INCLUDING NUC-9701

FIELD OF THE INVENTION

The present invention generally relates to a novel process for the preparation of 8-chloroadenosine derivatives, and particularly NUC-9701 (8-chloroadenosine-5’-0- [naphthyl(benzyloxy-L-alaninyl)] phosphate) an anticancer drug of the ProTide class of 8- chloroadenosine.

BACKGROUND OF THE INVENTION

Drugs of the ProTide class are masked phosphate derivatives of nucleosides. They have been shown to be particularly potent therapeutic agents in the fields of both antivirals and oncology. Drugs of the ProTide class, more specifically, are prodrugs of monophosphorylated nucleosides. These compounds appear to avoid many of the inherent and acquired resistance mechanisms which limit the utility of the parent nucleosides (see, for example, ‘Application of ProTide Technology to Gemcitabine: A Successful Approach to Overcome the Key Cancer Resistance Mechanisms Leads to a New Agent (NUC-1031) in Clinical Development’·, Slusarczyk et al; J. Med. Chem .; 2014, 57, 1531-1542).

ProTide derivatives of purine nucleosides such as 8-chloroadenosine and related compounds have also shown excellent activity in vitro against a range of solid tumours, leukaemias and lymphomas.

WO 2017/207989 discloses 8-chloroadenosine (8-CI-A) derivatives and their use in treating cancer. 8-CI-A is a nucleoside analogue that has cytotoxic activity in many cancers, including leukaemia. 8-CI-A is currently under phase I clinical study for chronic lymphocytic leukaemia. 8-CI-A is composed of an adenine base which has a chlorine group attached at position 8. 8-CI-A is a very potent drug, which exerts cytotoxicity by causing cell cycle arrest at G2/M phase, mediating catastrophic mitotic division, thereby inducing apoptosis and autophagy. 8-CI-A itself is not a particularly potent anticancer agent but can be used to make further prodrug-type delivery systems. NUC-9701 is an example of such a system: -9701

Drugs of the ProTide class are typically prepared as a mixture of two diastereoisomers, epimeric at the phosphate centre. The diastereoisomers of NUC-9701 , for example, have the following structures (in which Np is a 1 -napthyl):

It is an aim of certain embodiments of the present invention to enable access to 2’, 3’- protected 8-CI-A that can be converted into 8-CI-A delivery agents that can deliver either 8- Cl-A or a phosphate nucleotide of 8-CI-A to a patient.

It is an aim of certain embodiments of the present invention to provide a process of providing the 2’, 3’- protected 8-CI-A and/or 8-CI-A delivery agents which is scalable, economic and/or efficient, e.g. more scalable, economic and/or efficient than known methods.

It is an aim of certain embodiments of this invention to provide a process of providing NUC-9701 in substantially diastereoisomerically pure form.

It is an aim of certain embodiments of this invention to provide a process of providing the (S _p ) and/or (R _p )-epimer(s) of NUC-9701 in substantially diastereoisomerically pure form(s) which is scalable, economic and/or efficient, e.g. more scalable, economic and/or efficient than methods using HPLC. Thus, it is an aim of certain embodiments of this invention to provide a method of providing the (S _p ) and/or (R _p )-epimer(s) in substantially diastereoisomerically pure form(s) which is suitable for large scale manufacture.

It is an aim of certain embodiments of this invention to provide a simple process i.e. a process which involves a minimum number of process steps and or reagents of providing the (S _p ) and/or (R _p )-epimer(s) in substantially diastereoisomerically pure form(s).

Another aim of certain embodiments of this invention is to provide a process which ensures the separated (S _p )- or (R _p )-epimer are provided in substantially diastereoisomerically pure form and at the same time meet or exceed the necessary criteria stipulated by organisations such as the US FDA concerning the amounts and nature of any trace impurities which arise from synthesis and separation.

Certain embodiments of the present invention meet some or all of the above stated aims.

SUMMARY OF THE INVENTION

In accordance with an aspect of the present invention there is provided a process for the preparation of a compound of formula (Ilia) or (lllb)

the process comprising step 2): step 2) chlorinating a compound of formula (IV):

to provide a compound of formula (Ilia) or (lllb);

wherein P ¹ and P ² , are each independently a protecting group;

P ³ , P ⁴ and P ⁵ are each independently selected from H and a protecting group;

P ^5a is independently a protecting group.

The process may further comprise step 1):

step 1) introducing P ¹ and P ² to a compound of formula (V):

to provide a compound of formula (IV).

P ⁵ may be a protecting group. The product of step 2) may be a compound of formula (Ilia). The process may further comprise step 2a):

step 2a) removing the protecting group P ^5a from the compound of formula (Ilia) to provide a compound of formula (lllb). P ⁵ may be H. The product of step 2) may be a compound of formula (lllb).

The compound of formula (lllb) may

Also provided is a process for the preparation of NUC-9701 :

The process for the preparation of NUC-9701 may comprise the process of the first aspect. The process for the preparation of NUC-9701 may further comprise step 1). The process for the preparation of NUC-9701 may further comprise step 3) and step 4):

step 3) coupling a compound of formula (lllb) with a compound of formula (II)

wherein LG is a leaving group, in the presence of a base (B1), to provide a compound of formula (I):

step 4) removing protecting groups P ¹ and P ² and, where P ³ and P ⁴ are protecting groups, removing P ³ and P ⁴ from (I) to provide NUC-9701.

DETAILED DESCRIPTION

Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

A protecting group for a hydroxyl group (e.g. P ¹ , P ² and P ⁵ ) may be independently selected from optionally substituted -Si(Ci-C6-alkyl)3, optionally substituted -C(0)-Ci-C ₆ -alkyl, optionally substituted -C(0)-aryl, optionally substituted -C(0)-0Ci-C ₆ -alkyl, -C(0)-0-allyl, - C(0)-0-CH ₂ -fluorenyl, optionally substituted -C(aryl)3 _, optionally substituted -(C1-C3- alkylene)-aryl, optionally substituted -C(0)0CH ₂ -aryl and -Ci-C ₄ -alkyl-0-Ci-C ₄ -alkyl.

According to some embodiments, P ¹ and P ² are each independently selected from optionally substituted -Si(Ci-C6-alkyl)3, optionally substituted -C(0)-Ci-C ₆ -alkyl, optionally substituted -C(0)-aryl, optionally substituted -C(0)-0Ci-C ₆ -alkyl, -C(0)-0-allyl, -C(0)-0- CH2-fluorenyl, optionally substituted -C(aryl)3 _, optionally substituted -(Ci-C3-alkylene)-aryl, optionally substituted -C(0)0CH ₂ -aryl and -Ci-C ₄ -alkyl-0-Ci-C ₄ -alkyl; or P ¹ and P ² together form a -C(R ¹ )2- group, wherein R ¹ is at each occurrence Ci-C4-alkyl; or wherein the two R ¹ groups together with the carbon atom to which they are attached together form a C3-C7-cycloalkyl ring; or wherein the two R ¹ groups together form a group selected from =0 and =S.

P ¹ and P ² may be the same.

P ¹ and P ² may each be selected from optionally substituted -Si(Ci-C6-alkyl)3, optionally substituted -C(0)-0Ci-C ₆ -alkyl and optionally substituted -C(0)0CH ₂ -aryl, -C(0)-0-allyl. Preferably, P ¹ and P ² are each selected from -C(0)0-fBu, -C(0)0-benzyl and -C(0)0CH ₂ -allyl. Thus, P ¹ and P ² may each be -C(0)0CH ₂ -aryl. P ¹ and P ² may each be -C(0)0-fBu.

Alternatively, P ¹ and P ² may each be selected from optionally substituted -C(0)-Ci-C ₆ - alkyl and optionally substituted -C(0)-aryl, e.g. P ¹ and P ² may each be selected from benzoyl and acetyl.

In a further alternative, P ¹ and P ² may each be optionally substituted -Si(Ci-C6-alkyl)3. P ¹ and P ² may each be -Si(Ci-C4-alkyl)3. The alkyl groups may be unsubstituted. P ¹ and P ² may each be f-butyldimethylsilyl. Preferably, however, P ¹ and P ² together form an -C(R ¹ )2- group. R ¹ may at each occurrence be CrC4-alkyl. In some embodiments, P ¹ and P ² together form a -C(Me)2- group.

A protecting group for an amino group (e.g. P ³ and P ⁴ ) may at each occurrence be independently selected from -C(0)OCrC ₆ -alkyl, optionally substituted -C(0)OCH ₂ - aryl, -C(0)-0-allyl, -C(0)-0-CH ₂ -fluorenyl, optionally substituted -C(aryl)3 _, optionally substituted -(Ci-C3-alkylene)-aryl, optionally substituted -C(0)-Ci-C ₆ -alkyl, optionally substituted -C(0)-aryl, -S(0) ₂ -Ci-C ₆ -alkyl, optionally substituted -S(0) ₂ -aryl and optionally substituted -Si(Ci-C6-alkyl)3.

P ³ may be independently selected from -C(0)OCi-C ₆ -alkyl, optionally substituted -C(0)OCH ₂ -aryl, -C(0)-0-allyl, -C(0)-0-CH ₂ -fluorenyl, optionally substituted - C(aryl)3 _, optionally substituted -(Ci-C3-alkylene)-aryl, optionally substituted -C(0)-Ci-C ₆ - alkyl, optionally substituted -C(0)-aryl, -S(0) ₂ -Ci-C ₆ -alkyl, optionally substituted -S(0) ₂ - aryl and optionally substituted -Si(Ci-C6-alkyl)3.

P ³ may be independently selected from -C(0)OCi-C ₆ -alkyl, optionally substituted -C(0)OCH ₂ -aryl, -C(0)-0-allyl, optionally substituted -C(aryl)3 _, and optionally substituted -Si(Ci-C6-alkyl)3. Preferably, P ³ is selected from -C(0)0-tBu, -C(0)0-benzyl and -C(0)OCH ₂ -allyl. Thus, P ³ may be -C(0)OCH ₂ -aryl.

Alternatively, P ³ may be independently selected from optionally substituted -C(0)-Ci-C ₆ -alkyl and optionally substituted -C(0)-aryl, e.g. P ³ may be independently selected from benzoyl and acetyl.

In another alternative, P ³ is H.

Likewise, P ⁴ may be independently selected from H, -C(0)OCi-C ₆ -alkyl, optionally substituted -C(0)OCH ₂ -aryl, -C(0)-0-allyl, -C(0)-0-CH ₂ -fluorenyl, optionally substituted -C-(aryl)3 _, optionally substituted -(Ci-C3-alkylene)-aryl, optionally substituted - C(0)-Ci-C ₆ -alkyl, optionally substituted -C(0)-aryl, -S(0) ₂ -Ci-C ₆ -alkyl, optionally substituted -S(0) ₂ -aryl and optionally substituted -Si(Ci-C6-alkyl)3.

In another alternative, P ⁴ is H.

It may be that each of P ³ and P ⁴ are H. It may be that each of P ³ and P ⁴ are H and P ¹ and P ² together form a -C(Me)2- group. According to some embodiments, P ³ is not -C(0)-aryl. According to some embodiments, P ⁴ is not -C(0)-aryl. According to some embodiments, neither of P ³ and P ⁴ are -C(0)-aryl. According to some embodiments, P ³ is not benzoyl. According to some embodiments, P ⁴ is not benzoyl. According to some embodiments, neither of P ³ and P ⁴ are benzoyl.

According to some embodiments, P ⁵ is selected from H and a protecting group. In some embodiments, P ⁵ is a silyl protecting group. P ⁵ may be independently selected from optionally substituted -Si(CrC6-alkyl)3, optionally substituted -C(0)-CrC ₆ -alkyl, optionally substituted -C(0)-aryl, optionally substituted -C(0)-OCrC ₆ -alkyl, -C(0)-0-allyl, -C(0)-0- Chh-fluorenyl, optionally substituted -C(aryl)3 _, optionally substituted -(Ci-C3-alkylene)-aryl, optionally substituted -C(0)OCH ₂ -aryl and -Ci-C ₄ -alkyl-0-Ci-C ₄ -alkyl.

P ⁵ may be independently selected from optionally substituted -Si(CrC6-alkyl)3, optionally substituted -C(0)-OCrC ₆ -alkyl and optionally substituted -C(0)OCH ₂ -aryl, -C(0)-0-allyl. Preferably, P ⁵ is selected from -C(0)0-fBu, -C(0)0-benzyl and -C(0)OCH ₂ -allyl. Thus, P ⁵ may be -C(0)OCH ₂ -aryl. P ⁵ may be -C(0)0-fBu.

Alternatively, P ⁵ may be independently selected from optionally substituted -C(0)-Ci-C ₆ - alkyl and optionally substituted -C(0)-aryl, e.g. P ⁵ may be independently selected from benzoyl and acetyl.

In a further alternative, P ⁵ may be optionally substituted -Si(CrC6-alkyl)3. P ⁵ may be -Si(CrC4-alkyl)3. The alkyl groups may be unsubstituted. P ⁵ may be t- butyldimethylsilyl. In another alternative, P ⁵ is H.

According to some embodiments, P ⁵ is not -Si(Ci-C6-alkyl)3 _. According to some embodiments, P ⁵ is not -Si(Et)3 _.

According to some embodiments, P ^5a is a protecting group. In some embodiments, P ^5a is a silyl protecting group. P ^5a may be independently selected from optionally substituted -Si(CrC6-alkyl)3, optionally substituted -C(0)-CrC ₆ -alkyl, optionally substituted -C(0)-aryl, optionally substituted -C(0)-OCrC ₆ -alkyl, -C(0)-0-allyl, -C(0)-0-CH ₂ -fluorenyl, optionally substituted -C(aryl)3 _, optionally substituted -(Ci-C3-alkylene)-aryl, optionally substituted -C(0)OCH ₂ -aryl and -Ci-C ₄ -alkyl-0-Ci-C ₄ -alkyl.

P ^5a may be independently selected from optionally substituted -Si(CrC6-alkyl)3, optionally substituted -C(0)-OCrC ₆ -alkyl and optionally substituted -C(0)OCH ₂ -aryl, -C(0)-0-allyl. Preferably, P ^5a is selected from -C(0)0-fBu, -C(0)0-benzyl and -C(0)0CH ₂ -allyl. Thus, P ^5a may be -C(0)0CH ₂ -aryl. P ^5a may be -C(0)0-fBu.

Alternatively, P ^5a may be independently selected from optionally substituted -C(0)-Ci-C ₆ - alkyl and optionally substituted -C(0)-aryl, e.g. P ^5a may be independently selected from benzoyl and acetyl.

In a further alternative, P ^5a may be optionally substituted -Si(Ci-C6-alkyl)3. P ^5a may be -Si(Ci-C4-alkyl)3. The alkyl groups may be unsubstituted. P ^5a may be t- butyldimethylsilyl.

According to some embodiments, P ^5a is not -Si(Ci-C6-alkyl)3 _. According to some embodiments, P ^5a is not -Si(Et)3 _.

In some embodiments, the compound of formula (V) is

he compound of formula (IV) is

In some embodiments, the compound of formula (IV) is

he compound of formula (lllb) is

In some embodiments, the compound of formula (lllb) is

LG may be selected from halo, alkyl sulfonyl, aryl sulfonyl, heteroaryloxy or substituted phenoxy. LG may be halo, e.g. chloro or bromo. LG may be OTf or OTs. LG may be a substituted phenoxy. Thus, the compound of formula (II) may be a compound of formula (VI): wherein R ¹ may be selected from the group comprising: halo, trifluoromethyl, cyano and nitro; and a is an integer selected from 1 , 2, 3, 4 and 5. R ¹ may be halo. R ¹ may be F. a may be 5. Thus, the compound of formula (VI) may be

In some embodiments, step 1) may be carried out in the presence of an acid e.g. perchloric acid, p-toluenesulfonic acid, sulfuric acid or methanesulfonic acid. In some embodiments, step 1) may be carried out in a solvent S1. Step 1) may be conducted in an organic solvent. Organic solvents include but are not limited to ethers (e.g. tetrahydrofuran, dioxane, diethyl ether, methyl-f-butylether); ketones (e.g. acetone and methyl isobutyl ketone); halogenated solvents (e.g. dichloromethane, chloroform and 1 ,2-dichloroethane); and amides (e.g. DMF, NMP); or mixtures thereof. The solvent may be acetone. In some embodiments, step 1) may be carried out in the presence of HCIO ₄ . Preferably, step 1) may be carried out with HCIO ₄ in acetone. In some embodiments, step 2) may be carried out using NCS. In some embodiments, step 2) may be carried out in the presence of an acid e.g. sulfuric acid, acetic acid, perchloric acid, phosphoric acid, oxalic acid, TFA, or methanesulfonic acid. In some embodiments, step 2) may be carried out in a solvent S1. In some embodiments, step 2) may be carried out in the presence of TFA. In some embodiments, step 2) may be carried out using NCS in H2SO4. In some embodiments, step 2) may be carried out using NCS in DMF and TFA. In some embodiments, step 3) may be carried out using a racemic ligand. In some embodiments, step 3) may be carried out in a solvent S1. Where step 3) is conducted in the presence of a Grignard reagent, the organic solvent is preferably an ether. Most preferably, the solvent is THF. In some embodiments, step 3) may be carried out using 1.0 to 1.5 eq of racemic ligand. In some embodiments, step 3) may be carried out using 1.5 to 2 eq of a Grignard reagent. In some embodiments, step 3) may be carried out using 1.1 eq of racemic ligand and 1.5 eq of Grignard reagent e.g. f-BuMgCI. In some embodiments, step 3) may be carried out using 1.2 eq of racemic ligand and 1.75 eq of Grignard reagent e.g. f-BuMgCI. In some embodiments, step 3) may be carried out using

In some embodiments, step 4) may be carried out in an acid e.g. formic acid. Step 4) may be carried out in the presence of water.

Where a protecting group is acid sensitive (e.g. trityl, C(0)OtBu, MOM, MEM, 2,4- dimethoxybenzyl, 2,3-dimethoxybenzyl, -C(Me)2-) the deprotection step can be conducted using a suitable acid. The acid may be a Bronsted acid (e.g. TFA, phosphoric acid, HCI, or formic acid) or a Lewis acid (e.g. ZnBr2, CeCh). Lewis acids (e.g. ZnBr2) are less preferred. HCI is likewise less preferred. Preferably, the acid is TFA. In an alternative preferable embodiment, the acid is formic acid.

Where a protecting group is base sensitive (e.g. acetyl, benzoyl, C=0) the deprotection step can be conducted using a suitable base, e.g. aqueous NH3 or aqueous NaOH. Base sensitive groups may be less preferred.

Where a protecting group is a silyl group (e.g. triethylsilyl or t-butyldimethylsilyl, the deprotection step can be conducted using a suitable acid (e.g. TFA) or using a suitable fluorine source (e.g. tetrabutylammonium fluoride, fluorosilicic acid, HF).

Where a protecting group is a benzyl group or a C(0)Obenzyl group, the deprotection step can be conducted using H2 and a suitable catalyst (e.g. Pd/C). Such protecting groups may be less preferred. Where a protecting group is a 4-methoxy-benzyl, 2,3-dimethoxybenzyl, 2,4- dimethoxybenzyl or C(0)0-(4-methoxybenzyl) the deprotection step can be performed using a suitable oxidizing agent (e.g. meta-chloroperbenzoic acid).

Where a protecting group is -C(0)-0-allyl, the deprotection step can be performed using (PPh ₃ ) ₄ Pd.

Where a protecting group is -C(0)-0-CH ₂ -fluorenyl, the deprotection step can be performed using piperidine.

The deprotection step may be conducted in an organic solvent or a mixture thereof. Exemplary organic solvents include, but are not limited to halogenated solvents (e.g. dichloromethane, chloroform, dichloroethane); alcohols (e.g. methanol, ethanol, isopropanol) and ethers (e.g. tetrahydrofuran, diethyl ether).

Where the deprotection step is carried out in the presence of an acid (e.g. TFA, the organic solvent is preferably a halogenated solvent, e.g. dichloromethane.

The base (B1) might be a nitrogen base. Nitrogen bases include /V-alkylimidazoles, (e.g. /V-methyl imidazole (NMI)), imidazole, optionally substituted pyridines, (e.g. collidine, pyridine, 2,6-lutidine) and trialkylamines (e.g. triethylamine, and diisopropylethylamine). Alternatively, the base (B1) may be an organometallic base or metal hydride base (e.g. NaH). Thus, the base may be a Grignard reagent (i.e. an alkylmagnesium halide). Exemplary Grignard reagents include f-butylmagnesium halides such as fBuMgCI, fBuMgBr. Preferably, the base is fBuMgCI.

In some embodiments, the process is a method of making the (R _p )-diastereoisomer of NUC-9701 in diastereomerically enriched form and the compound of formula (II) is the (R _p )-diastereoisomer in diastereomerically enriched form.

In some embodiments, the process is a method of making the (S _p )-diastereoisomer of NUC-9701 in diastereomerically enriched form and the compound of formula (II) is the (S _p )-diastereoisomer in diastereomerically enriched form.

The following abbreviations are used throughout this specification:

DMF - A/,/\/-dimethylformamide eq. - molar equivalents

MEM - 2-methoxyethoxymethyl MOM - methoxymethyl Ms - methanesulfonate

NCS - N-chlorosuccinimide NMI - /V-methyl imidazole

NMP - A/-methyl-2-pyrrolidone Np - 1 -naphthyl

RT - room temperature TBDMS - te/f-butyldimethylsilyl

Tf - trifluoromethylsulfonate (triflate) TFA - trifluoroacetic acid

THF - tetrahydrofuran Ts - toluenesulfonate (tosylate)

V is used to denote volume (in ml_) per weight (in g) starting material. So, if there was 1 g of starting material, 10 V would mean 10 mL of the indicated liquid.

EXAMPLES

The present invention is further illustrated by the following examples, which are provided by way of illustration only and should not be construed to limit the scope of the invention.

Example 1 : Acetonide protection

Adenosine 1 (100 g) and acetone (4 L, 40 v/w) were stirred for 5 minutes at 20-30 °C for 5 minutes. Perchloric acid (40 mL, 0.4 v/w) was added to the reaction mixture and stirred at 20-30 °C for 1 h, monitoring by UPLC. The reaction mass was quenched with 7 % sodium bicarbonate solution (1 L, 10 v/w) at 20-30 °C and cooled to -5 °C to 5 °C. The reaction mass was stirred for 1 hour. The obtained product was filtered, the filter cake was washed with acetone (200 mL, 2.0 v/w) and dried under vacuum for 8 h to yield compound 2.

Yield: 89 g, 77 %

HPLC purity: 97.96 % Example 2: Chlorination

To a mixture of compound 2 (10 g, 1 eq) and DMF (100 ml_, 10 v/w) was added sulfuric acid (0.9 mL, 0.5 eq) and the mixture was stirred at 20-30 °C for 15 minutes. To the reaction mixture was first added N-chlorosuccinimide (7.38 g, 1.5 eq) and, after 6 hours, a further 0.2 eq of NCS was added. The reaction mixture was stirred at 20-30 °C for 22 hours, monitored by HPLC. The reaction mass was quenched into a 7 % sodium bicarbonate solution (10 v/w) and 35 % sodium chloride solution (20 v/w) and stirred at 20- 30 °C for 15 minutes. To the reaction mixture was added ethyl acetate (20 v/w) and the mixture was stirred at 20-30 °C for 10 minutes. The aqueous layer was extracted with ethyl acetate (20 v/w). The organic layer was washed with DM water (2 ^c 10 v/w) and dried over anhydrous Na2SC>4. The reaction mass was cooled to -5 °C to 5 °C and stirred for 1 h. The obtained solid was filtered and washed with ethyl acetate (2 vol) and dried under vacuum to afford the solid compound 3.

Yield: 4.1 g, 37 %

HPLC purity: 96.56 %

Example 3: Ligand coupling followed by acetonide deprotection

NUC-9701

To a mixture of compound 3 (5 g, 1 eq) and THF (50 ml_, 10 v/w) was added racemic ligand 4 (8.9 g, 1.1 eq). The reaction mass was stirred at 20-30 °C for 10 minutes, then cooled to -5 °C to 5 °C and stirred for 10 minutes. ¾uMgCI (11 ml_, 2.0 M in THF, 1.5 eq) was added to the reaction mass slowly at below 10 °C. The reaction mass was stirred at - 5 °C to 5 °C for 1 hour. The reaction was monitored by HPLC. The reaction mass was quenched with 10 % ammonium chloride solution (50 ml_, 10 v/w) at below 10 °C. After quenching, the temperature of the reaction mixture was raised to 20-30 °C and stirred for 10 minutes. The product was extracted with ethyl acetate (2 x 50 ml_, 2 x 10 v/w). The combined organic layer was washed with water (50 ml_, 10 v/w). The organic layer was first washed with 7 % NaHCC>3 solution (50 ml_, 10 v/w) and then washed with 10 % sodium chloride solution (50 ml_, 10 v/w) at 20-30 °C. The organic layer was then dried over anhydrous sodium sulfate (5 g, 1 w/w) and filtered. The sodium sulfate was then washed with ethyl acetate (5 ml_, 1 v/w). The organic layer was concentrated under reduced pressure at below 45 °C to give the crude product, the compound 5, which was further deprotected by a treatment with a mixture of formic acid/water (3:2) at rt for 16 h. The crude product was concentrated under reduced pressure and purified to yield the compound NUC-9701 as an off-white solid.