on silica gel to give 29-1 (8.9 g, 13.2 mmol, 89.14% yield) as pale yellow solid. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ=7.59 (d, J=7.6 Hz, 2H, exchange with D2O), 7.50-7.43 (m, 5H), 7 .35-7.29 (m, 4H), 7.22-7.17 (m, 6H), 7.14-7.06 (m, 2H), 6.71 (d, J=9.2 Hz , 2H), 5.88-5.79 (m, 1H), 5.55 (s, 1H), 5.39 (d, J=7.2 Hz, 1H), 5.22 (dd, J= 1.6, 17.2 Hz, 1H), 5.08 (dd, J=1.2, 10.4 Hz, 1H), 4.88-4.76 (m, 2H), 4.31-4 .28 (m, 1H), 4.09-4.04 (m, 1H), 3.58 (s, 3H), 3.29-3.25 (m, 1H), 3.20-3.14 (m, 1H), 2.79 (d, J=9.2 Hz, 1H, exchange with D2O), 2.41 (s, 3H), 1.61 (d, J=4.4 Hz, 1H) . ESI-LCMS: m/z 673 [M+H]+.

Preparation of intermediate compound (29-2). To a solution of 29-1 (8.5 g, 12.6 mmol) in pyridine (80 ml) was added BzCl (2.1 g, 15.1 mmol) dropwise at 0°C. After stirring the mixture for 1 hour at room temperature. 300 ml of H ₂ O and 500 ml of EA were added. The aqueous layer was extracted using EA, the combined organic layer was washed with brine, dried over Na ₂ SO ₄ and concentrated to give crude product 29-2 (11.30 g) as a yellow oil. ESI-LCMS: m/z 777 [M+H] ⁺ .

Preparation of intermediate compound (29-3). 2 n. NaOH (60 ml, MeOH:H ₂ O=4:1) was added dropwise at 0°C to a solution of crude product 29-2 (12.0 g, 15.4 mmol) in pyridine (120 ml). The mixture was stirred at 0°C for 15 minutes and subsequently neutralized with a saturated aqueous NH ₄ Cl solution. 500 ml of H ₂ O and 800 ml of EA were added. The aqueous phase was extracted using EA, the combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated to give the crude product, which was purified on silica gel to give 29-3 (6.5 g, 9.8 mmol, yield 66, 2% in two stages) as a white solid. 1H-NMR (400 MHz, CDCl ₃ ): δ=8.47 (d, J=7.6 Hz, 1H), 7.92 (d, J=7.6 Hz, 1H), 7.65-7 .60 (m, 1H), 7.54-7.50 (m, 6H), 7.47-7.44 (m, 2H), 7.297.22 (m, 6H), 6.85-6.78 (m, 2H), 5.90-5.80 (m, 1H), 5.74 (s, 1H), 5.28-5.23 (m, 1H), 5.14-5.11 (m , 1H), 4.30-4.25 (m, 3H), 4.18-4.15 (m, 1H), 3.79 (s, 3H), 3.54-3.48 (m, 1H ), 3.13-3.07 (m, 1H), 2.84 (d, J=10.8 Hz, 1H), 1.92 (d, J=4.4 Hz, 1H). ESI-LCMS: m/z 659 [M+H] ⁺ .

Receipt (29-4). To a solution of 29-3 (3.8 g, 5.8 mmol) and DCI (816 mg, 6.9 mmol) in dry DCM (40 ml) was added CEP[N(iPr) ₂ ] ₂ (2.4 g, 8.1 mmol) in an Ar atmosphere. The mixture was stirred at room temperature. for 1 hour. The reaction mixture was washed with 10% aqueous NaHCO ₃ and brine, dried over Na ₂ SO ₄ and concentrated to give the crude product, which was purified by preparative HPLC. 29-4 (3.9 g, 4.5 mmol, 78.69% yield) was obtained as a white solid. 1H NMR (400 MHz, CDCl ₃ ): δ=8.67 (d, J=7.6 Hz, 1H), 7.94-7.91 (m, 2H), 7.64 (t, J=7, 2 Hz, 1H), 7.56-7.42 (m, 8H), 7.28-7.20 (m, 6H), 6.81-6.77 (m, 2H), 5.87-5 .78 (m, 1H), 5.74 (d, J=4.0 Hz, 1H), 5.25-5.20 (m, 1H), 5.11-5.08 (m, 1H), 4.43-4.34 (m, 1H), 4.27-4.21 (m, 2H), 4.15-4.09 (m, 1H), 3.94-3.84 (m, 1H ), 3.79 (d, J=1.6 Hz, 3H), 3.733.56 (m, 3H), 3.46-3.42 (m, 1H), 3.22-3.03 (m, 1H), 2.87 (t, J=10.0 Hz, 1H), 2.69 (t, J=6.4 Hz, 1H), 2.63-2.54 (m, 1H), 1, 72 (dd, J=4.4, 105.6 Hz, 1H), 1.30-1.22 (m, 12H). ³¹ P-NMR (162 MHz, CDCl ₃ ): 149.00, 148.08. ERILCMS: m/z 859 [M+H] ⁺ .

Example 26. Synthesis of oligonucleotides.

Modified oligonucleotides are synthesized on an ABI-394 synthesizer using a 93-step cycle modified with respect to the unlocking, coupling and waiting stages. Solid

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Claims (23)

the support is 3'-NHTr-5'-LCAA-CPG. Each oligonucleotide can be synthesized separately using the methods described herein. Modified oligonucleotides can be purified by anion exchange HPLC. Buffer solutions are, for example, 20 mM sodium phosphate in 10% CH3CN, pH 8.5 (buffer A) and 20 mM sodium phosphate in 10% CH3CN, 1.8 M NaBr, pH 8.5 (buffer B) . Fractions containing full-length oligonucleotides are pooled, desalted and lyophilized. Subsequently, the purified dry oligomer was desalted using Sephadex G-25 M (Amersham Biosciences). The cartridge is conditioned three times with 10 ml of deionized water. Finally, the purified oligomer, thoroughly dissolved in RNase-free water, is applied dropwise to the cartridge, eluting very slowly. The salt-free oligomer is eluted using deionized water directly into a screw cap vial. Approximately 0.10 OD of the oligomer is dissolved in water and then pipetted into special vials for ion exchange HPLC and LCMS. Analytical HPLC and liquid chromatography with electroionization mass spectrometry (ES LC-MS) determine the integrity of the oligonucleotides. Purity and molecular weight were determined by HPLC analysis (60°C, IEX - Thermo DNAPac PA-100, A - 25 mM sodium phosphate 10% acetonitrile pH 11, B - 1.8 M NaBr 25 mM sodium phosphate 10% acetonitrile pH 11 ; RPIP- Waters XBridge OST C18, A- 100 mM HFIP 7 mM TEA B- 7:3 methanol/acetonitrile) and ESI-MS analysis using Promass Deconvolution for Xcalibur (Novatia, Newtown, PA, USA). CLAIM 1. The compound represented by formula (I): where X represents NH; Y is -OCF3, -O(CR ⁴ ₂ )aCR ⁴ 3, -O(CR ⁴ 2)bOCR ⁴ 3 or -O(CR ⁴ 2)b-CR ⁴ =CR ⁴ 2; Z represents H; B is a nucleotide base selected from the group consisting of adenine (A), guanine (G), thymine (T), cytosine (C), uracil (U), 5-methylcytosine (5-me-C), 6- N-benzoyladenine (A ^Bz ), 4-N-benzoylcytosine (C ^Bz ) and 2-N-isobutyrylguanine (Gi ^Bu ); each R ¹ is independently C1-6 alkyl or C3-6 cycloalkyl; R ² represents -CH2CH2CN or C1-6 alkyl; R ³ is H, trityl, monomethoxytrityl (MMTr) or tritolyl; each R ⁴ is independently H or F; a is equal to 1 or 2; and b is 1, 2 or 3. 2. The compound according to claim 1, in which Y represents -OEt or -OCH ₂ CH ₂ OCH ₃ . 3. A compound according to claim 1, in which R ¹ represents C ₂ _ ₃ alkyl. 4. A compound according to claim 1, wherein R ¹ is isopropyl. 5. The compound according to claim 1, wherein R ³ represents trityl. 6. A compound according to claim 1, wherein R ³ is monomethoxytrityl (MMTr) or tritolyl. 7. A compound according to claim 1, wherein B is adenine (A), guanine (G) or cytosine (C). 8. A compound according to claim 1, wherein B is 6-N-benzoyladenine (A ^Bz ), 4-N-benzoylcytosine (C ^Bz ) or 2-N-isobutyrylguanine (G ^{1 Bu} ). 9. A composition for use as a synthetic precursor to oligonucleotides, wherein the composition contains a compound according to claim 1 and one or more solvents. 10. A method for producing a purified and desalted oligonucleotide, the method comprising the steps of (i) unblocking the compound of claim 1 to produce an unblocked intermediate, (ii) combining the unlocked intermediate with a nucleotide monomer to form a crude oligonucleotide, (iii) purifying the crude oligonucleotide using anion exchange high performance liquid chromatography (HPLC) to obtain purified oligonucleotide - 60 045540 tide and (iv) desalting the purified oligonucleotide to obtain a purified and desalted oligonucleotide, where deblocking involves removing the R ³ group. 11. The compound represented by formula (II): where X represents NH; Z represents H; B is a nucleotide base selected from the group consisting of adenine (A), guanine (G), cytosine (C), uracil (U), 6-X-benzoyladenine (A ^Bz ), 4-M-benzoylcytosine (C ^Bz ) and 2-Nisobutyrylguanine (G ^1Bu ); each R ¹ independently represents C3 alkyl or C3.6 cycloalkyl; R ² represents -CH ₂ CH ₂ CN or Cb ₆ alkyl; R ³ represents H, trityl, monomethoxytrityl (MMTg) or tritolyl; A represents -(CR'R')i. ₂ -; and each R' is independently H or Me. 12. Connection by π. 11, in which R ¹ represents C _{2 .3} alkyl. 13. Connection by π. 11, in which R ¹ represents isopropyl. 14. Connection by π. 11, in which R ³ represents trityl. 15. A compound according to claim 11, wherein R ³ is monomethoxytrityl (MMTg) or tritolyl. 16. The compound according to π.11, in which B represents adenine (A) or guanine (G). 17. The compound according to claim 11, wherein B is 6-X-benzoyladenine (A ^Bz ), 4-Nbenzoylcytosine (C ^Bz ) or 2-X-isobutyrylguanine (G ^1Bu ). 18. A composition for use as a synthetic precursor to oligonucleotides, where the composition contains a compound at π. 11 and one or more solvents. 19. A method for obtaining a purified and desalted oligonucleotide, the method includes the steps of (i) unblocking the compound at π. 11 to obtain an unlocked intermediate, (i) coupling the unlocked intermediate with a nucleotide monomer to obtain a crude oligonucleotide, (iii) purifying the crude oligonucleotide by anion exchange high performance liquid chromatography (HPLC) to obtain a purified oligonucleotide, and (iv) desalting the purified oligonucleotide to obtain purified and desalted oligonucleotide, where unblocking includes removal of the R ³ group. 20. The compound according to claim 1, where Y is -OCF ₂ CH ₂ OCH ₃ . 21. The compound according to claim 1, where Y is -OCH ₂ CH ₂ OCF ₃ . 22. The compound according to claim 1, where Y is -O(CR ⁴ 2)b-CR ⁴ =CR ⁴ 2. 23. A compound according to claim 1, wherein Y is -OCF ₃ . Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky lane, 2