EA046381B1 - ECTONUCLEOTIDASE INHIBITORS AND METHODS OF THEIR APPLICATION - Google Patents

Description

BACKGROUND OF THE INVENTION

CD73, also called 5'-nucleotidase (5'-NT) or ecto-5'-nucleotidase (Ecto 5'NTase), is a membrane-bound cell surface enzyme whose primary role is to catalyze the conversion of extracellular nucleotides (eg, AMP) to their corresponding nucleosides (eg adenosine). CD73 is found in most tissues and is expressed in lymphocytes, endothelial cells and epithelial cells. It is also widely expressed in many tumor cell lines and is particularly upregulated in cancerous tissues (Antonioli et al., Nat. Rev. Cancer, 13: 842-857, 2013).

In tandem with CD39 (ecto-ATPase), CD73 generates adenosine from ATP/AMP, which is often released from damaged or inflamed cells into the extracellular environment. Extracellular adenosine produced by CD73 interacts with G protein-coupled receptors on target cells. An important downstream effect of this signaling is enhanced immunosuppression through a number of pathways. For example, CD73 is a co-signaling molecule on T lymphocytes. Under normal conditions, extracellular adenosine levels promote a self-limiting immune response that prevents excessive inflammation and tissue damage. For tumors, the effect of abnormally elevated CD73 is that the resulting increased levels of adenosine, catalyzed by CD73, result in inhibition of the immune system's antitumor response.

Although CD73 plays a role in cancer immunosuppression, higher CD73 expression is associated with various stages of tumor progression, including tumor vascularization, invasiveness, and metastasis, as well as shorter survival time in breast cancer patients. Some of these observations result from the enzyme-independent function of CD73 as an adhesion molecule required for the binding of lymphocytes to the endothelium.

Overall, CD73 has become an important target for the development of new cancer treatments, either as single drugs or in combination with other anticancer drugs. Indeed, combining anti-CD73 monoclonal antibodies with antibodies for other chemotherapeutic targets improves response and survival in animal models of cancer (Allard et al., Clin. Cancer Res., 19:5626-35, 2013).

Many of the current cancer treatments and chemotherapy agents cannot successfully treat all patients or all symptoms in treated patients, and many of these treatments are associated with unwanted side effects. As some cancers develop resistance to various chemotherapy agents, alternative cancer treatments are needed. Thus, there is a need for additional compounds and methods for treating cancer and other diseases.

The essence of the invention

Described herein are compounds of formula (I):

(I) or pharmaceutically acceptable salts and/or prodrugs thereof, wherein Y is

Het represents heterocyclyl or heteroaryl;

R ^1a is selected from H, halogen, hydroxy, cyano, azido, amino, amido, C _1-6 alkyl, hydroxyC _1-6 alkyl, amino-C _1-6 alkyl, -OC(O)-OC _1-6 alkyl, C _1-6 acyloxy, C _|-6 a1koxy, C _2-6 alkenyl and C _2-6 alkynyl;

R ^1b is selected from H, halogen, C _1-6 alkyl, hydroxy-C _1-6 alkyl, amino-C _1-6 alkyl, C _2-6 alkenyl and C _2-6 alkynyl;

R ^2a is selected from halogen, hydroxy, cyano, azido, amino, C _1-6 alkyl, hydroxy-C _1-6 alkyl, amino-C _1-6 alkyl, C _1-6 acyloxy, -O-C(O)- O-C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyl and C _2-6 alkynyl;

R ^2b is selected from halogen, C _1-6 alkyl, C _2-6 alkenyl and C _2-6 alkynyl, preferably substituted or unsubstituted C ₂ alkynyl, most preferably unsubstituted C ₂ alkynyl;

- 1 046381

R ³ is selected from H and alkyl;

R ⁴ is selected from H, alkyl, CN, aryl, heteroaryl, -C(O)OR ⁹ , -C(O)NR ¹¹ R ¹² , -S(O) ₂ R ¹⁰ , -P(O)(OR ¹¹ ) (OR ¹² ) and -P(O)(OR ¹¹ )(NR ¹³ R ¹⁴ );

R ⁵ is selected from H, cyano, alkyl, cycloalkylalkyl, heterocyclylalkyl, aralkyl, heteroaralkyl and -C(O)OR ⁹ ;

R ⁶ is selected from -C(O)OR ⁹ , -C(O)NR ¹⁶ R ¹⁷ and -P(O)(OR ¹¹ )(OR ¹² );

R ⁹ is independently selected from H, alkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, aryl, aralkyl, heteroaryl and heteroaralkyl;

R ¹⁰ is independently selected from alkyl, alkenyl, alkynyl, amino, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, aryl, aralkyl, heteroaryl and heteroaralkyl; and each of R ¹¹ and R ¹² is independently selected from H, alkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl; or

R ¹¹ and R ¹² together with the carbon atom to which they are attached form a 5-7 membered heterocyclyl;

R ¹³ represents H or alkyl;

R ¹⁴ represents alkyl or aralkyl;

each R ¹⁵ is independently selected from hydroxy, alkoxyacyloxy and NR ¹³ R ¹⁴ ;

each R ¹⁶ and R ¹⁷ is independently selected from H, hydroxy, alkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl; or

R ¹⁶ and R ¹⁷ together with the nitrogen atom to which they are attached form a 5-7 membered heterocyclyl.

In certain preferred embodiments of formula (I), the included compounds meet conditions a) and b); or a) and c); Where:

a) the connection is not

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b) if R ⁴ and R ⁶ are each -C(O)OH and R ⁵ is benzyl substituted on the phenyl ring with a heterocyclyl or heteroaryl substituent, then the phenyl ring substituent is selected from unsubstituted or substituted pyrrolidinyl, piperazinonyl, piperidonyl, tetrahydropyrimidonyl , pyridonyl and pyridyl; And

c) if R ⁴ is -C(O)OH or tetrazolyl, R ⁶ is -C(O)OH, and R ⁵ is benzyl substituted on the phenyl ring by a second phenyl ring, then either a benzylphenyl ring or a second phenyl ring the ring is substituted with -C(O)OR ⁹ where R ⁹ represents H or alkyl.

R ⁴

R ⁵ ^l

In some embodiments, Y is ^k . In other embodiments, osuoo ^R 'kll II / ^p \p-|— o15 \//

R ¹⁵ of Y represents

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In certain preferred embodiments: a) the connection is not

b) R ^2b is selected from halogen, C _2-6 αlkyl, C _2-6 αlkenyl and ^^alkynyl, preferably substituted or unsubstituted C ₂ alkynyl, most preferably unsubstituted C ₂ alkynyl; and either

c) R ⁵ is benzyl substituted on the phenyl ring with a substituent selected from unsubstituted or substituted piperidonyl, tetrahydropyrimidonyl, pyridonyl and pyridyl, or

d) R ⁵ is benzyl substituted on the phenyl ring with a second phenyl ring substituted by -C(O)OR ⁹ where R ⁹ is H or alkyl.

In some embodiments, the present invention provides a pharmaceutical composition suitable for use in a subject for the treatment or prevention of cancer, comprising an effective amount of any of the compounds described herein (e.g., a compound of the invention, such as a compound of formula (I) ) or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable excipients. In some embodiments, pharmaceuticals may be used to treat or prevent a condition or disease described herein.

Disclosed herein are methods of treating diseases and conditions in which CD73 inhibition is effective, comprising administering to a subject in need thereof an effective amount of a compound described herein (e.g., a compound of formula (I)

- 4 046381 or any of the embodiments described herein). In some embodiments, the human patient requires such treatment. These diseases include, but are not limited to, malignancies such as lung cancer, kidney cancer, skin cancer, breast cancer and ovarian cancer. Other diseases and conditions that can be treated using the methods described herein include, but are not limited to, neurological, neurodegenerative and CNS disorders such as depression and Parkinson's disease, cerebral and cardiac ischemic diseases, sleep disorders, fibrosis, immune and inflammatory disorders.

Provided herein are combination formulations of compounds of formula (I) with monoclonal antibodies and other chemotherapeutic agents that may enhance therapeutic benefits beyond adjuvant therapy alone.

Brief description of graphic materials

Fig. 1A shows an increase in the % of CD8+ cells from CD45+ cells in EG7 tumors of mice treated with Compound 9.

Fig. 1B demonstrates the reversal of efficacy with Compound 9 when CD8+ cells are depleted with anti-CD8 antibody.

Fig. 2A shows a reversal of AMP-mediated CD8+ T cell suppression using the CD73 inhibitor Compound 9, including an EC ₅₀ of 11.6 nM for CD8+ T cell proliferation.

Fig. 2B shows EC ₅₀ =9.6 nM for CD8+ T cell activation.

Fig. 2C shows EC ₅₀ =4.5 nM for IFN-gamma production.

Fig. 2D shows EC ₅₀ =5.6 nM for Granzyme B production.

Fig. 3A demonstrates the effect of Compound 9 on the proliferation of certain cell lines, including comparable % survival of EG7 cells, a mouse T-cell lymphoma cell line.

Fig. 3B shows comparable % survival of A375 cells, a human melanoma cell line.

Fig. 3C shows comparable % of human CD8+ T cells separated.

Fig. 4A shows the potency of compound 9 assessed against CD73- and CD73-expressing SK-MEL-28 cells.

Fig. 4B shows EC50=0.17 nM for recombinant human CD73.

Fig. 4C shows EC ₅₀ =0.38 nM for human plasma CD73.

Fig. 4D shows EC ₅₀ =0.21 nM for surface quantitation of human CD73 cells.

Fig. 5A shows the level of compound 9 in mouse plasma

Fig. 5B shows inhibition of CD73 in mouse plasma.

Fig. 6A demonstrates the effectiveness of compound 9 against EG7 tumors.

Fig. 6B demonstrates the effectiveness of compound 9 against CT26 tumors.

Fig. 7A demonstrates tumor volume reduction with compound 9 monotherapy as well as combination therapy with an anti-PD-LT antibody.

Fig. 7B-7E show separate repeats of this measurement for each dosage. In fig. 7B shows the carrier. In fig. 7C shows an anti-PD-Ll antibody. In fig. 7D shows connection 9. FIG. 7E shows compound 9+anti-PD-L1.

Fig. 8A shows tumor volume reduction with compound 9 monotherapy as well as combination therapy with oxaliplatin.

Fig. 8B-8E show separate repeats of this measurement for each dosage. In fig. 8B shows the carrier. In fig. 8C represents oxaliplatin. In fig. 8D shows connection 9. FIG. 8E shows compound 9+oxaliplatin.

In fig. 9A demonstrates a reduction in tumor volume with compound 9 monotherapy as well as with combination therapy with doxorubicin.

Fig. 9B-9E show separate repeats of this measurement for each dosage. In fig. 9B shows the carrier. In fig. 9C represents doxorubicin. In fig. 9D shows connection 9. FIG. 9E shows compound 9+doxorubicin.

In fig. 10A demonstrates sub-nanomolar inhibition of serum CD73 activity in head and neck squamous cell carcinoma (HNSCC) with compound 9.

Fig. 10B demonstrates sub-nanomolar inhibition of serum CD73 activity in ovarian cancer by compound 9.

Fig. 10C demonstrates sub-nanomolar inhibition of serum CD73 activity in triple negative breast cancer (TNBC) with compound 9.

Fig. 10D demonstrates sub-nanomolar inhibition of serum CD73 activity in esophageal cancer by compound 9.

Fig. 11 shows normalized levels of CD73 mRNA expression in tumor and normal tissues.

Fig. 12A shows tumor volume reduction with compound 9 monotherapy as well as

- 5 046381 combination therapy with docetaxel in the EG7 tumor model.

Fig. 12B-12E show individual repeats of tumor volume reduction.

Fig. 12B shows the media. In fig. 12C shows compound 9. FIG. 12D shows docetaxel. In fig. 12E shows compound 9+docetaxel.

Fig. 13A demonstrates that compound 9 reduces the growth of established EG7 tumors.

Fig. 13B-13D show individual repeats of tumor volume reduction.

In fig. 13B represents the carrier. Fig. 13C shows dosing of compound 9 started on day 1. FIG. 13D shows dosing of compound 9 started on day 6.

Detailed Description of the Invention

Definitions.

Unless otherwise specified, all technical and scientific terms used herein have the meanings commonly understood by one skilled in the art as understood herein. The following references provide one skilled in the art with a general definition of many of the terms used in this specification: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings assigned to them below unless otherwise specified.

In some embodiments, chemical structures are presented along with the corresponding chemical name. In case of discrepancies, the chemical structure rather than the name shall prevail.

In this specification, the terms contains, containing, including and having, and the like may have the meaning ascribed to them in US Patent Law and may mean includes, including, and the like; the expressions essentially consisting of or consisting essentially in a like manner have the meanings set forth in the United States Patent Law, and the term is open-ended to permit the presence of more than what is stated if the essential or new characteristics of what is stated do not substantially change when the presence of more than what is specified, but excludes embodiments of the prior art.

Unless specifically stated or obvious from the context, as used herein, the term or is understood to include. Unless specifically stated or obvious from the context as used herein, the singular forms are understood to include both the singular and the plural.

The term acyl is a common one in the art and refers to the group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.

The term acylamino is a common one in the art and refers to an amino group substituted by an acyl group and can be represented, for example, by the formula hydroyur6ylC(O^H-.

The term acyloxy is a common one in the art and refers to the group represented by the general formula hydroyur6ylC(O)O-, preferably alkylC(O)O-.

The term alkoxy refers to an alkyl group, preferably a lower alkyl group, having an oxygen atom attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, t-butoxy and the like.

The term alkoxyalkyl refers to an alkyl group substituted with an alkoxy group and can be represented by the general formula alkyl-O-alkyl.

The term alkenyl, as used herein, refers to an aliphatic group containing at least one double bond, and is intended to include both unsubstituted alkenyls and substituted alkenyls, the latter of which refers to alkenyl moieties having substituents replacing hydrogen on one or more carbon atoms of the alkenyl group. Such substituents may be on one or more carbon atoms that are included or not included in one or more double bonds. Moreover, such substituents include all those provided for alkyl groups, as discussed below, except where stability does not permit. For example, replacement of an alkenyl group with one or more alkyl, carbocyclyl, aryl, heterocyclyl or heteroaryl groups is contemplated.

An alkyl group or alkane is a straight or branched non-aromatic hydrocarbon that is fully saturated. Typically, a straight or branched chain alkyl group has from 1 to 20 carbon atoms, preferably from 1 to 10, unless otherwise specified. Examples of straight and branched alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, pentyl and octyl. A C1-C6 straight or branched chain alkyl group is also referred to as a lower alkyl group.

Moreover, the term alkyl (or lower alkyl), as used throughout the specification, examples and claims, is intended to include both unsubstituted alkyls and substituted alkyls, the latter of which refers to alkyl moieties having substituents replacing hydrogen on one or more carbon atoms of the hydrocarbon skeleton. Such substitutes, if not

- 6 046381 otherwise stated may include, for example, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl or acyl), thiocarbonyl (such as thioester, thioacetate or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety. Those skilled in the art will appreciate that the substituent moieties on the hydrocarbon chain may themselves be substituted if desired. For example, substituted alkyl substituents may include substituted and unsubstituted forms of amino, azido, imino, amido, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate) and silyl groups, as well as ethers, alkylthio, carbonyls (including ketones, aldehydes, carboxylates and esters), -CF3, -CN and the like. Examples of substituted alkyls are described below. Cycloalkyls may be further substituted with alkyls, alkenyls, alkoxy, alkylthio, aminoalkyls, carbonyl-substituted alkyls, -CF3, -CN and the like.

Term _Cx . _y , when used in combination with a chemical moiety such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, is intended to include groups that contain x to y carbon atoms in the chain. For example, the term C _x-y alkyl refers to substituted or unsubstituted saturated hydrocarbon groups, including straight chain alkyl and branched chain alkyl, that contain x to y carbon atoms in the chain, including haloalkyl groups such as trifluoromethyl and 2,2 ,2-trifluoroethyl, etc. C0 alkyl represents hydrogen, where the group is in the terminal position, the bond is internal. The terms C2 _- alkenyl and C2 _- alkynyl refer to substituted or unsubstituted unsaturated aliphatic groups similar in length and possible substitution on alkyls described above, but which contain at least one double or triple bond, respectively.

The term alkylamino, as used herein, refers to an amino group substituted with at least one alkyl group.

The term alkylthio, as used herein, refers to a thiol group substituted by an alkyl group, and can be represented by the general formula alkyl S-.

The term alkynyl, as used herein, refers to an aliphatic group containing at least one triple bond, and is intended to include both unsubstituted alkynyls and substituted alkynyls, the latter of which refers to alkynyl moieties having substituents replacing hydrogen on one or more carbon atoms of the alkynyl group. Such substituents may be on one or more carbon atoms that are included or not included in one or more triple bonds. Moreover, such substituents include all those provided for alkyl groups, as discussed above, except where stability does not permit. For example, replacement of an alkenyl group with one or more alkyl, carbocyclyl, aryl, heterocyclyl or heteroaryl groups is contemplated.

The term amide, as used herein, refers to the group

II R ³ ® xL,/ ''rM where each R ³⁰ independently represents hydrogen or a hydrocarbyl group, or two R ³⁰ taken together with the N atom to which they are attached form a heterocycle containing from 4 to 8 atoms in the ring structure .

The terms amine and amino are known in the art and refer to both unsubstituted and substituted amines and their salts, for example, the group that may be represented by

where each R ³¹ independently represents hydrogen or a hydrocarbyl group, or two R ³¹ taken together with the N atom to which they are attached form a heterocycle containing from 4 to 8 atoms in a ring structure. The term aminoalkyl, as used herein, refers to an alkyl group substituted with an amino group.

The term aralkyl, as used herein, refers to an alkyl group substituted with an aryl group.

The term aryl as used herein includes substituted or unsubstituted single ring aromatic groups in which each ring atom is a carbon. Preferably, the ring is a 5-7 membered ring, more preferably a 6 membered ring. The term aryl also includes polycyclic ring systems having two or more cyclic rings in which two or more carbon atoms are shared by two adjacent rings.

- 7 046381 rings, where at least one of the rings is aromatic, for example, the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls and/or heterocyclyls. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline and the like.

The term carbamate is common in the art and belongs to the group

wherein R ³² and R ³³ independently represent hydrogen or a hydrocarbyl group such as an alkyl group, or R ³² and R ³³ taken together with the intermediate atom(s) form a heterocycle containing from 4 to 8 atoms in a ring structure.

The terms carbocycle and carbocyclic, as used herein, refer to a saturated or unsaturated ring in which each ring atom is a carbon. The term carbocycle includes both aromatic carbocycles and non-aromatic carbocycles. Non-aromatic carbocycles include both cycloalkane rings, in which all carbon atoms are saturated, and cycloalkene rings, which contain at least one double bond.

The term carbocycle includes 5-7 membered monocyclic and 8-12 membered bicyclic rings. Each ring of the bicyclic carbocycle can be selected from saturated, unsaturated and aromatic rings. A carbocycle includes bicyclic molecules in which one, two, or three or more atoms are common to two rings. The term fused carbocycle refers to a bicyclic carbocycle in which each of the rings shares two adjacent atoms with the other ring. Each fused carbocycle ring can be selected from saturated, unsaturated and aromatic rings. In an exemplary embodiment, an aromatic ring, such as phenyl, may be fused to a saturated or unsaturated ring, such as cyclohexane, cyclopentane, or cyclohexene. Any combination of saturated, unsaturated and aromatic bicyclic rings, as far as valency allows, is included in the definition of carbocyclic. Examples of carbocycles include cyclopentane, cyclohexane, bicyclo[2,2,1]heptane, 1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4,2,0]oct-3-ene, naphthalene and adamantane . Examples of fused carbocycles include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4,2,0]octane, 4,5,6,7-tetrahydro-N-indene and bicyclo[4,1,0] hept-3-ene. Carbocycles may be substituted at any or more positions capable of bearing a hydrogen atom.

A cycloalkyl group is a cyclic hydrocarbon that is fully saturated. Cycloalkyl includes monocyclic and bicyclic rings. Typically, a monocyclic cycloalkyl group contains from 3 to about 10 carbon atoms, more typically from 3 to 8 carbon atoms, unless otherwise noted. The second bicyclic cycloalkyl ring may be selected from saturated, unsaturated and aromatic rings. Cycloalkyl includes bicyclic molecules in which one, two or three or more atoms are common to two rings. The term fused cycloalkyl refers to a bicyclic cycloalkyl in which each of the rings shares two adjacent atoms with the other ring. The second ring of the fused bicyclic cycloalkyl may be selected from saturated, unsaturated and aromatic rings. A cycloalkenyl group is a cyclic hydrocarbon containing one or more double bonds.

The term carbocyclylalkyl, as used herein, refers to an alkyl group substituted with a carbocyclic group.

The term carbonate is a common one in the art and refers to the group -OCO2-R ³⁴ , where R ³⁴ represents a hydrocarbyl group.

The term carboxy, as used herein, refers to the group represented by the formula -CO2H.

The term ester, as used herein, refers to the group -C(O)OR ³⁵ where R ³⁵ represents a hydrocarbyl group.

The term ether, as used herein, refers to a hydrocarbyl group connected via an oxygen atom to another hydrocarbyl group. Accordingly, the ether substituent of the hydrocarbyl group may be hydrocarbyl-O-. Ethers can be either symmetrical or unsymmetrical. Examples of ethers include, but are not limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethers include alkoxyalkyl groups, which can be represented by the general formula alkyl-O-alkyl.

The terms halo and halogen as used herein mean halogen and include chlorine, fluorine, bromine and iodine.

The terms hetaralkyl and heteroaralkyl, as used herein, refer to an alkyl group substituted with a hetaryl group.

The term heteroalkyl, as used herein, refers to a saturated or unsaturated chain of carbon atoms and at least one heteroatom, where the two heteroatoms are not adjacent.

The terms heteroaryl and hetaryl include substituted or unsubstituted aromatic ones

- 8 046381 ring structures, preferably 5-7 membered rings, more preferably 5-6 membered rings, where the ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms . The terms heteroaryl and hetaryl also include polycyclic ring systems containing two or more cyclic rings in which two or more carbon atoms are common to two adjacent rings, where at least one of the rings is heteroaromatic, for example, the other cyclic rings may be cycloalkyls , cycloalkenyls, cycloalkynyls, aryls, heteroaryls and/or heterocyclyls. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine and the like.

The term heteroatom, as used herein, means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen and sulfur.

The terms heterocyclyl, heterocycle and heterocyclic refer to substituted or unsubstituted non-aromatic ring structures, preferably 3-10 membered rings, more preferably 3-7 membered rings, where the ring structures include at least one heteroatom, preferably one to four a heteroatom, more preferably one or two heteroatoms. The terms heterocyclyl and heterocyclic also include polycyclic ring systems containing two or more cyclic rings in which two or more carbon atoms are common to two adjacent rings, where at least one of the rings is heterocyclic, for example, the other cyclic rings may be cycloalkyls , cycloalkenyls, cycloalkynyls, aryls, heteroaryls and/or heterocyclyls. Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams and the like.

The term heterocyclylalkyl, as used herein, refers to an alkyl group substituted with a heterocyclic group.

The term hydrocarbyl, as used herein, refers to a group that is bonded through a carbon atom that does not have an =O or =S substituent, and typically contains at least one carbon-hydrogen bond and predominantly a carbon skeleton, but, optionally, may include heteroatoms. Thus, groups like methyl, ethoxyethyl, 2-pyridyl and trifluoromethyl are considered hydrocarbyl for the purposes of this application, but not substituents such as acetyl (which has an =O substituent on the linking carbon) and ethoxy (which is attached via an oxygen atom , not a carbon atom). Hydrocarbyl groups include, but are not limited to, aryl, heteroaryl, carbocycle, heterocyclyl, alkyl, alkenyl, alkynyl, and combinations thereof.

The term hydroxyalkyl, as used herein, refers to an alkyl group substituted with a hydroxy group.

The term lower, when used in combination with a chemical moiety such as acyl, acyloxy, alkyl, alkenyl, alkynyl or alkoxy, is intended to include groups where the substituent has ten or fewer non-hydrogen atoms, preferably six or fewer. Lower alkyl, for example, refers to an alkyl group that contains ten or fewer carbon atoms, preferably six or fewer. In some embodiments, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents as defined herein are, respectively, lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, whether present themselves alone or in combination with other substituents, such as hydroxyalkyl and aralkyl in the specifications (in this case, for example, the atoms in the aryl group are not counted when counting the carbon atoms in the alkyl substituent).

The terms polycyclyl, polycycle, and polycyclic refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls) in which two or more atoms are shared by two adjacent rings, e.g., the rings are fused rings . Each of the rings of the polycycle may be substituted or unsubstituted. In some embodiments, each ring of the polycycle contains from 3 to 10 ring atoms, preferably from 5 to 7.

The term silyl refers to a silicon group with three hydrocarbyl moieties attached to it.

The term substituted refers to moieties having substituents replacing hydrogen on one or more backbone carbon atoms. It is understood that substitution or substituted includes the implicit condition that such substitution corresponds to the permissible valency of the substituted atom and substituent, and that the substitution results in a stable compound, for example, that does not spontaneously undergo transformation such as rearrangement, cyclization, elimination, etc. As used herein, the term substituted is intended to include all acceptable substituents on organic compounds. In broad terms, acceptable substituents include acyclic and cyclic, branched and straight-chain, carbocyclic and heterocyclic, aromatic and non-aromatic substituents, and organic compounds. Acceptable substituents may be one or more and may be the same or different for the respective organic compounds.

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For purposes of this invention, heteroatoms, such as nitrogen, may have hydrogen substituents and/or any permissible organic substituents described herein that satisfy the valency of the heteroatoms. Substituents may include any of the substituents described herein, for example, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl or acyl), thiocarbonyl (such as thioether, thioacetate or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety. Those skilled in the art will appreciate that the substituents themselves can be substituted if desired. Unless specifically stated to be unsubstituted, references to chemical moieties in this specification are understood to include substituted variants. For example, reference to an aryl group or moiety implicitly includes both substituted and unsubstituted variants.

The term sulfate is known in the art and refers to the group -OSO3H or a pharmaceutically acceptable salt thereof.

The term sulfonamide is known in the art and refers to the group represented by the general formulas

where R ³⁶ and R ³⁷ independently represent hydrogen or hydrocarbyl such as alkyl, or R ³⁶ and R ³⁷ taken together with the intermediate atom(s) form a heterocycle containing from 4 to 8 atoms in the ring structure.

The term sulfoxide is a common one in the art and refers to the group -S(O)-R ³⁸ where R ³⁸ is hydrocarbyl.

The term sulfonate is accepted in the art and refers to the group -SO ₃ H or a pharmaceutically acceptable salt thereof.

The term sulfone is common in the art and refers to the group -S(O) ₂ -R ³⁹ , where R ³⁹ represents hydrocarbyl.

The term thioalkyl, as used herein, refers to an alkyl group substituted with a thiol group.

The term thioester, as used herein, refers to the group C(O)SR ⁴⁰ or -SC(O)R ⁴⁰ , where R ¹⁰ represents hydrocarbyl.

The term thioether, as used herein, is equivalent to an ether where the oxygen atom is replaced by a sulfur atom.

The term urea is common in the art and can be represented by the general formula

where R ⁴¹ and R ⁴² independently represent hydrogen or hydrocarbyl such as alkyl, or any presence of R ⁴¹ taken together with R ⁴² and intermediate atom(s) forms a heterocycle containing from 4 to 8 atoms in the ring structure.

The term protecting group refers to a group of atoms which, when attached to a reactive functional group in a molecule, masks, reduces or prevents the reactivity of the functional group. Typically, the protecting group can be selectively removed as desired during the synthesis. Examples of protecting groups can be found in the reviews of Greene and Wuts, Protective Groups in Organic Chemistry, ^3rd Ed., 1999, John Wiley & Sons, NY and Harrison et al., Compendium of Synthetic Organic Methods, Vols. 1-8, 1971-1996, John Wiley & Sons, NY. Representative nitrogen protecting groups include, but are not limited to, formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl (CBZ), tert-butoxycarbonyl (Boc), trimethylsilyl (TMS), 2-trimethylsilyl-ethanesulfonyl (TES), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (FMOC), nitro-veratriloxycarbonyl (NVOC) and the like. Representative hydroxyl protecting groups include, but are not limited to, those where the hydroxyl group is either acylated (esterified) or alkylated, such as benzyl and trityl ethers, as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers (e.g., TMS or TIPS groups), glycol ethers such as ethylene glycol and propylene glycol derivatives and allyl ethers.

In some embodiments, the compounds of the present invention may be racemic. In some embodiments, the compounds of the present invention may be enriched in one enantiomer. For example, a compound of the invention may have an ee. more than

- 10 046381 approximately 30% e.i., approximately 40% e.i., approximately 50% e.i., approximately 60% e.i., approximately 70% e.i., approximately 80% e.i. , approximately 90% e.i. or even approximately 95% e.i. or more. In some embodiments, the compounds of the invention may have more than one stereocenter. In some such embodiments, the compounds of the invention may be enriched in one or more diastereomers. For example, a compound of the invention may have a di of greater than about 30% d.i., about 40% d.i., about 50% d.i., about 60% d.i., about 70% d.i. approximately 80% d.i., approximately 90% d.i. or even approximately 95% d.i. or more.

In some embodiments, the therapeutic drug may be fortified to provide predominantly one enantiomer of a compound (eg, Formula (I)). An enantiomerically enriched mixture may contain, for example, at least about 60 mole percent of one enantiomer, or more preferably at least about 75, about 90, about 95, or even about 99 mole percent. In some embodiments, a compound enriched in one enantiomer is substantially free of the other enantiomer, where essentially free means that the substance in question is less than about 10%, or less than about 5%, or less than about 4%, or less less than about 3%, or less than about 2%, or less than about 1% compared to the amount of the other enantiomer, for example, in a composition or mixture of compounds. For example, if a composition or mixture of compounds contains about 98 g of the first enantiomer and about 2 g of the second enantiomer, it can be said to contain about 98 mole percent of the first enantiomer and only about 2% of the second enantiomer.

In some embodiments, the therapeutic drug may be fortified to provide a predominant content of one diastereomer of a compound (eg, Formula (I)). A diastereomerically enriched mixture may contain, for example, at least about 60 mole percent of one diastereomer, or, more preferably, at least about 75, about 90, about 95, or even about 99 mole percent.

The term subject to which administration is applied includes, but is not limited to, humans (i.e., men or women of any age group, e.g., pediatric subject (e.g., infants, children, adolescents) or adults (e.g., young adult, middle-aged adult or elderly)) and/or other primates (eg, cynomolgus monkeys, rhesus monkeys); mammals, including commercially important mammals such as cattle, pigs, horses, sheep, goats, cats and/or dogs; and/or birds, including commercial birds such as chickens, ducks, geese, quail and/or turkeys. The preferred subjects are humans.

As used herein, a therapeutic agent that prevents a disorder or condition refers to a compound that, in a statistical sample, results in a reduction in the occurrence of the disorder or condition in a treated sample relative to an untreated control sample, or delays the occurrence of, or reduces the severity of one or more more symptoms of a disorder or condition relative to an untreated control sample.

The term treatment includes prophylactic and/or therapeutic treatment. The term prophylactic or therapeutic treatment is known in the art and includes administering to a subject one or more of the described compositions. When administered before the clinical manifestation of an undesirable condition (e.g., a disease or other undesirable condition of a subject), the treatment is prophylactic (i.e., protects the subject from developing the undesirable condition), and when administered after the manifestation of an undesirable condition, the treatment is therapeutic (i.e., intended to reduce, alleviate, or stabilization of an existing undesirable condition or its side effects).

The term prodrug is intended to cover compounds that, under physiological conditions, are converted to therapeutically active agents of the present invention (eg, a compound of formula I). A general method for preparing a prodrug is to include one or more selected moieties that are hydrolyzed under physiological conditions to release the desired molecule. In other embodiments, the prodrug is converted by enzymatic activity in a subject. For example, esters or carbonates (eg, esters or carbonates of alcohols or carboxylic acids) are preferred prodrugs of the present invention. In some embodiments, some or all of the compounds of formula (I) in the composition presented above may be replaced by a corresponding suitable prodrug, for example, where the hydroxyl in the parent compound is an ester of either a carbonate or a carboxylic acid.

An effective amount, as used herein, refers to an amount that is sufficient to achieve the desired biological effect. A therapeutically effective amount, as used herein, refers to an amount that is sufficient to achieve the desired therapeutic effect. For example, a therapeutically effective amount may refer to an amount that is sufficient to improve at least one sign or symptom of the cancer.

- 11 046381

Response to a treatment may include, but is not limited to, a decrease or amelioration of negative symptoms, a decrease in the progression of the disease or its symptoms, an increase in favorable symptoms or clinical outcomes, a decrease in side effects, stabilization of the disease, or partial or complete cure of the disease.

In some embodiments, the present invention provides a compound of formula (I):

or pharmaceutically acceptable salts and/or prodrugs thereof, wherein Y is

Het represents heterocyclyl or heteroaryl;

R ^1a is selected from halogen, hydroxy, cyano, azido, amino, amido, C _1-6 alkyl, hydroxyC _1-6 alkyl, amino^^alkyl, -OC(O)-OC _1-6 αlkyl, C _1-6 acyloxy , C _1- ^kokcu, ^^alkenyl, and ^^alkynyl;

R ^1b is selected from H, halogen, C _1-6 αlkyl, hydroxy^alkyl, amino^alkyl, A^alkenyl and A^alkynyl;

R2 ^a is selected from halogen, hydroxy, cyano, azido, amino, Aralkyl, hydroxy-C _1-6 αlkyl, amino-A^alkyl, A^acyloxy, -O—C(O)—O—C ₁ - ₆ αlkyl, C ₁ - ₆ αlkoxy, ^^alkenyl and ^^alkynyl;

R2 ^b is selected from halogen, C ₁ - ₆ alkyl, A^alkenyl and ^^alkynyl, preferably substituted or unsubstituted C ₂ alkynyl, most preferably unsubstituted C ₂ alkynyl;

R ³ is selected from H and alkyl;

R ⁴ is selected from H, alkyl, CN, aryl, heteroaryl, -C(O)OR ⁹ , -C(O)NR ¹¹ R ¹² , -S(O)2R ¹⁰ , -P(O)(OR ¹¹ )( OR ¹² ) and -P(O)(OR ¹¹ )(NR ¹³ R ¹⁴ );

R ⁵ is selected from H, cyano, alkyl, cycloalkylalkyl, heterocyclylalkyl, aralkyl, heteroaralkyl and -C(O)OR ⁹ ;

R ⁶ is selected from -C(O)OR ⁹ , -C(O)NR ¹⁶ R ¹⁷ and -P(O)(OR ¹¹ )(OR ¹² );

R ⁹ is independently selected from H, alkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, aryl, aralkyl, heteroaryl and heteroaralkyl;

R ¹⁰ is independently selected from alkyl, alkenyl, alkynyl, amino, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, aryl, aralkyl, heteroaryl and heteroaralkyl; and each of R ¹¹ and R ¹² is independently selected from H, alkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl; or

R ¹¹ and R ¹² together with the carbon atom to which they are attached form a 5-7 membered heterocyclyl; And

R ¹³ represents H or alkyl;

R ¹⁴ represents alkyl or aralkyl;

each R ¹⁵ is independently selected from hydroxy, alkoxyacyloxy and NR ¹³ R ¹⁴ ;

each R ¹⁶ and R ¹⁷ is independently selected from H, hydroxy, alkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl; or

R ¹⁶ and R ¹⁷ together with the nitrogen atom to which they are attached form a 5-7 membered heterocyclyl.

- 12 046381

In certain preferred embodiments of formula (I), the included compounds meet conditions a) and b); or a) and c); Where:

a) the connection is not

b) if R ⁴ and R ⁶ are each -C(O)OH and R ⁵ is benzyl substituted on the phenyl ring with a heterocyclyl or heteroaryl substituent, then the phenyl ring substituent is selected from unsubstituted or substituted pyrrolidinyl, piperazinonyl, piperidonyl, tetrahydropyrimidonyl , pyridonyl and pyridyl; And

c) if R ⁴ is -C(O)OH or tetrazolyl, R ⁶ is -C(O)OH, and R ⁵ is benzyl substituted on the phenyl ring by a second phenyl ring, then either a benzylphenyl ring or a second phenyl ring the ring is substituted with -C(O)OR ⁹ where R ⁹ represents H or alkyl.

- 13 046381

In some embodiments, the present invention provides a compound of formula (II):

(P) or pharmaceutically acceptable salts and/or prodrugs thereof, where

Het represents heterocyclyl or heteroaryl;

R ^1a is selected from halogen, hydroxy, cyano, azido, amino, amido, C _1-6 alkyl, hydroxyC _1-6 alkyl, amino-C _1-6 alkyl, -OC(O)-OC _1-6 alkyl, C _{1 -6} acyloxy, C _1-6 alkoxy, C _2-6 alkenyl, and C _2-6 alkynyl;

R ^1b is selected from H, halogen, C _1-6 alkyl, hydroxy-C _1-6 alkyl, amino-C _1-6 alkyl, C _2-6 alkenyl and C _2-6 alkynyl;

R2 ^a is selected from halogen, hydroxy, cyano, azido, amino, C _1-6 alkyl, hydroxy-C _1-6 alkyl, amino-C _1-6 alkyl, C _1-6 acyloxy, -OC(O)-OC _{1 -6} alkyl, C _1-6 alkoxy, C _2-6 alkenyl and C _2-6 alkynyl;

R2 ^b is selected from H, halogen, C _1-6 alkyl, C _2-6 alkenyl and C _2-6 alkynyl;

R ³ is selected from H and alkyl;

R ⁴ is selected from alkyl, aryl, heteroaryl, -C(O)OR ⁹ , -C(O)NR ¹¹ R ¹² , -S(O)2R ¹⁰ , -P(O)(OR ¹¹ )(OR ¹² ) and -P(O)(OR ¹¹ )(NR ¹³ R ¹⁴ );

R ⁵ is selected from H, cyano, alkyl, cycloalkylalkyl, heterocyclylalkyl, aralkyl, heteroaralkyl and -C(O)OR ⁹ ;

R ⁶ is selected from -C(O)OR ⁹ , -C(O)NR ¹¹ R ¹² and -P(O)(OR ¹¹ )(OR ¹² );

R ⁹ is independently selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, aryl, aralkyl, heteroaryl and heteroaralkyl;

R ¹⁰ is independently selected from alkyl, alkenyl, alkynyl, amino, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, aryl, aralkyl, heteroaryl and heteroaralkyl; and each of R ¹¹ and R ¹² is independently selected from H, alkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl; or

R ¹¹ and R ¹² together with the carbon atom to which they are attached form a 5-7 membered heterocyclyl; And

R ¹³ represents H or alkyl; And

R ¹⁴ represents alkyl or aralkyl; provided that a), b) and c) or a), b) and d);

a) the connection is not

- 14 046381

ci or

Cl

b) R ^2b is selected from halogen, C _2-6 alkyl, C _2-6 alkenyl and C _2-6 alkynyl, preferably substituted or unsubstituted C ₂ alkynyl, most preferably unsubstituted C ₂ alkynyl;

c) if R ⁴ and R ⁶ are each -C(O)OH and R ⁵ is benzyl substituted on the phenyl ring with a heterocyclyl or heteroaryl substituent, then the phenyl ring substituent is selected from unsubstituted or substituted piperidonyl, tetrahydropyrimidonyl, pyridonyl and pyridyl ; And

d) if R ⁴ is -C(O)OH or tetrazolyl, R ⁶ is -C(O)OH, and R ⁵ is benzyl substituted on the phenyl ring by a second phenyl ring, then either a benzylphenyl ring or a second phenyl the ring is substituted with -C(O)OR ⁹ where R ⁹ represents H or alkyl.

- 15 046381

In certain preferred embodiments: a) the connection is not

; ^and , or

N

C.I.

b) R ^2b is selected from halogen, C _2-6 alkyl, C _2-6 alkenyl and C _2-6 alkynyl, preferably substituted or unsubstituted C ₂ alkynyl, most preferably unsubstituted C ₂ alkynyl; and either

c) R ⁵ is benzyl substituted on the phenyl ring with a substituent selected from unsubstituted or substituted piperidonyl, tetrahydropyrimidonyl, pyridonyl and pyridyl, or

d) R ⁵ is benzyl substituted on the phenyl ring with a second phenyl ring substituted by -C(O)OR ⁹ where R ⁹ is H or alkyl.

The following paragraphs describe various embodiments of the compounds of formula (I) or II, which may be combined in any combination according to the formulas defined above.

In certain embodiments, R ^1a is H or hydroxy. In certain embodiments, R ^1b is H or hydroxyl. In other embodiments, R ^1a is hydroxy and R ^1b is H.

In some embodiments, R ^2a is C1-6alkyl. In certain embodiments, R ^2b is C2.6 alkyl, C _2-6 alkenyl and C _2-6 alkynyl, preferably substituted or unsubstituted C ₂ alkynyl such as ethynyl. In certain preferred embodiments, R ^2a is Me and R ^2b is ethynyl. In some variants

- 16 046381 implementation R ^2a represents hydroxy and R ^2b represents ethyl or vinyl. In other preferred embodiments, R ^2a is hydroxy and R ^2b is ethynyl. In some embodiments, R ^2b is propynyl, butynyl,

either unsubstituted or substituted

In certain embodiments, R ³ is H.

In some embodiments, a compound of formula (I) has the following structure:

In some embodiments, a compound of formula (II) has the following structure:

In some embodiments, R ^1a has the α configuration. For example, a compound of formula (I) may have structure (IA):

(IA)

Also, the compound of formula (II) may have the structure (IIAa):

(II Aa)

In alternative embodiments, R ^1a has the β configuration. In some such embodiments, the compound of formula (I) has structure (IB):

rZ

(IB)

In some such embodiments, the compound of formula (II) has the structure (IIBa):

In additional embodiments of compounds of formula (I), for example, as described by you

- 17 046381 more, R ^2a represents the α configuration. For example, a compound of formula (I) may have structure (IC):

In some such embodiments, the compound of formula (II) has the structure (IICa):

In alternative embodiments, R ^2a has the β configuration. In some such embodiments, the compound of formula (I) has structure (ID):

In some such embodiments, the compound of formula (II) has the structure (IIDa):

In some preferred embodiments, the compound of formula (I) has structure (IE):

In some such embodiments, the compound of formula (II) has structure (IIEa):

In particularly preferred such embodiments, R ^1a is hydroxy, R ^2a is hydroxy, and R ^2b is selected from methyl, ethyl, vinyl and ethynyl, most preferably ethynyl. In the most preferred embodiments of the compounds of formula (IE), R ^1a is hydroxy, R ^2a is hydroxy, and R ^2b is ethynyl. In some preferred embodiments of a compound of formula (IIEa), R ^1a is

- 18 046381 hydroxy, R ^2a is hydroxy and R ^2b is ethynyl.

R ^{4 rs} -J /b.

In certain embodiments, Y is ^R

In some embodiments, R ⁴ is selected from -C(O)OR ⁹ , -C.'(O)NRR ¹² , -S(O)2R ¹⁰ , and -P(O)(OR ¹¹ )(OR ¹² ). In some embodiments, R ⁴ is -C(O)OR ⁹ and R ⁹ is H or alkyl. In other embodiments, R ⁴ is -C(O)NR ¹¹ R ¹² . In certain embodiments, R ¹¹ and R ¹² are each independently selected from H and alkyl; or R ¹¹ and R ¹² together with the nitrogen atom to which they are attached form a 5-7 membered heterocyclyl. In other embodiments, R ⁴ is -S(O)2R ¹⁰ and R ¹⁰ is alkyl or aryl.

In some embodiments, R ⁶ is -C(O)OR ⁹ and R ⁹ is H or alkyl, such as H or C _1-6 alkyl. In other embodiments, R ⁶ is -C(O)NR ¹¹ R ¹² . In certain embodiments, R ¹¹ and R ¹² are each independently selected from H and alkyl; or R ¹¹ and R ¹² together with the nitrogen atom to which they are attached form a 5-7 membered heterocyclyl.

In certain preferred embodiments, R ⁴ and R ⁶ are each -C(O)OH, most preferably R ⁵ is benzyl, for example, as discussed in more detail below.

In certain embodiments, R ⁵ is selected from H, alkyl, aralkyl, and heteroaralkyl. In certain such embodiments, each alkyl, aralkyl, and heteroaralkyl in R ⁵ is unsubstituted or substituted with one or more substituents selected from halogen, alkyl, alkoxy, carbonyl, amino, amido, cycloalkyl, heterocyclyl, and heteroaryl. In other embodiments, the alkyl, aralkyl, and heteroaralkyl substituents on R ⁵ are selected from halogen, haloalkyl, alkoxy, carbonyl, aryl, heterocyclyl, and heteroaryl. In certain embodiments, R ⁵ is aralkyl, for example, substituted on the aryl ring with a 5-7 membered heterocyclyl or 5-7 membered heteroaryl. In certain specific embodiments, R ⁵ is selected from H, methyl, ethyl, -CH2-ethynyl, and -CH2-vinyl. In other embodiments, R ⁵ is selected from benzyl, -CH2-pyridyl, _-CH2 -pyridazinyl, _-CH2 -oxazolyl, -CH2 _- thiophenyl, -CH2 _- furanyl, _-CH2 -thiazolyl, and _-CH2 -benzothiazolyl. , preferably benzyl and -CH ₂ -thiophenyl.

In certain preferred embodiments, R ⁵ is benzyl substituted on the phenyl ring (eg, at the para position) with a heterocyclyl or heteroaryl substituent, preferably wherein the phenyl ring substituent is selected from substituted piperidonyl, tetrahydropyrimidonyl, pyridonyl, and pyridyl. In some embodiments, the phenyl ring substituent is piperazinonyl. In certain such embodiments, piperidonyl, tetrahydropyrimidonyl, pyridonyl, or pyridyl is substituted with one or more of alkyl, hydroxyalkyl, or alkoxyalkyl.

In certain embodiments, R ⁵ is aralkyl or heteroaralkyl with a para substituent on the aryl or heteroaryl ring selected from heterocyclyl, heteroaryl and aryl; And

R ^2b represents methyl, ethyl or C _2-6 alkynyl.

- 19 046381

In certain preferred embodiments, R ⁵ is benzyl substituted on the phenyl ring (for example, at the 4 position)

- 20 046381

In certain embodiments, ^R5 is benzyl substituted on the phenyl ring (e.g., at the 4 position)

In some embodiments

represents

- 21 046381

- 22 046381

- 23 046381

- 24 046381

- 25 046381

O o r ¹ ® || ||

R ¹⁵ o 10

In some embodiments, Y is In certain embodiments, each R ¹⁵ is hydroxy.

In certain embodiments, Het is selected from 6-10 membered aryl, 5-8 membered heterocyclyl, 5-8 membered monocyclic, or 5-10 membered bicyclic heteroaryl and may be unsubstituted or substituted with one or more substituents selected from halogen, alkyl, haloalkyl, alkoxy, carbonyl, amino, amido, alkylthio, alkoxycarbonyl, cycloalkyl, aryl, heterocyclyl and heteroaryl. In some embodiments, Het substituents are selected from halo, haloalkyl, amino, and heterocyclyl. In certain embodiments, Het is a nitrogen-containing heterocyclyl or heteroaryl, preferably attached to the core ring via a nitrogen atom of the heterocyclyl or heteroaryl ring. In some variants

implementation Het represents

In other embodiments, Het represents

- 26 046381 by yourself

In other embodiments, Het is _R a

where Z represents CH or N;

R ^a is selected from H, halogen, hydroxy, alkyl, thiophenyl, -NR7R ⁸ , aralkyl, aryl and heteroaryl, preferably H, Cl, -NR7R ⁸ , and phenyl;

R ^b is selected from halogen, alkyl, haloalkyl, hydroxyalkyl, alkylthio, amido, carbonyl, amido and heteroaryl;

R ⁷ is selected from H, hydroxy, alkyl, aralkyl, heteroaralkyl, cycloalkyl and heterocyclyl and

R ⁸ represents H or alkyl or

R ⁷ and R ⁸ , together with the nitrogen atom to which they are attached, form a 4-7 membered heterocyclyl ring.

In some embodiments, Het is

In certain embodiments, Z is CH. In other embodiments, Z is N.

In certain embodiments, R ^a is selected from H, halogen, alkyl, thienyl, -NR7R ⁸ , aryl and heteroaryl, preferably H, Cl, -NR ⁷ R ⁸ , and phenyl; In some embodiments, R ^a is -NR ⁷ R ⁸ .

In certain embodiments, R ^b is selected from halogen, alkyl, hydroxyalkyl, haloalkyl, amido, carbonyl, amido, and heteroaryl. In some embodiments, R ^b is selected from Cl, -CF3, carbonyl, and -CONH2.

In some embodiments, Het is

In some embodiments, R ⁷ is selected from H, alkyl, aralkyl, heteroaralkyl, cycloalkyl, and heterocyclyl. In certain embodiments, R ⁷ is alkyl or cycloalkyl, for example when the alkyl or cycloalkyl is unsubstituted or substituted with one or more substituents selected from hydroxy, alkoxy, aryl, amino and cycloalkyl. In other embodiments, R ⁷ is aralkyl or heteroaralkyl, for example where the aralkyl or heteroaralkyl is unsubstituted or substituted with halogen or alkyl.

In some embodiments, R ⁸ is selected from H, methyl, and ethyl.

In other embodiments, R ⁷ and R ⁸ , together with the nitrogen atom to which they are attached, form a heterocyclyl ring, for example, selected from azetidinyl, morpholino, pyrrolidinyl and azepanyl.

Methods of application.

Provided herein are methods of inhibiting CD73 in a cell, comprising contacting the cell with a compound of the invention, such as a compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, the cell is contacted in a subject in need thereof, thereby treating the adenosine-mediated disease or disorder.

Also described herein are methods of treating a disease or disorder mediated by adenosine, comprising administering a compound of the invention, such as a compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, methods of treating cancer are described herein comprising administering a compound of this invention, such as a compound of formula (I), or a pharmaceutically acceptable salt thereof.

Adenosine acts on a variety of immune cells to cause immunosuppression, and the immunosuppressive effects of ectonucleotidases, which increase adenosine levels, are also associated with increased infection of mammalian cells by parasites, fungi, bacteria and viruses. In addition to its immunosuppressive effects, adenosine also plays a role in modulating the cardiovascular system (as a vasodilator and cardiac depressant), the central nervous system (CNS) (causing sedative, anxiolytic and antiepileptic effects), the respiratory system (causing bronchoconstriction), the kidneys (having biphasic action; causing vasoconstriction at low concentrations and vasodilation at high doses), fat cells (inhibiting lipolysis) and platelets (as an antiplatelet agent). In addition, adenosine also promotes fibrosis (excess matrix production) in various tissues. Therefore, improved treatments targeting CD73 will provide drugs to treat a wide range of conditions beyond malignancy, including cerebral and cardiac ischemic disease, fibrosis, immune and inflammatory disorders (eg, inflammatory bowel disease), neurological, neurodegenerative and CNS disorders, and diseases (eg depression, Parkinson's disease) and sleep disorders.

In some embodiments, the adenosine-mediated disease or disorder is selected from cerebral ischemic disease, malignancy, coronary artery disease, depression, fibrosis, immune disorder, inflammatory disorder (eg, inflammatory bowel disorder), neurological disorder or disease, neurodegenerative disorder, or diseases (eg Parkinson's disease), disorders and diseases of the central nervous system and sleep disorders.

The methods described herein can be used to treat a wide range of malignancies, including bladder cancer, bone cancer, brain cancer (including glioblastoma), breast cancer, heart cancer, cervical cancer, colon cancer, colorectal cancer, cancer esophageal, fibrosarcoma, stomach cancer, gastrointestinal cancer, head and neck cancer, Kaposi's sarcoma, kidney cancer (including renal cell adenocarcinoma), leukemia, liver cancer, lung cancer (including non-small cell lung cancer, small cell lung cancer and mucoepidermoid cancer lung), lymphoma, melanoma, myeloma, ovarian cancer (including ovarian adenocarcinoma), pancreatic cancer, penile cancer, prostate cancer, testicular germ cell cancer, thymoma and thymic carcinoma.

In some embodiments, the subject suffers from a malignancy selected from breast cancer, brain cancer, colon cancer, fibrosarcoma, kidney cancer, lung cancer, melanoma, ovarian cancer, and prostate cancer. In some embodiments, the subject suffers from a malignancy selected from breast cancer, colon cancer, fibrosarcoma, melanoma, ovarian cancer, and prostate cancer. In other embodiments, the subject suffers from a malignancy selected from brain cancer, breast cancer, kidney cancer, lung cancer, melanoma, and ovarian cancer. In some embodiments, the subject has squamous cell carcinoma of the head and neck region, ovarian cancer, breast cancer, or esophageal cancer. In other embodiments, the subject suffers from pancreatic cancer, esophageal cancer, stomach cancer, head and neck cancer, colon cancer, lung cancer, or kidney cancer. In still other embodiments, the subject suffers from breast cancer. In some embodiments, the breast cancer is adenocarcinoma of the breast. In some embodiments, the breast cancer is triple negative breast cancer.

In some embodiments, methods of treating or preventing cancer may be demonstrated by one or more responses such as increased apoptosis, inhibition of tumor growth, decreased tumor metastasis, inhibition of tumor metastasis, decreased microvascular density, decreased neovascularization, inhibition of tumor migration, tumor regression, and increase subject's survival.

In some embodiments, the adenosine-mediated disease or disorder is a disease or disorder mediated by CD73 activity. In some embodiments, the compounds of the invention, such as compounds of formula (I), can be used as CD73 inhibitors.

In some embodiments, the methods described herein treat or prevent cardiovascular disease using CD73 inhibitors. Mu

- 28 046381 tan genes encoding CD73 lead to extensive calcification of lower extremity arteries and small joint capsules, which is associated with an increased risk of cardiovascular disease (Hilaire et al., N. Engl. J. Med, 364(5): 432 -442, 2011).

In some embodiments, the methods described herein treat or prevent cancer using CD73 inhibitors. CD73 small interfering RNA and anti-CD73 monoclonal antibodies have shown significant effects in the treatment or prevention of cancer (Antonioli et al., Nat. Rev. Cancer, 13: 842-857, 2013). There is a strong correlation between CD73 expression and the ability of cancer cells to migrate, invade and adhere to the extracellular matrix (ECM) (Antonioli 2013; Antonioli et al., Trends Cancer, 2(2): 95-109, 2016).

In some embodiments, treatment or prevention of cancer with CD73 inhibitors may be demonstrated by one or more responses selected from tumor-specific T cell activation, clonal expansion, and homing (Antonioli 2016). In other embodiments, the methods described herein increase the number of effector T lymphocytes (eg, cytolytic effector T lymphocytes).

Combined treatment

In some embodiments, a method of treating or preventing cancer may include administering a CD39 inhibitor in conjunction with one or more other chemotherapeutic agents. In one embodiment, the CD73 inhibitor is a compound of the invention, such as a compound of formula (I). Other chemotherapeutic agents may include CD73-specific monoclonal antibodies, which enhance the effects of other antibodies and drugs due to increased overall immune system activity (lower T-regulatory function and higher T-effector function, etc.) (Antonioli 2016).

In some embodiments, a method of treating or preventing cancer may include administering a compound of the invention along with one or more other chemotherapeutic agents.

Chemotherapeutic agents that can be co-administered with the compounds of the invention include the following: 1-amino-4-phenylamino-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (acid blue 25), 1-amino-4- [4-hydroxyphenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate, 1-amino-4-[4-aminophenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate, 1- amino-4-[1-naphthylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate, 1-amino-4-[4-fluoro-2-carboxyphenylamino]-9,10dioxo-9,10- dihydroanthracene-2-sulfonate, 1-amino-4-[2-antracetylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate, ABT-263, afatinib dimaleate, axitinib, aminoglutethimide, amsacrine, anastrozole, APCP , asparaginase, AZD5363, Bacillus Calmette-Guerin (BCG) vaccine, bicalutamide, bleomycin, bortezomib, β-methylene-ADP (AOPCP), buserelin, busulfan, cabazitaxel, cabozantinib, campothecin, capecitabine, carboplatin, carfilzomib, carmustine, ceritinib, chlorambucil , chloroquine, cisplatin, cladibine, clodronate, cobimethinbin, colchicine, crizotinib, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, demethoxyviridine, dexamethasone, dichloroacetate, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, eribu lin, erlotinib, estradiol, estramustine , etoposide, everolimus, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gefitinib, gemcitabine, genistein, goserelin, GSK1120212, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, Ixa bepilon, lenalidomide, letrozole, leucovorin, leuprolide , levamisole, lomustine, lonidamine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, metformin, methotrexate, miltefosine, mitomycin, mitotane, mitoxantrone, MK-2206, mutamycin, No. (4-sulfamoylphenylcarbamothioyl)pivalamide, NF 279, NF449, nilutamide , nocodazole, octreotide, olaparib, oxaliplatin, paclitaxel, pamidronate, pazopanib, pemexetred, pentostatin, perifosine, PF-04691502, plicamycin, pomalidomide, porfimer, PPADS, procarbazine, quercetin, raltitrexed, ramucirumab, reactive blue 2, rituximab, rolofilin, romidepsin , rucaparib, selumetinib, sirolimus, sodium 2,4-dinitrobenzenesulfonate, sorafenib, streptozocin, sunitinib, suramin, talazoparib, tamoxifen, temozolomide, temsirolimus, teniposide, testosterone, thalidomide, thioguanine, thiotepa, titanocene dichloride, tonaphylline, topotecan, trametinib, trastuzumab , tretinoin, veliparib, vinblastine, vincristine, vindesine, vinorelbine and vorinostat (SAHA). In other embodiments, chemotherapeutic agents that may be administered with the compounds of the invention include: ABT-263, dexamethasone, 5-fluorouracil, PF-04691502, romidepsin, and vorinostat (SAHA). In other embodiments, chemotherapeutic agents that may be administered with the compounds of the invention include: 1-amino-4-phenylamino-9,10-dioxo-9,10dihydroanthracene-2-sulfonate (blue 25 acid), 1-amino- 4-[4-hydroxyphenyl-amino]-9,10-dioxo-9,10dihydroanthracene-2-sulfonate, 1-amino-4-[4-aminophenylamino]-9,10-dioxo-9,10-dihydroanthracene-2sulfonate, 1-amino-4-[1-naphthylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate, 1-amino-4-[4fluoro-2-carboxyphenylamino]-9,10-dioxo-9, 10-dihydroanthracene-2-sulfonate, 1-amino-4-[2anthracenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate, APCP, β-methylene-ADP (AOPCP),

- 29 046381 capecitabine, cladribine, cytarabine, fludarabine, doxorubicin, gemcitabine, N-(4sulfamoylphenylcarbamothioyl) pivalamide, NF279, NF449, PPADS, quercetin, reagent blue 2, rolofilin sodium 2,4-dinitrobenzenesulfonate, sumarin and tonapophylline.

Many combination drugs have been developed for the treatment of malignancy. In some embodiments, the compounds of the invention (eg, compounds of formula (I)) may be administered in conjunction with combination therapy. Examples of combination drugs with which the compounds of the invention can be administered are included in Table. 1.

Table 1

Example of combinatorial therapy for cancer treatment

Name Therapeutic agents ABV doxorubicin, bleomycin, vinblastine ABVD doxorubicin, bleomycin, vinblastine, dacarbazine AC (breast) doxorubicin, cyclophosphamide AS (sarcoma) doxorubicin, cisplatin AS (neuroblastoma) cyclophosphamide, doxorubicin ACE cyclophosphamide, doxorubicin, etoposide AC cyclophosphamide, doxorubicin AD doxorubicin, dacarbazine AR doxorubicin, cisplatin ARAC-DNR cytarabine, daunorubicin B-CAVe bleomycin, lomustine, doxorubicin, vinblastine BCVPP carmustine, cyclophosphamide, vinblastine, procarbazine, prednisone VEASORR bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, prednisone, filgrastim VER bleomycin, etoposide, cisplatin BIP bleomycin, cisplatin, ifosfamide, mesna VOMR bleomycin, vincristine, cisplatin, mitomycin SA cytarabine, asparaginase SAVO cisplatin, methotrexate, bleomycin, vincristine CAF cyclophosphamide, doxorubicin, fluorouracil CAL-G cyclophosphamide, daunorubicin, vincristine, prednisone, asparaginase CAMP cyclophosphamide, doxorubicin, methotrexate, procarbazine SAR cyclophosphamide, doxorubicin, cisplatin CAV cyclophosphamide, doxorubicin, vincristine CAVE ADD CAV and etoposide CA-VP16 cyclophosphamide, doxorubicin, etoposide SS cyclophosphamide, carboplatin CDDP/VP-16 cisplatin, etoposide CEF cyclophosphamide, epirubicin, fluorouracil SERR(B) cyclophosphamide, etoposide, prednisone, together or without/bleomycin CEV cyclophosphamide, etoposide, vincristine CF cisplatin, fluorouracil or carboplatin fluorouracil CHAP cyclophosphamide or cyclophosphamide, altretamine,

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doxorubicin, cisplatin ChlVPP chlorambucil, vinblastine, procarbazine, prednisone CHOP cyclophosphamide, doxorubicin, vincristine, prednisone CHOP-BLEO addition of bleomycin to CHOP CISCA cyclophosphamide, doxorubicin, cisplatin CLD-BOMP bleomycin, cisplatin, vincristine, mitomycin CMF methotrexate, fluorouracil, cyclophosphamide CMFP cyclophosphamide, methotrexate, fluorouracil, prednisone CMF VP cyclophosphamide, methotrexate, fluorouracil, vincristine, prednisone CMV cisplatin, methotrexate, vinblastine CNF cyclophosphamide, mitoxantrone, fluorouracil CNOP cyclophosphamide, mitoxantrone, vincristine, prednisone COB cisplatin, vincristine, bleomycin CODE cisplatin, vincristine, doxorubicin, etoposide COMLA cyclophosphamide, vincristine, methotrexate, leucovorin, cytarabine COMP cyclophosphamide, vincristine, methotrexate, prednisone Cooper scheme cyclophosphamide, methotrexate, fluorouracil, vincristine, prednisone COP cyclophosphamide, vincristine, prednisone COPE cyclophosphamide, vincristine, cisplatin, etoposide COPP cyclophosphamide, vincristine, procarbazine, prednisone SR (chronic lymphocytic leukemia) chlorambucil, prednisone OC (ovarian cancer) cyclophosphamide, cisplatin CVD cisplatin, vinblastine, dacarbazine CVI carboplatin, etoposide, ifosfamide, mesna With VP cyclophosphamide, vincristine, prednisone CVPP lomustine, procarbazine, prednisone CYVADIC cyclophosphamide, vincristine, doxorubicin, dacarbazine D.A. daunorubicin, cytarabine DAT daunorubicin, cytarabine, thioguanine

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DAV daunorubicin, cytarabine, etoposide DCT daunorubicin, cytarabine, thioguanine DHAP cisplatin, cytarabine, dexamethasone D.I. doxorubicin, ifosfamide DTIC/T amoxifen dacarbazine, tamoxifen DVP daunorubicin, vincristine, prednisone EAP etoposide, doxorubicin, cisplatin E.C. etoposide, carboplatin EFP etoposide, fluorouracil, cisplatin ELF etoposide, leucovorin, fluorouracil EMA 86 mitoxantrone, etoposide, cytarabine E.P. etoposide, cisplatin EVA etoposide, vinblastine FAC fluorouracil, doxorubicin, cyclophosphamide FAM fluorouracil, doxorubicin, mitomycin FAMTX methotrexate, leucovorin, doxorubicin FAP fluorouracil, doxorubicin, cisplatin F-CL fluorouracil, leucovorin FEC fluorouracil, cyclophosphamide, epirubicin FED fluorouracil, etoposide, cisplatin FL flutamide, leuprolide FZ flutamide, goserelin acetate implant HDMTX methotrexate, leucovorin Hexa-CAF altretamine, cyclophosphamide, methotrexate, fluorouracil ShMTX/6-MR methotrexate, mercaptopurine, leucovorin I.E. ifosfamide, etoposide, mesna IfoVP ifosfamide, etoposide, mesna IPA ifosfamide, cisplatin, doxorubicin M-2 vincristine, carmustine, cyclophosphamide, prednisone, melphalan MAC-III methotrexate, leucovorin, dactinomycin, cyclophosphamide MASS methotrexate, doxorubicin, cyclophosphamide, lomustine

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MACOP-B methotrexate, leucovorin, doxorubicin, cyclophosphamide, vincristine, bleomycin, prednisone MAID mesna, doxorubicin, ifosfamide, dacarbazine m-BACOD bleomycin, doxorubicin, cyclophosphamide, vincristine, dexamethasone, methotrexate, leucovorin M.B.C. methotrexate, bleomycin, cisplatin M.C. mitoxantrone, cytarabine M.F. methotrexate, fluorouracil, leucovorin MICE ifosfamide, carboplatin, etoposide, mesna MINE mesna, ifosfamide, mitoxantrone, etoposide mini BEAM carmustine, etoposide, cytarabine, melphalan MOBP bleomycin, vincristine, cisplatin, mitomycin MOP mechlorethamine, vincristine, procarbazine MOPP mechlorethamine, vincristine, procarbazine, prednisone MOPP/ABV mechlorethamine, vincristine, procarbazine, prednisone, doxorubicin, bleomycin, vinblastine MP (plural myeloma) melphalan, prednisone MP (prostate cancer) mitoxantrone, prednisone MTX/6-MO methotrexate, mercaptopurine MTX/6-MP/VP methotrexate, mercaptopurine, vincristine, prednisone MTX-CDDPAdr methotrexate, leucovorin, cisplatin, doxorubicin MV (breast cancer) mitomycin, vinblastine MV (acute myelocytic leukemia) mitoxantrone, etoposide M-UASMethotrexate vinblastine, doxorubicin, cisplatin MVP Mitomycin vinblastine, cisplatin MVPP mechlorethamine, vinblastine, procarbazine, prednisone NFL mitoxantrone, fluorouracil, leucovorin NOVP mitoxantrone, vinblastine, vincristine ORA vincristine, prednisone, doxorubicin ORRA addition of procarbazine to OPA.

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ASD cisplatin, doxorubicin PAC-I cisplatin, doxorubicin, cyclophosphamide PA-CI cisplatin, doxorubicin PCV lomustine, procarbazine, vincristine PFL cisplatin, fluorouracil, leucovorin ROS prednisone, vincristine, lomustine ProMACE prednisone, methotrexate, leucovorin, doxorubicin, cyclophosphamide, etoposide ProMACE/cytaBOM prednisone, doxorubicin, cyclophosphamide, etoposide, cytarabine, bleomycin, vincristine, methotrexate, leucovorin, co-trimoxazole PRoMACE/MOPP prednisone, doxorubicin, cyclophosphamide, etoposide, mechlorethamine, vincristine, procarbazine, methotrexate, leucovorin Pt/VM cisplatin, teniposide PVA prednisone, vincristine, asparaginase PVB cisplatin, vinblastine, bleomycin PVDA prednisone, vincristine, daunorubicin, asparaginase SMF streptozocin, mitomycin, fluorouracil TAD mechlorethamine, doxorubicin, vinblastine, vincristine, bleomycin, etoposide, prednisone TTT methotrexate, cytarabine, hydrocortisone Topo/CTX cyclophosphamide, topotecan, mesna VAB-6 cyclophosphamide, dactinomycin, vinblastine, cisplatin, bleomycin VAC vincristine, dactinomycin, cyclophosphamide VACadr vincristine, cyclophosphamide, doxorubicin, dactinomycin, vincristine VAD vincristine, doxorubicin, dexamethasone VATH vinblastine, doxorubicin, thiotepa, fluoxymesterone VBAP vincristine, carmustine, doxorubicin, prednisone VBCMP vincristine, carmustine, melphalan, cyclophosphamide, prednisone

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V.C. vinorelbine, cisplatin VCAP vincristine, cyclophosphamide, doxorubicin, prednisone V.D. vinorelbine, doxorubicin VelP vinblastine, cisplatin, ifosfamide, mesna VIP etoposide, cisplatin, ifosfamide, mesna V.M. mitomycin, vinblastine VMCP vincristine, melphalan, cyclophosphamide, prednisone V.P. etoposide, cisplatin V-TAD etoposide, thioguanine, daunorubicin, cytarabine 5+2 cytarabine, daunorubicin, mitoxantrone 7+3 cytarabine together with daunorubicin or idarubicin or mitoxantrone "8 in 1" methylprednisolone, vincristine, lomustine, procarbazine, hydroxyurea, cisplatin, cytarabine, dacarbazine

In some embodiments, chemotherapeutic agents that can be administered with the compounds of the invention, such as a compound of formula (I), include a CD39 inhibitor. CD39 or ecto-nucleoside triphosphate diphosphohydrolase 1 (E-NTPDase1 or ENTPD 1) is a membrane-bound enzyme that catalyzes the conversion of extracellular adenosine triphosphate (ATP) and/or ADP (adenosine diphosphate) to adenosine monophosphate (AMP). In one embodiment, the CD39 inhibitor is polyoxometalate-1 (POM-1).

In other embodiments, chemotherapeutic agents that may be administered with compounds of the invention, such as a compound of formula (I), include known CD73 inhibitors. In some embodiments, the CD73 inhibitor is an anthraquinone derivative (Baqi et al., J. Med. Chem., 53(5): 2076-2086, 2010, incorporated herein by reference). In other embodiments, the CD73 inhibitor is a sulfonic acid derivative (Raza et al., Med. Chem., 8: 1133-1139, 2012, incorporated herein by reference). In other embodiments, the CD73 inhibitor is selected from 1-amino-4-phenylamino-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (acid blue 25), 1-amino-4-[4-hydroxyphenyl-amino] -9,10-dioxo-9,10-dihydroanthracene-2-sulfonate, 1-amino-4-[4-aminophenylamino] -9,10-dioxo-9,10-dihydroanthracene-2-sulfonate, 1-amino-4 -[1-naphthylamino]9,10-dioxo-9,10-dihydroanthracene-2-sulfonate, 1-amino-4-[4-fluoro-2-carboxyphenylamino]-9,10-dioxo-9,10dihydroanthracene-2- sulfonate, 1-amino-4-[2-anthracenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate, sodium 2,4-dinitrobenzenesulfonate, N(4-sulfamoylphenylcarbamothioyl)pivalamide, APCP, β-methyleneADP (AOPCP), PPADS, Nf279, NF449, quercetin, reactive blue 2 and sumarin (Baqi 2010; Raza 2012).

In some embodiments, the combination of a compound of the invention, such as a compound of formula (I), with a second CD73 inhibitor or CD39 inhibitor may have a synergistic effect in the treatment of cancer and other adenosine-mediated diseases or disorders. Without wishing to be bound by theory, this synergy may be observed because CD39 and CD73 are often found on different cell types. The hypoxic tumor microenvironment also induces increased levels of CD39 and CD73.

In some embodiments, chemotherapeutic agents that can be co-administered with compounds of the invention, such as a compound of formula (I), include an adenosine receptor inhibitor. In other embodiments, the adenosine receptor inhibitor is selected from rolophylline, tonapophylline, ATL-444, istradefylline, MSX-3, preladenant, SCH-58261, SCH-412348, SCH-442416, ST-1535, VER-6623, VER-6947 VER-7835 , vipadenanta and ZM-241385. In some embodiments, the adenosine receptor inhibitor targets the A _2A receptor, since this subtype is predominantly expressed on most immune cells.

In other embodiments, chemotherapeutic agents that can be co-administered with compounds of the invention, such as a compound of formula (I), include a nucleoside drug. In certain embodiments, the nucleoside drug is selected from gemcitabine, capecitabine, cytarabine, fludarabine, and cladribine.

In other embodiments, the combination therapy includes a compound of the invention, such as a compound of formula (I), administered together with an anthracycline. In other embodiments, the combination therapy includes a compound of the invention, such as a compound of formula (I), administered together with doxorubicin. Combination treatment with anti-CD73 antibody and doxorubicin has demonstrated significant chemotherapeutic effects (Young et al., Cancer Discov., 4(8): 1-10, 2014, incorporated herein by reference).

In some embodiments, the combination therapy includes a compound of the invention

- 35 046381 tenia, such as a compound of formula (I), administered together with an A2A receptor inhibitor and an anthracycline. In some embodiments, the anthracycline is doxorubicin. Combination treatment with anti-CD73 antibody, A2A receptor inhibitor and doxorubicin demonstrated enhanced chemotherapeutic effect (Antonioli 2013).

In certain embodiments, co-therapy of this invention includes co-administration with other types of chemotherapeutic agents, such as immuno-oncology agents. Cancer cells often have specific cell surface antigens that can be recognized by the immune system. Thus, immuno-oncology agents such as monoclonal antibodies can selectively bind to cancer cell antigens and influence cell death. Other immuno-oncology agents may suppress tumor-mediated inhibition of the native immune response or otherwise activate the immune response and thus facilitate tumor recognition by the immune system. Typical antibody-based immuno-oncology agents include, but are not limited to, abagovomab, adecatumumab, afutuzumab, alemtuzumab, anatumomab mafenatox, apolizumab, blinatumomab, BMS-936559, catumaxomab, durvalumab, epacadostat, epratuzumab, indoximod, inotuzumab, ozogamicin, intelum umab, ipilimumab, isatuximab, lambrolizumab, MED14736, MPDL3280A, nivolumab, obinutuzumab, ocaratuzumab, ofatumumab, olatatumab, pembrolizumab, pidilizumab, rituximab, ticilimumab, samizumab and tremelimumab. In some embodiments, the antibody-based immuno-oncology agents are selected from anti-CD73 monoclonal antibodies (mAbs), anti-CD39 mAbs, anti-PD-1 mAbs, and anti-CTLA4 mAbs. Thus, in some embodiments, the methods of the invention include co-administration of one or more immuno-oncology agents, such as the agents mentioned above.

In some embodiments, the combination therapy includes a compound of the present invention, such as a compound of formula (I), administered in conjunction with anti-PD-1 antibody therapy and anti-CTLA4 antibody therapy. Combination treatment with anti-CD73 monoclonal antibodies (mAb), anti-PD-1 mAb, and anti-CTLA4 mAb has shown significant chemotherapeutic effects (Young 2014; Antonioli 2013).

In some embodiments, the combination therapy includes co-administration of a compound of the present invention, such as a compound of formula (I), with an αnmi-PD-1 antibody. In certain embodiments, the combination therapy includes co-administration of a compound of the present invention, such as a compound of formula (I), with oxaliplatin. In other embodiments, the combination therapy includes co-administration of a compound of the present invention, such as a compound of formula (I), with doxorubicin.

In some embodiments, a compound of the invention may be administered in conjunction with non-chemical treatments for cancer. In certain embodiments, a compound of the invention may be administered in conjunction with radiation therapy. In some embodiments, a compound of the invention may be administered in conjunction with surgery, thermal ablation, focused ultrasound therapy, cryotherapy, or any combination thereof.

In some embodiments, the compounds of the invention may be co-administered with one or more other compounds of the invention. In addition, such combinations may be administered in conjunction with other therapeutic agents, such as other agents suitable for the treatment of cancer, immunological or neurological diseases, such as the agents mentioned above. In some embodiments, coadministration of one or more additional chemotherapeutic agents with a compound of the invention provides a synergistic effect. In some embodiments, coadministration of one or more additional chemotherapeutic agents provides an additive effect.

Pharmaceutical compositions.

In certain embodiments, the present invention provides a pharmaceutical preparation suitable for use in a human patient, comprising any of the compounds demonstrated above (for example, a compound of the present invention, such as a compound of formula (I), and one or more pharmaceutically acceptable excipients In some embodiments, pharmaceutical preparations may be used to treat or prevent a condition or disease described herein.Any of the compounds described may be used in the manufacture of medicaments for the treatment of any disease or condition described herein.

The compositions and methods of the present invention can be used to treat a subject in need thereof. In some embodiments, the subject is a mammal, such as a human or non-human mammal. When administered to a subject, such as a human, the composition or compound is preferably administered in the form of a pharmaceutical composition containing, for example, a compound of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline, or other solvents or carriers such as glycols, glycerol, oils such as olive oil, or injections

- 36 046381 adjustable organic esters. In a preferred embodiment, when such pharmaceutical compositions are intended for administration to humans, especially for invasive routes of administration (i.e., routes such as injection or implantation that bypass transport or diffusion across an epithelial barrier), the aqueous solution contains no or substantially no pyrogens. pyrogens. For example, excipients may be selected to effect delayed release of the agent or to selectively target one or more cells, tissues, or organs. The pharmaceutical composition may be in unit dose form such as a tablet, capsule (including particulate capsule and gelatin capsule), granule, lyophilisate for reconstitution, powder, solution, syrup, suppository, injection or the like. The composition may also be present in a transdermal delivery system, for example, a transdermal patch. The composition may also be present in a solution suitable for topical use, such as eye drops.

The pharmaceutically acceptable carrier may contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or increase absorption of a compound, such as a compound of the invention. Such physiologically acceptable agents include, for example, carbohydrates such as glucose, sucrose or dextrans, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The drug or pharmaceutical composition may be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (drug) may also be a liposome or other polymer matrix, which may include, for example, a compound of the invention. Liposomes, for example, which include phospholipids or other lipids, are non-toxic, physiologically acceptable and metabolizable carriers that are relatively easy to prepare and administer.

The expression pharmaceutically acceptable is used herein to designate those compounds, substances, compositions and/or unit dosage forms that are medically suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reaction or other problems or complications. , commensurate with a reasonable benefit/risk ratio.

The phrase pharmaceutically acceptable carrier, as used herein, refers to a pharmaceutically acceptable substance, composition or carrier medium, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be acceptable in terms of being compatible with the other ingredients of the formulation and not harmful to the patient. Some examples of substances that can serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; (4) tragacanth powder; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic sodium chloride solution; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances used in pharmaceutical preparations.

The pharmaceutical composition may be administered to a subject by any of a number of routes of administration, including, for example, oral administration (e.g., drops, as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including break-open capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (for example, sublingually); anally, rectally, or vaginally (eg, as a pessary, cream, or foam); parenterally (including intramuscularly, intravenously, subcutaneously or intrathecally, such as a sterile solution or suspension); intranasally; intraperitoneally; subcutaneously; transdermally (eg, as a patch applied to the skin); and topically (eg, as a cream, ointment, or spray applied to the skin or as eye drops). The compound may also be formulated for inhalation. In certain embodiments, the compound may be simply dissolved or suspended in sterile water. In some embodiments, the compound may be simply dissolved or suspended in sterile water. Nos. 6110973, 5763493, 5731000, 5541231, 5427798, 5358970 and 4172896, as well as in the patents cited therein.

The compositions may be conveniently presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient that can be combined with a carrier substance to form a unit dosage form will vary depending on the subject being treated and the particular route of administration.

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The amount of active ingredient that can be combined with a carrier substance to form a unit dosage form is generally that amount of the compound that provides a therapeutic effect. Typically, of one hundred percent, this amount will be from about 1 to about ninety-nine percent active ingredient, preferably from about 5 to about 70%, most preferably from about 10 to about 30%.

Methods for preparing these formulations or compositions include the step of combining an active compound, such as a compound of the invention, with a carrier and, optionally, one or more additional ingredients. Typically, the formulations are prepared by uniformly and thoroughly combining the active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, molding the product.

Compositions of the invention suitable for oral administration may be in the form of capsules (including break-open capsules and gelatin capsules), starch capsules, pills, tablets, lozenges (using a flavored base, usually sucrose and gum arabic or tragacanth), freeze-dried preparations, powders, granules or as a solution or suspension in an aqueous or non-aqueous liquid, or as a liquid oil-in-water or water-in-oil emulsion, or as an elixir or syrup, or as lozenges (using an inert base such as gelatin and glycerin, or sucrose and gum arabic) and/or in the form of mouthwashes and the like, each of which contains a predetermined amount of a compound of the present invention as an active ingredient. The compositions or compounds may also be administered as a bolus, electuary, or paste.

In the preparation of solid oral dosage forms (capsules (including particle-coated capsules and gelatin capsules), tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate and/or any of the following: (1) fillers or diluents such as starches, lactose, sucrose, glucose, mannitol and/or silicic acid; (2) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or gum arabic; (3) humectants such as glycerin; (4) leavening agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, some silicates and sodium carbonate; (5) dissolution retarding agents such as paraffin; (6) absorption accelerators such as quaternary ammonium compounds; (7) wetting agents such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents such as kaolin and bentonite clay; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof; (10) complexing agents such as modified and unmodified cyclodextrins; and (11) dyes. In the case of capsules (including break-open capsules and gelatin capsules), tablets and pills, the pharmaceutical compositions may also contain buffering agents. In addition, solid compositions of this type can be used as fillers in soft and hard gelatin capsules, using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like.

A tablet may be prepared by compression or molding, optionally with one or more additional ingredients. Compressed tablets can be prepared by using a binder (eg, gelatin or hydroxypropyl methylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (eg, sodium starch glycolate or cross-linked sodium carboxymethylcellulose), a surface active or dispersing agent. Molded tablets can be prepared by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

Tablets and other solid dosage forms of pharmaceutical compositions, such as dragees, capsules (including particle-coated capsules and gelatin capsules), pills and granules, may optionally be filled or formulated with coatings and shells, such as enteric coatings and other coatings well known in the field of pharmaceuticals. In addition, they can be formulated to provide a slow or controlled release of the active ingredient, using, for example, hydroxypropyl methylcellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They can be sterilized, for example, by filtration through a bacteria-retaining filter or by administering sterilizing agents in the form of sterile solid compositions, which can be dissolved in sterile water or some other sterile injection medium immediately before use. Such compositions may further optionally contain opacifiers and may be compositions that release the active ingredient(s) only or, preferably, in a specific part of the gastrointestinal tract, optionally in a delayed form. Examples of encapsulated compositions that can be used include polymeric substances and waxes. Additionally, the active ingredient may be in microencapsulated form, if appropriate, with one or more of the excipients described above.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient

- 38 046381 liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed, peanut, corn, wheat germ, olive, castor and sesame oils), glycerin, tetrahydrofuryl alcohol, polyethylene glycols and sorbitan fatty acid esters, and mixtures thereof.

In addition to inert diluents, oral compositions may further contain excipients such as humectants, emulsifiers and suspending agents, sweeteners, flavoring agents, colors, fragrances and preservatives.

Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol esters of sorbitan, microcrystalline cellulose, aluminum meta-hydroxide, bentonite, agar-agar and tragacanth, as well as mixtures thereof.

Formulations of pharmaceutical compositions for rectal, vaginal or urethral administration can be presented in the form of suppositories, which can be prepared by mixing one or more active compounds with one or more suitable non-irritating excipients or carriers, including, for example, cocoa butter, polyethylene glycol, wax or salicylate for suppositories that are solid at room temperature but liquid at body temperature and therefore will melt in the rectum or vaginal cavity and release the active compound.

Formulations of pharmaceutical compositions for administration to the oral cavity may be presented in the form of a mouthwash, or an oral spray, or an oral ointment.

Alternatively or additionally, the compositions may be formulated for delivery via a catheter, stent, wire, or other intraluminal device. Delivery through such devices may be particularly effective for delivery to the bladder, urethra, ureter, rectum or intestine.

Formulations suitable for vaginal administration further include pessaries, tampons, creams, gels, pastes, foams or spray preparations containing such carriers as are known in the art to be suitable.

Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and implants. The active ingredient may be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffering agents or propellants that may be necessary.

Ointments, pastes, creams and gels may contain, in addition to the active compound, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acids, talc and zinc oxide or mixtures of them.

Powders and sprays may contain, in addition to the compound provided herein, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder or a mixture of these substances. Sprays may additionally contain conventional propellants such as chlorofluorocarbons and volatile unsubstituted hydrocarbons such as butane and propane.

Transdermal patches have the additional advantage of providing controlled delivery of the compound of the present invention to the body. Such dosage forms can be prepared by dissolving or dosing the compound in an appropriate medium. Additionally, absorption enhancers can be used to increase the delivery of the compound through the skin. The rate of such entry can be controlled using a rate-controlling membrane or by dispersing the compound in a polymer matrix or gel.

Ophthalmic compositions, ophthalmic ointments, powders, solutions and the like are also considered to be within the scope of the present invention. Examples of ophthalmic formulations are described in US Publications 2005/0080056, 2005/0059744, 2005/0031697 and 2005/004074 and US Patent No. 6,583,124, the contents of which are incorporated herein by reference. If desired, liquid ophthalmic formulations may have properties similar to or compatible with tear fluids, aqueous humor, or vitreous humor. The preferred route of administration is topical administration (eg, topical administration such as eye drops or administration through an implant).

As used herein, the phrases parenteral administration and parenterally administered mean modes of administration other than enteral and topical administration, typically by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal , transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal and intra-articular injection and infusion. Pharmaceutical compositions,

- 39 046381 suitable for parenteral administration, may contain one or more active compounds in combination with one or more pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions or sterile powders, which can be reconstituted immediately before use with sterile injectable solutions or dispersions , which may contain antioxidants, buffers, bacteriostatic agents, solutes that maintain isotonicity of the drug with the blood of the intended recipient, or suspending agents or thickeners.

Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerin, propylene glycol, polyethylene glycol and the like) and suitable mixtures thereof, vegetable oils such as olive oil and injectable organic complexes esters such as ethyl oleate. Proper flow can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

In addition, these carriers may contain adjuvants such as preservatives, humectants, emulsifiers and dispersing agents. Prevention of exposure to microorganisms can be achieved by the administration of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid and the like. It may also be desirable to include agents in the compositions to maintain isotonicity, such as sugars, sodium chloride and the like. In addition, prolonged absorption of the injectable pharmaceutical form can be achieved by the administration of agents that delay absorption, such as aluminum monostearate and gelatin.

In some cases, to prolong the effect of the drug, it is desirable to slow the absorption of the drug by subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of crystalline or amorphous material with low water solubility. The rate of absorption of a drug is further dependent on its rate of dissolution, which in turn may depend on crystal size and crystalline form. Alternatively, slowing the absorption of a parenterally administered dosage form is achieved by dissolving or suspending the drug in an oil carrier medium.

Injection depot forms are prepared by forming microencapsulated matrices of the compounds of interest in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, as well as the nature of the particular polymer used, the rate of drug release can be adjusted. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Preparations for injection depots are also obtained, including the drug in liposomes, microemulsions, which are compatible with body tissue.

For use in the methods of this invention, the active compounds may be taken alone or in the form of a pharmaceutical composition containing, for example, from 0.1 to 99.5% (more preferably from 0.5 to 90%) of the active ingredient in combination with a pharmaceutically acceptable carrier.

Methods of administration may also be provided by rechargeable or biodegradable devices. In recent years, various slow-release polymer devices have been developed and tested in vivo for controlled drug delivery, including protein biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and insoluble polymers, can be used to provide sustained release of the compound at a specific target site during implant formation.

The actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that will be effective to achieve the desired therapeutic response in a particular patient, composition and route of administration, without toxicity to the patient.

The dosage level selected will depend on a variety of factors, including the potency of the particular compound or combination of compounds used or its ester, salt or amide, route of administration, time of administration, release rate of the particular compound(s), duration of treatment, other drugs, compounds and/or or substances used in combination with the particular compound(s), age, sex, weight, condition, general health and prior medical history of the patient being treated, and similar factors well known in medicine.

A physician or veterinarian of ordinary skill in the art can readily determine and administer a therapeutically effective amount of the desired pharmaceutical composition. For example, a physician or veterinarian may begin dosages of a pharmaceutical composition or compound at levels lower than those required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. By a therapeutically effective amount is meant a concentration of a compound sufficient to achieve the desired thera

- 40 046381 chanting effect. It is generally understood that the effective amount of a compound will vary depending on the weight, gender, age and medical history of the subject. Other factors that influence the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, the other type of therapeutic agent administered with the compound of the invention. A larger total dose can be delivered in multiple doses of the agent. Methods for determining potency and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13 ed., 1814-1882, incorporated herein by reference).

Generally, a suitable daily dose of the active compound used in the compositions and methods of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such effective dose will generally depend on the factors described above.

If desired, an effective daily dose of the active compound may be administered in one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally in unit dosage forms. In some embodiments of the present invention, the active compound can be administered two or three times a day. In preferred embodiments, the active compound will be administered once daily.

In some embodiments, the dosing follows a 3+3 schedule. The traditional 3+3 design does not require dose-toxicity curve modeling beyond the classical assumption for cytotoxic drugs that toxicity increases with dose. This rule-based design works with cohorts of three patients; the first group is treated at an initial dose believed to be safe based on extrapolation from animal toxicology data, and subsequent groups are treated at increased dose levels that have been recorded in advance. In some embodiments, three doses of a compound of formula (I) range from about 100 to about 1000 mg orally, such as from about 200 to about 800 mg, such as from about 400 to about 700 mg, such as from about 100 to about 400 mg , for example, from about 500 to about 1000 mg, and in addition, for example, from about 500 to about 600 mg. Dosing may be three times daily when taken without food, or twice daily when taken with food. In some embodiments, the three doses of a compound of formula (I) range from about 400 to about 800 mg, such as from about 400 to about 700 mg, such as from about 500 to about 800 mg, and in addition, for example, from about 500 to about 600 mg twice daily. In certain preferred embodiments, a dose greater than about 600 mg is administered twice daily.

If none of the three patients in the cohort experiences dose-limiting toxicity, an additional three patients will be treated at the next higher dose level. However, if one of the first three patients experiences dose-limiting toxicity, three more patients will be treated at the same dose level. Dose escalation continues until at least two patients in a group of three to six patients experience dose-limiting toxicity (ie, > about 33% of patients with dose-limiting toxicity at that dose level). The recommended dose for phase II trials is usually defined as a dose level just below the toxic dose level.

In some embodiments, the dosage regimen may be from about 40 mg/m ² to about 100 mg/m ² , such as from about 50 mg/m ² to about 80 mg/m ² , and in addition, for example, from about 70 mg/ ^m2 to about 90 mg/ ^m2 IV over 3 weeks of a 4-week cycle.

In some embodiments, the compounds of the invention may be used alone or in conjunction with another type of therapeutic agent. As used herein, the expression co-administration refers to any form of administration of two or more different therapeutic compounds such that the second compound is administered while the previously administered therapeutic compound is still effective in the body (e.g., two compounds are simultaneously effective in a subject that may involve synergistic effects of the two compounds). For example, different therapeutic compounds may be administered either in the same composition or in separate compositions, either simultaneously or sequentially. In some embodiments, the different therapeutic compounds may be administered within one hour, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, or weeks from each other. Thus, a subject who receives such treatment may benefit from the combined effects of various therapeutic compounds.

In some embodiments, coadministration of a compound of the present invention with one or more additional therapeutic agents (e.g., one or more additional chemotherapeutic agents) provides improved efficacy compared to each individual administration of a compound of the present invention (e.g., a compound of formula (I) or Ia ) or one or more therapeutic agents. In some such embodiments, coadministration provides an additive effect, where the additive effect refers to the sum of each of the effects of individual administration of a compound of the invention and one or more additional therapeutic agents.

- 41 046381

The present invention includes the use of pharmaceutically acceptable salts of the present invention in the compositions and methods of the present invention. In some embodiments, the provided salts of the invention include, but are not limited to, alkyl, dialkyl, trialkyl, or tetraalkylammonium salts. In some embodiments, the provided salts of the invention include, but are not limited to, salts of L-arginine, benetamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N- methylglucamine, hydrabamine, N-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine and zinc. In some embodiments, the provided salts of the invention include, but are not limited to, Na, Ca, K, Mg, Zn or other metal salts.

Pharmaceutically acceptable acid addition salts can also exist in the form of various solvates, for example, with water, methanol, ethanol, dimethylformamide and the like. Mixtures of such solvates can also be prepared. The source of such solvate may be a crystallization solvent corresponding to the solvent of preparation or crystallization, or random for such a solvent.

The compositions may contain wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as colorants, release agents, coatings, sweeteners, flavors, preservatives and antioxidants.

Examples of pharmaceutically acceptable antioxidants are: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol and the like; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like.

The invention as generally described will now be more readily understood with reference to the following examples, which are included only for the purpose of illustrating certain aspects and embodiments of the present invention and are not intended to limit the invention.

General methods of synthesis.

Compound numbers 1-129 used in the general synthesis section below refer only to structures of the genus in that section and do not apply to compounds described elsewhere in this application. The compounds described herein can be prepared by the methods depicted in the reaction schemes below.

The starting materials and reagents used in the preparation of these compounds are either available from a commercial supplier such as Aldrich Chemical Co., Bachem, etc., or can be prepared by methods well known in the art. The schemes are merely illustrative of some of the methods by which the compounds described herein can be synthesized, and various modifications to these schemes can be made and will be offered by POSITA with reference to this disclosure. The starting materials and intermediates, as well as the final reaction products, can be isolated and purified, if desired, using conventional methods, including, but not limited to, filtration, distillation, crystallization, chromatography, and the like, and can be characterized using conventional means , including physical constants and spectral data.

Unless otherwise indicated, the reactions described herein take place at atmospheric pressure over a temperature range of about -78°C to about 150°C.

General schemes.

Compounds of formula (I) having the structure

where Z, R ^u , R ^v , R ^w , R ^x , R ⁵ and R ⁹ are similar to the variables Z, R ^a , R ^b , R ^w , R ⁵ , R ⁹ and R ^x are CH2P(O)(OR ¹⁵ )2 or -OP(O)(OH)CH ₂ P(O)(OR ¹⁵ )2 as defined in the Summary of the Invention, and can be synthesized as illustrated and described in Scheme 1.

- 42 046381

Scheme 1

Ketone A-3 is prepared from the commercially available diol A-1 by selectively protecting the primary alcohol with a suitable group such as TBDPS, TBDMS, Ac and Bz, followed by oxidation of the secondary alcohol to A-2, where R is H, alkyl, TMC or heterocycles. Stereoselective addition of the appropriate ethynyl nucleophile A-4, such as Grignard reagents or Li reagents, to ketone A-3 produces propargyl alcohol A-5. Removal of the acetonide protecting group is performed using dilute aq. acids such as TFA, HCl, H ₂ SO ₄ , HClO ₄ , PPTS, CSA or other Lewis acids. Acylation of triol A-6 with reagents such as Ac ₂ O, acetyl chloride and BzCl, in the presence of a base such as pyridine and the catalytic 4-DMAP results in the formation of a triester such as A-7 triacetate. Glycosylation under conditions (NIC-bisDtrimethylsilylDacetamide and TMSOTf) or (TfOH and DBU) in a solvent (MeCN, dichloroethane or toluene) between donor A-7 and acceptor A-8, such as 2-chloroadenine, 6-amino-2-chloroadenine, 2,6-dichloroadenine, 5,7-dichloro-Chimidazo-D-EDpyridine, 5-chloro-3H-imidazo[4,5-b]pyridine, uracil, thymine, cytosine or guanine, leads to the formation of nucleoside product A-9 . In the case where R ^u is NH2, it is protected as N(Boc) ₂ by Boc ₂ O in the presence of TEA and catalytic 4-DMAP. Removal of the protecting group in A-9, in the case where P is a TBDMS or TBDPS group, treatment with TBAF to obtain the primary alcohol A-10, which then undergoes an insertion reaction with diazo reagent A11 in the presence of a catalyst such as Rh ₂ (OAc) ₄ or Cu(OAc) ₂ leads to the formation of A-12. Alkylation with an electrophile A-13, such as an alkyl halide, triflate, tosylate or mesylate, in the presence of a base such as Cs ₂ CO ₃ , K ₂ CO ₃ , LiHMDS, DBU or NaH, results in the formation of A-14. The ester groups in A-14 are finally removed with a base such as LiOH, NaOH and KOH in water to give A-15 in formula (Ia).

Alternatively, the alkynyl group at the 3' position in intermediate A-5 can be replaced with either alkyl or vinyl groups using the appropriate alkyl or vinyl lithium reagents and Grignard reagents in step 3 of Scheme 1.

- 43 046381

Scheme 2

Compounds of formula (Ia) can be prepared according to Scheme 2. A suitable protecting group such as P representing a silyl group (TBDPS or TBDMS) in precursor B-1 can be selectively removed using a reagent such as TBAF or HF in THF, while the P ¹ protecting group such as the Ac, Bz and MOM group remains. The resulting primary alcohol B-2 can react with diazo reagent A-11 in a solvent such as benzene, toluene, DCM and dichloroethane in the presence of a metal catalyst such as Rh2(OAc)4 to form intermediate B-3. Alkylation of B-3 with electrophile A-13, such as a halide, triflate, mesylate or sulfonate, is carried out in the presence of a base such as K ₂ CO ₃ , Cs ₂ CO ₃ , LiHMDS, NaH and DBU to produce intermediate B-4. Removal of the acetonide protecting group in B-4 is accomplished by acid treatment such as aq. TFA, HCl, H2SO4 or HClO ₄ in a solvent such as DCM, acetone, THF or dioxane to produce diol B-5. Acylation of B-5 with a reagent such as Ac2O or acetyl chloride in the presence of pyridine, TEA or DIPEA and catalytic 4-DMAP results in the formation of triacetate B-6 (for P1=OAc) as the glycosylation donor. This intermediate B-6 interacts with heterocycle B-7, a glycosylation acceptor such as 2-chloroadenine, 6-amino-2-chloroadenine, 2,6-dichloroadenine, 5,7-dichloro-1H-imidazo[4,5-b ]pyridine, 5-chloro3H-imidazo[4,5-b]pyridine, uracil, thymine, cytosine and guanine under the influence of conditions such as [N,O-6uc(trimethylsilyl)acetamide and TMSOTf] or (TfOH and DBU) in solvent (MeCN, dichloroethane or DME) to form the intermediate nucleoside B-8. Finally, removal of the ester protecting groups in B by treatment with aq. LiOH, NaOH and KOH in a solvent such as THF, dioxane, MeOH or EtOH results in the desired end product of formula (Ia).

Scheme 3

- 44 046381

Compounds of formula (Ia) can be prepared according to Scheme 3. 2,6-Dichloroadenine C-1 prepared according to Scheme 1 can undergo several synthetic transformations. Selective nucleophilic substitution of the 6-chloro group in precursor 1 with a R ^u -H(C2) nucleophile such as amines, alkoxides or thiolates in a solvent such as DMF, THF, dioxane, alcohols or NMP results in the formation of the C-3 intermediate . The C-1 precursor can also undergo a coupling reaction, such as the Suzuki, Stille, or Negishi reaction, with an appropriate reagent such as boronic acids (C-4), boronic acid esters, tin reagents (C-5), or zinc reagents (C -6) to obtain intermediate C-7, respectively. Treatment of both intermediates C-3 and C-7 aq. LiOH, NaOH, KOH, NaOMe, NaOEt or KOEt in a solvent such as THF, dioxane, MeOH or EtOH produces the desired end products of formula (Ia).

Scheme 4

Compounds of formula (Ia) can be prepared by C-1 according to Scheme 4. In this method, by treating A-7 with 2,6-dichloropurine, TMSOTf and N,O-bis(trimethylsilyl)acetamide via the Vorbruggen reaction, the protected nucleoside is obtained D-2. Selective removal of the tert-butyldiphenylsilyl moiety from the 5'-hydroxyl group results in the formation of the D-3 alcohol. When combined with the desired substituted acyl diazoreactant D-4, the substituted nucleoside D-5 is obtained. A variety of diazo reagents can be used in this reaction. Some examples include those in which R ^w is CO2R ⁹ , SOR ⁹ , SO2R ⁹ , P(O)(OR ⁹ ) ₂ , and CN and R ⁹ are defined as in the Summary of the Invention. If an alkyl substituent ^R5 is desired, it can be successfully introduced by an alkylation reaction in which a nucleophile such as R5-X (X=halide, OTf, OM or OT) is used with a base such as cesium carbonate in a polar aprotic solvent , such as THF or DMF to produce the key intermediate C-1. A substituent such as an amine can be added to a purine base by replacing the chlorine at the 6-position to produce intermediate D-7 with various D-6 amines in a solvent such as EtOH, THF or dioxane. After final deprotection of D-7 by aqueous hydrolysis with a base such as lithium hydroxide, the target compound of the structure of formula (1) is obtained.

- 45 046381

Scheme 5

Compounds of formula (Ia) can be prepared according to Scheme 5. Alkylation of the B-3 precursor from Scheme 2 above with an E-1 electrophile such as 4-iodobenzyl halide (Br, Cl or I), or the corresponding OTf, OM or OT base, such as K ₂ CO ₃ , Cs ₂ CO ₃ , NaH or LiHMDS in a solvent such as DMF or THF leads to the formation of intermediate E-2, which can combine with various boronic acids such as (2-oxo-1, 2-dihydropyridin-3-yl) boronic acid (E-3) demonstrated herein. The resulting pyridone product E-4 is alkylated with various alkyl halides (A-13) in the presence of a base such as K ₂ CO ₃ , Cs ₂ CO ₃ , NaH or LiHMDS in a solvent such as DMF and THF to give E-5. The acetonide protecting group in E-5 is removed by treatment with aq. TFA, HCl, H ₂ SO ₄ or AcOH in a solvent such as DCM, acetone, dioxane or THF to produce an E-6 diol, which is acetylated with Ac ₂ O or acetyl chloride with a catalytic amount of 4-DMAP and a base such as pyridine, TEA or DIPEA in a solvent such as DCM to produce a mixture of E-7 anomers as a glycosylation donor. Intermediate E-7 can react with either the heterocyclic acceptor 2,6dichloroadenine (D-1) or the N-substituted 6-amino-2-chloroadenine (E-9), which is formed by replacing the 6-chloro group in D-1 suitable amines (D-7). Both glycosylations can occur under activation conditions such as [(N,O-bis-(trimethylsilyl)acetamide and TMSOTf] or (TfOH and DBU) in a solvent (MeCN, dichloroethane or toluene), between donor E-7 and acceptors D- 1 or E-9, to obtain the corresponding nucleoside products, E-8 or E-10, respectively. Nucleoside E-8 is converted to E-10 by nucleophilic substitution with various amines (D-7). Finally, the desired compounds of formula (Ia) are obtained from E-10 by removing the protecting groups from all its ester groups by treating aq with LiOH, NaOH and KOH in a solvent such as THF, dioxane, MeOH or EtOH.

- 46 046381

Scheme 6

Compounds of formula (Ia) can be prepared according to Scheme 6. Alkylation with benzyl halide F-4 with various alkyl side chains (R ⁵ ) and precursor B-3 in the presence of a base such as K2CO ₃ , Cs ₂ CO ₃ , LiHMDS or NaH, in a suitable solvent such as DMF or THF leads to the formation of intermediate F-5. Triacetate F-7 is formed by a two-step conversion from F-5 by deprotection (aq. TFA in DCM) and acylation (Ac2O or acetyl chloride in pyridine) as described by the above schemes. Glycosylation between F-7 and various acceptors such as 2,6-dichloroadenine (D-1) under activation conditions [(N,O-bis(trimethylsilyl)acetamide and TMSOTf] or (TfOH and DBU) in solvent (MeCN , dichloroethane or toluene) to produce nucleoside F-8, which is converted to amino analogues of F-10 with various amines (F-9) in the presence of a base such as pyridine, TEA or DIPEA, in a suitable solvent such as dioxane, DMF or THF Finally, the desired molecules in formula (1) are prepared from F-10 by treating aq. LiOH, NaOH and KOH in a solvent such as THF, dioxane, MeOH or EtOH. Alternatively, the intermediate F-10 can also be directly obtained by glycosylation between donor F-7 and other acceptors such as N-substituted 6amino-2-chloroadenines (F-11).

The desired benzyl halides F-4 are prepared from 4-(((tert-butyldimethylsilyl)oxy)methyl)aniline (F-1) through a five-step conversion. The cyclic urea ring is initially formed from aniline F-1 reacting with 3-chloropropyl isocyanate, followed by cyclization with a base such as NaH, NaOH or LiHMDS in a solvent such as DMF or THF to form F-2. Intermediate F-2 is then converted to F-3 by removing the TBDMS group with TBAF and proceeds to the final product by converting the primary alcohol to bromide with CBr ₄ and PPh ₃ in a solvent such as DCM or THF.

Alternatively, the key intermediate F-5 is also prepared from precursor B-3 according to Scheme 7. Alkylation of precursor B-3 with halides such as 4-nitrobenzyl bromide in the presence of a base such as K ₂ CO ₃ or Cs ₂ CO ₃ in DMF leads to the formation of a nitro intermediate, which is then reduced to aniline with Fe in aq. _NH4Cl . Formation of cyclourea is accomplished from aniline with 3-chloropropyl isocyanate in the presence of a base such as TEA in THF, followed by intramolecular ring closure with treatment of a base such as NaH or LiHMDS. Introduction of N-alkyl side chains is accomplished using an electrophile such as A-13 to produce the key intermediate F-5.

- 47 046381

Scheme 7

Formula (Ib) can be prepared in accordance with Scheme 8. The desired diazo reagent G-2, R ^w of which is an aryl or heteroaryl group, can be prepared from the ester of G-1 with a suitable sulfonyl azide reagent, such as 4-acetmidobenzenesulfonyl azide, in the presence of a base such as Et ₃ N and DBU in MeCN or dioxane. Combining diazo reagent G-2 and alcohol A-9 from Scheme 1 via an inclusion reaction catalyzed by a Rh or Cu catalyst such as Rh ₂ (OAc)4 in a solvent such as toluene, DCM or dichloroethane produces product G-3 . Alkylation of G-3 with an A-13 electrophile such as an alkyl halide, triflate, tosylate or mesylate in the presence of a base such as Cs ₂ CO ₃ , K ₂ CO ₃ , LiHMDS, DBU or NaH results in the formation of G-4. The ester groups in G-4 are finally removed with aq. bases such as LiOH, NaOH and KOH to obtain the desired product of formula (Ib).

Scheme 8

Compounds of formula (Ib) can be prepared according to Scheme 9. Primary alcohol B-2 can be reacted with diazoreagent G-2 from Scheme 8 in the presence of a metal catalyst such as Rh ₂ (OAc) ₄ in a solvent such as benzene, toluene , DCM or dichloroethane, to form intermediate H-1. Alkylation of H-1 with an A-13 electrophile such as a halide, triflate, mesylate or sulfonate is carried out in the presence of a base such as K ₂ CO ₃ , Cs ₂ CO ₃ , LiHMDS, NaH and DBU to produce intermediate H-2. Removal of the acetonide protecting group in H-2 is accomplished by acid treatment such as aq. TFA, HCl, H ₂ SO ₄ or HClO ₄ or CSA in a solvent such as DCM, acetone, THF or dioxane to produce diol H-3. Acylation of H-3 with a reagent such as Ac ₂ O or acetyl chloride in the presence of pyridine, TEA or DIPEA and catalytic 4-DMAP results in the formation of H-4 triacetate as the glycosylation donor. This H-4 intermediate reacts with heterocycle B-7, a glycosylation acceptor such as 2-chloroadenine, 6-amino-2-chloroadenine, 2,6-dichloroadenine, 5,7-dichloro-III-imidazo[4,5-b] pyridine, 5-chloro3H-imidazo[4,5-b]pyridine, uracil, thymine, cytosine and guanine under conditions such as [N,O-6uc(trimethylsilyl)acetamide and TMSOTf] or (TfOH and DBU) in solvent ( MeCN, dichloroethane or DME) to form the nucleoside intermediate H-5. Finally, removal of the ester protecting groups in H5 by treatment with aq. LiOH, NaOH and KOH in a solvent such as THF, dioxane, MeOH or EtOH leads to the formation of the desired end product of formula (Ib).

- 48 046381

Scheme 9

Compounds of formula (Ic) can be prepared according to Scheme 10. Tertiary alcohol I-2, where R ¹ is methyl (prepared according to the procedure described in Franchetti, P. et al. J. Med. Chem. 2005, 48, 4983-4989) or other alkyl groups; ethynyl (prepared according to the procedure described in Hulpia, F. et al. Bioorg. Med. Chem. Lett. 2016, 26, 19701972) or other alkynyl groups; and vinyl groups were converted to I-4 either directly by treatment with an acetylation reagent such as Ac ₂ O and a catalytic amount of H ₂ SO ₄ in AcOH or by a two-step process involving removal of the acetonide protecting group with treatment with aq. TFA or other acid in DCM first, followed by acetylation of the resulting diol I-3 with a reagent such as Ac ₂ O or acetyl chloride. By glycosylation of I-4 with a heteroaromatic glycosyl acceptor such as the heterocycle B-7 described in Scheme 2, where R ^u represents H, Cl, NH2, an N-alkyl group such as 2-chloroadenine, 6-amino-2chloroadenine, 2,6-dichloroadenine, 5,7-dichloro-1H-imidazo[4,5-b]pyridine, 5-chloro-3H-imidazo[4,5-b]pyridine, uracil, thymine, cytosine and guanine under such conditions as [N,O-bis(trimethylsilyl)acetamide and TMSOTf] or (TfOH and DBU) in a solvent (MeCN, dichloroethane or DME) to give nucleoside intermediate I-5. Removal of the silyl protecting group on I-5 using a fluoride source such as TBAF in THF results in the formation of the primary alcohol I-6, which is then converted to the triol I-7 using aq. LiOH or NaOH in a solvent such as THF, MeOH or EtOH. Finally, treatment of I-7 with methylenebis(phosphonic dichloride) and trimethylphosphate before subsequent treatment with triethylammonium carbonate affords the desired end product of formula (Ic).

Scheme 10

Compounds of formula (Ic) can be prepared according to Scheme 11. The primary alcohol I-6 (where R ^u =Cl) is alkylated with an electrophile J-1, such as (diethoxyphosphoryl)methyltrifluoromethanesulfonate or diethyl(iodomethyl)phosphonate, in the presence of a base such as TEA, DIPEA, NaH and Cs ₂ CO ₃ , in a solvent such as THF, DMF, dioxane or NMP to give intermediate J-2. Installation of an amino group on J-4 by nucleophilic substitution of the chloro group on J-2 with the compound R7R8NH (J-3), where ^R7 and ^R8 are H or alkyl groups, in the presence of a base such as TEA or DIPEA, in a solvent such as dioxane , THF or DMF. A two-step deprotection sequence (TMSBr and aq. LiOH or NaOH) is required to convert J-4 to the desired product of formula (Ic).

Scheme 11 but

DIEA,dioxane

J-3 r ⁷ r ⁸ nh

Formula (1c)

1. TMSBr, MeCN

2. ester hydrolysis

AcO Oas

Those skilled in the art will appreciate that the starting materials and reaction conditions may vary, and the reaction sequence and additional steps used to prepare the compounds covered by the present invention are demonstrated by the following examples. In some cases, it may be necessary to protect certain reactive functions to achieve some of the above conversions. In general, the need for such protecting groups, as well as the conditions necessary for the addition and removal of such groups, will be apparent to those skilled in organic chemistry. The descriptions of all articles and references mentioned in this application, including patents, are incorporated herein by reference.

The preparation of the compounds of the present invention is further illustrated by the following examples, which should not be construed as limiting the scope or spirit of the invention to the specific procedures and compounds described therein.

Synthetic examples

Example 1. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl) methoxy)-2-benzylmalonic acid

Example 1

Stage 1.

To a mixture of (3aR,5R,6aS)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-2,2-dimethyldihydrofuro[2,3d][1,3]dioxol-6(3aH)-one (10 g , 23.44 mmol, 1 equiv.) in THF (100 ml) was added ethynylmagnesium bromide (0.5 M, 328.19 ml, 7 equiv.) at 15°C under N ₂ atmosphere. The mixture was stirred for 16 hours before adding more ethynylmagnesium bromide (0.5 M, 125 ml, 3 eq.). The mixture was stirred for an additional 3 hours before diluting with saturated aq. NH ₄ Cl solution (250 ml) and extra

- 50 046381 were treated using EtOAc (3x250 ml). The combined organic layer was washed with brine (250 ml), dried over Na ₂ SO ₄ , filtered and evaporated to dryness. The crude product was purified by silica gel flash column chromatography (petroleum ether: EtOAc=1:0-4:1) to give (3aR,5R,6R,6aR)-5-(((tert-butyldiphenylsilyl)oxy)methyl) -6-ethynyl-2,2dimethyl-tetrahydrofuro[2,3^][1,3]dioxol-6-ol (19.47 g, 92% yield) as a yellow solid.

Stage 2.

To a solution of (3aR,5R,6R,6aR)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-6-ethynyl-2,2-dimethyltetrahydrofuro[2,3^][1,3]dioxol-6- ol (9.47 g, 20.92 mmol, 1 eq.) in DCM (100 ml) was added H2O (10 ml) and TFA (100 ml) at 0°C. The mixture was stirred at 25°C for 1 hour before quenching with saturated aq. NaHCO ₃ solution to pH 7 and then extracted using DCM (2x300 ml). The combined organic layer was washed with brine (200 ml), dried over Na2SO4, filtered and evaporated to dryness. The crude product was purified by silica gel flash column chromatography (petroleum ether: EtOAc=1:0-0:1) to give (3R,4S,5R)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-4-ethynyltetrahydrofuran-2 ,3,4-triol (5.17 g, 60% yield) as yellow gum.

Stage 3.

To a solution of (3R,4S,5R)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-4-ethynyltetrahydrofuran-2,3,4triol (5.17 g, 12.53 mmol, 1 eq.) in pyridine (50 ml) at 15°C, 4-DMAP (4.59 g, 37.60 mmol, 3 eq.) and Ac ₂ O (11.74 ml, 125.32 mmol, 10 eq.) were added. The mixture was stirred at 15°C for 16 hours, after which H2O (500 ml) was added to the mixture. The reaction mixture was extracted using EtOAc (3x200 ml). The combined organic layer was washed with brine (200 ml), dried over Na ₂ SO ₄ , filtered and evaporated to dryness. The crude product was purified by silica gel flash column chromatography (petroleum ether: EtOAc=1:0-1:1) to give (3R,4R,5R)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-4-ethynyltetrahydrofuran-2 ,3,4-triyl triacetate (7.19 g, 79% yield) as yellow gum.

Stage 4.

To a solution of (3R,4R,5R)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-4-ethynyltetrahydrofuran-2,3,4triyltriacetate (6.89 g, 12.79 mmol, 1 eq.) in MeCN (5 ml) at 0°C, 2-chloroadenine (2.39 g, 14.07 mmol, 1.1 equiv), DBU (5.78 ml, 38.37 mmol, 3 equiv) and TMSOTf (11 .56 ml, 63.96 mmol, 5 eq.). The mixture was stirred at 0°C for 0.5 h and then stirred at 65°C for 1 h before being diluted with saturated aq. NaHCO ₃ solution (500 ml). The aqueous phase was extracted using EtOAc (2x350 ml). The combined organic layer was washed with brine (350 ml), dried over Na ₂ SO ₄ , filtered and evaporated to dryness. The crude product was purified by silica gel flash column chromatography (petroleum ether: EtOAc=1:0-0:1) to give (2R,3R,4R,5R)-5-(6-amino-2-chloro-9H- purin-9-yl)-2-(((tert-butyldiphenylsilyl)oxy)methyl)-3ethynyltetrahydrofuran-3,4-diyldiacetate (4.52 g, 44% yield) as a yellow solid.

Stage 5.

To a solution of (2R,3R,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-2-(((tert-butyldiphenylsilyl)oxy)methyl)-3-ethynyltetrahydrofuran- 3,4-diyldiacetate (4.5 g, 6.94 mmol, 1 eq.) in DMF (50 ml) at 20°C was added TEA (4.83 ml, 34.71 mmol, 5 eq.), 4- DMAP (254 mg, 2.08 mmol, 0.3 eq.) and Boc ₂ O (7.58 g, 34.71 mmol, 5 eq.). The mixture was stirred at 20°C for 1 hour, after which H2O (250 ml) was added to the mixture. The reaction mixture was extracted using EtOAc (3x230 ml). The combined organic layer was washed with brine (250 ml), dried over Na2SO4, filtered and evaporated. The crude product was purified by silica gel flash column chromatography (petroleum ether: EtOAc=1:0-1:1) to give (2R,3R,4R,5R)-5-(6-(bis-(tert-butoxycarbonyl) amino)-2-chloro-9H-nurin-9-yl)-2(((tert-butyldiphenylsilyl)oxy)methyl)-3-ethynyltetrahydrofuran-3,4-diyldiacetate (3.26 g, 46% yield) as yellow foam.

Stage 6.

To a solution of (2R,3R,4R,5R)-5-(6-(bis-(tert-butoxycarbonyl)amino)-2-chloro-9H-purin-9-yl)-2-(((tert-butyldiphenylsilyl)oxy) methyl)-3-ethynyltetrahydrofuran-3,4-diyldiacetate (3.24 g, 3.82 mmol, 1 eq.) in THF (35 ml) at 0°C was added TBAF (1 M, 5.73 ml, 1. 5 eq.). The reaction mixture was stirred at 0°C for 1 hour and then diluted with H2O (150 ml). The reaction mixture was extracted using EtOAc (3x130 ml). The combined organic layer was washed with brine (150 ml), dried over Na2SO4, filtered and evaporated. The crude product was purified by silica gel flash column chromatography (petroleum ether: EtOAc=1:0-1:2) to give (2R,3R,4R,5R)-5-(6N,N'-(bis-(tert-butoxycarbonyl) )amino)-2-chloro-9H-purin-9-yl)-3-ethynyl-2-(hydroxymethyl)tetrahydrofuran-3,4-diyldiacetate (1.51 g, 54% yield) as a yellow foam.

Stage 7.

To a solution of (2R,3R,4R,5R)-5-(6-N,N'-(bis-(tert-butoxycarbonyl)amino)-2-chloro-9H-purin-9-yl)-3ethynyl-2- (hydroxymethyl)tetrahydrofuran-3,4-diyldiacetate (1.48 g, 2.43 mmol, 1 eq.) in toluene

- 51 046381 (10 ml) at 20°C in an atmosphere of N ₂ added Rh ₂ (OAc) ₄ (214 mg, 485.24 µmol, 0.2 eq.) and diethyl diazomalonate (903 mg, 4.85 mmol, 2 eq. .) in toluene (3 ml). The mixture was stirred at 95°C for 2 hours to obtain a green suspension, after which it was cooled to room temperature and evaporated to dryness. The crude product was purified by silica gel flash column chromatography (petroleum ether: EtOAc=1:0-3:1) to form diethyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5 -(6-N,N'-(bis(tert-butoxycarbonyl)amino)-2-chloro-9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (517 mg, 20% yield ) in the form of yellow foam.

Stage 8.

To a solution of diethyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-N,N'-(bis-(tert-butoxycarbonyl)amino)2-chloro-9H- purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (497.00 mg, 647.00 µmol, 1 eq.) in DMF (5 ml) at 25°C and added K ₂ CO ₃ ( 178.84 mg, 1.29 mmol, 2 eq.). The reaction mixture was stirred for 30 min and then benzyl bromide (221.32 mg, 1.29 mmol, 153.69 µl, 2 eq.) was added. The mixture was stirred at 25°C for 15.5 h before additional K2CO3 (100 mg) and BnBr (100 μl) were added to the mixture. The resulting mixture was stirred at 25°C for 24 hours before adding H2O (50 ml) to the mixture. The reaction mixture was extracted using EtOAc (2x50 ml). The combined organic layer was washed with brine (50 ml), dried over Na ₂ SO ₄ , filtered and evaporated. The crude product was purified by silica gel flash column chromatography (petroleum ether: EtOAc=1:0-3:1) to form diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3,4 -diacetoxy5-(6-(bis-(tert-butoxycarbonyl)amino)-2-chloro-9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (266 mg, 37% yield) in in the form of yellow foam.

Stage 9.

To a solution of diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-(bis-(tert-butoxycarbonyl)amino)-2-chloro-9H- purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (266 mg, 309.92 µmol, 1 eq.) in DCM (3 ml) was added with TFA (0.45 ml) at 0°C. The mixture was stirred at 25°C for 16 h before being treated with saturated aq. NaHCO solution to pH 7. The reaction mixture was extracted using DCM (2x50 ml). The combined organic layer was washed with brine (50 ml), dried over Na ₂ SO ₄ , filtered and evaporated to give crude diethyl 2-benzyl 2-(((2R,3R,4R,5R)-3,4-diacetoxy-5- (6-amino-2-chloro-9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (195 mg) as a yellow foam.

Stage 10.

A mixture of crude diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-amino-2-chloro-9Hpurin-9-yl)-3-ethynyltetrahydrofuran- 2-yl)methoxy)malonate (195 mg, 296.33 µmol, 1 eq.) in saturated NH ₃ in MeOH (3 ml) was stirred at 10°C for 16 h before evaporation directly to dryness. The crude product was purified by preparative TLC (EtOAc) to form diethyl 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3- ethynyl 3,4-dihydroxytetrahydrofuran-2-yl)methoxy)-2-benzylmalonate (91.7 mg, 49% yield) as a yellow foam.

Stage 11.

To a solution of diethyl-2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl) methoxy)-2-benzylmalonate (81 mg, 141.12 µmol, 1 eq.) in EtOH (2 ml) was added LiOH-H ₂ O (30 mg, 705.60 µmol, 5 eq.) in H2O (0.2 ml) at 10°C. The mixture was stirred at 50°C for 4 hours, then evaporated to dryness. The residue was dissolved in H2O (50 ml) and extracted using EtOAc (2x50 ml). The organic layers were removed and the aqueous phase was acidified to pH ~2.5 with 1 N aqueous HCl. The aqueous phase was extracted using EtOAc (3x50 ml).

The combined organic layer was washed with brine (50 mL), dried over Na ₂ SO ₄ , filtered and evaporated to give the title compound (55.4 mg, 74% yield) as a white solid.

1H NMR (400 MHz, DMSOO δ ppm 8.39 (s, 1H), 7.79 (br.s, 2H), 7.20 (br.d, J=7.03 Hz, 2H) , 7.01-7.12 (m, 3H), 5.82 (d, J=7.53 Hz, 1H), 4.87 (d, J=7.78 Hz, 1H), 4.16 ( dd, J=5.27, 2.51 Hz, 1H), 3.99-4.07 (m, 2H), 3.83 (br. d, J=8.03 Hz, 1H), 3.56 (s, 1H), 3.25 (dd, J=6.78 Hz, 2H); LC/MS [M+H] = 518.0.

- 52 046381

Example 2. Synthesis of 2-((((2S,3R,4R,5R))-5-(6-αmino-2-chloro-9H-purin-9-yl)-3-ethynyl-4-hydroxyteΊhydrofuran-2- yl)methoxy)-2-benzylmalonic acid

Example 2

Stage 1.

To a solution of (2R,3R,4R,5R)-5-(6-(bis-(tert-butoxycarbonyl)αmino)-2-chloro-9H-purin-9-yl)-2(((tert-butyldiphenylsilyl)oxy )methyl)-3-ethynyltetrahydrofuran-3,4-diyldiacetate (5 g, 5.89 mmol, 1 eq.) was added 2M NH ₃ in MeOH (50 ml) at 0°C. The reaction mixture was stirred at 25°C for 16 hours before it was evaporated. The crude product was purified by silica gel flash column chromatography (0-50% EtOAc in petroleum ether) to give tert-butyl-(9((2R,3R,4S,5R)-5-(((tert-butyldiphenylsilyl)oxy)methyl )-4-ethynyl-3,4-dihydroxytetrahydrofuran-2yl)-2-chloro-9H-purin-6-yl)carbamate (3.78 g, 90% yield) as a yellow foam.

Stage 2.

To a solution of tert-butyl-(9-((2R,3R,4S,5R)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-4-ethynyl-3,4dihydroxytetrahydrofuran-2-yl)-2-chloro 4-DMAP (258.63 mg, ₂ .12 mmol, 0.4 eq.) and TCDI (4.72 g, 26.46 mmol, 5 eq.). The reaction mixture was stirred at 25°C for 16 h before evaporation. The crude product was purified by silica gel flash column chromatography (0-33% EtOAc in petroleum ether) to give tert-butyl-(9-((3aR,4R,6R,6aR)-6-(((tert-butyldiphenylsilyl) oxy)methyl)-6a-ethynyl-2-thioxotetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-2-chloro-9H-purin6-yl)carbamate (1.4 g, yield 37 %) in the form of yellow foam.

Stage 3.

To a solution of tert-butyl-(9-((3aR,4R,6R,6aR)-6-(((tert-butyldiphenylsilyl)oxy)methyl)-6a-ethynyl-2thioxotetrahydrofuro[3,4^][1,3] dioxol-4-yl)-2-chloro-9H-purin-6-yl)carbamate (500 mg, 707.93 µmol, 1 eq.) in toluene (5 ml) was added AIBN (11.62 mg, 70.79 µmol, 0.1 equiv.) at 20-25°C. The reaction mixture was then heated to 60°C followed by the addition of (n-Bu) _3SnH (561.96 µL, 2.12 mmol, 3 eq). The reaction mixture was stirred at 60°C for 1.5 hours before it was evaporated. The crude product was purified by silica gel flash column chromatography (0-33% EtOAc in petroleum ether) to give tert-butyl-(9-((2R,3R,4R,5S)-5-(((tert-butyldiphenylsilyl) hydroxy)methyl)-4ethynyl-3-hydroxytetrahydrofuran-2-yl)-2-chloro-9H-purin-6-yl)carbamate (220 mg, 47% yield) as a white foam.

Stage 4.

To a solution of tert-butyl-(9-((2R,3R,4R,5S)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-4-ethynyl-3dihydroxytetrahydrofuran-2-yl)-2-chloro-9H -purin-6-yl)carbamate (220 mg, 339.39 mmol, 1 eq.) in DCM (1.4 ml) was added with TFA (0.7 ml, 9.45 mmol, 28 eq.) at 0°C. The reaction mixture was stirred at 25° for 1 hour before diluting with saturated aq. NaHCO ₃ solution (15 ml), and extracted using DCM (3x5 ml). The combined organic layer was washed with brine (10 ml), dried over Na ₂ SO ₄ , filtered and evaporated to give crude (2K,3E,4K^)-2-(6-amino-2-chloro-9H-purine-9- yl)-5(((tert-butyldiphenylsilyl)oxy)methyl)-4-ethynyltetrahydrofuran-3-ol (260 mg) as a yellow foam.

Stage 5.

To a solution of (2R,3R,4R,5S)-2-(6-amino-2-chloro-9H-purin-9-yl)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-4-ethynyltetrahydrofuran- 3-ol (260 mg, 474.36 µmol, 1 eq., crude) in DMF (2.5 ml) was added Boc ₂ O (931.75 mg, 4.27 mmol, 9 eq.), TEA (660, 25 µl, 4.74 mmol, 10 equiv) and 4-DMAP (5.80 mg, 47.44 µmol, 0.1 equiv) at 0°C. The reaction mixture was stirred at 25°C for 1.5 h, then diluted with H2O (20 ml) and extracted using EtOAc (3x5 ml). The combined organic layer was washed with brine (10 mL), dried over Na ₂ SO ₄ , filtered and evaporated to give crude (2R,3R,4S,5S)-2-(6-(N,N'-bis-((tert -butoxycarbonyl)amino)-2-chloro-9H-purine

- 53 046381

9-yl)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-4-ethynyltetrahydrofuran-3-yl-tert-butyl carbonate (560 mg) as orange gum.

Stage 6.

To a solution of crude (2R,3R,4S,5S)-2-(6-(N,N'-bis-((tert-butoxycarbonyl)amino)-2-chloro-9Hpurin-9-yl)-5-(( (tert-butyldiphenylsilyl)oxy)methyl)-4-ethynyltetrahydrofuran-3-yl-tert-butyl carbonate (560 mg, 660.02 µmol, 1 eq., crude) in THF (6 ml) at 0°C, add TBAF in THF (1 M, 1 ml, 1.52 eq.) The reaction mixture was stirred at 0°C for 1 hour. The reaction mixture was diluted with H ₂ O (10 ml) and extracted with EtOAc (3x5 ml). The combined organic layer was washed brine (10 ml), dried over Na ₂ SO ₄ , filtered and evaporated.The crude product was purified by preparative TLC (petroleum ether: EtOAc=2:1) to form (2R,3R,4S,5S)2-(6- (N,N'-bis-((tert-butoxycarbonyl)amino)-2-chloro-9H-purin-9-yl)-4-ethynyl-5-(hydroxymethyl)tetrahydrofuran-3-yl-tert-butyl carbonate (93 mg, yield 45% in three stages) in the form of a yellow foam.

Stage 7.

To a solution of (2R,3R,4S,5S)-2-(6-(N,N'-bis-((tert-butoxycarbonyl)amino)-2-chloro-9H-purin-9-yl-4ethynyl-5- (hydroxymethyl)tetrahydrofuran-3-yl-tert-butyl carbonate (93 mg, 152.45 µmol, 1 eq.) in toluene (1 ml) at 25°C under N2 was added (OAc) ₄ (6.74 mg, 15 .24 µmol, 0.1 eq.) The reaction mixture was heated to 90°C followed by the addition of diethyl 2-diazomalonate (85.14 mg, 457.34 µmol, 3 eq.) in toluene (1 ml). stirred at 90°C for 3 h before evaporation. '-bis-((tert-butoxycarbonyl)amino)-2-chloro-9H-purin-9-yl)-4((tert-butoxycarbonyl)oxy)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (180 mg ) in the form of gum.

Stage 8.

To a solution of crude diethyl-2-(((^, ⁿ ^.4K.5C)-5-(6-(CI-bis-((tert-butoxycarbonyl)amino)2-chloro-9H-purin-9-yl) -4-((tert-butoxycarbonyl)oxy)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (117.11 mg, 152.45 µmol, 1 eq.) in DMF (3 ml) _{was added} 421.39 mg, 3.05 mmol, 20 eq.) at 20-25° C. The reaction mixture was stirred for 0.5 h and then benzyl bromide (271.61 μl, 2.29 mmol, 15 eq.) was added. The reaction mixture was then stirred for an additional 16 hours, after which it was diluted with H2O (20 ml) and extracted using EtOAc (3x10 ml).The combined organic layer was washed with brine (15 ml), dried over Na ₂ SO ₄ , filtered and evaporated The crude product was purified by silica gel flash column chromatography (petroleum ether: EtOAc=1:0-2:1 gradient) to form diethyl-2-((^^^^)-5-(6-(Y^- bis-((tert-butoxycarbonyl)amino)-2-chloro-9H-purin-9-yl)-4-((tert-butoxycarbonyl)oxy)-3-ethynyltetrahydrofuran-2-yl)methoxy)-2-benzylmalonate (90 mg ) in the form of an almost white gum.

Stage 9.

To a solution of diethyl-2-((^^^^)-5-(6-(Y^-bis-((tert-butoxycarbonyl)amino)-2-chloro-9Hpurin-9-yl)-4-((tert TFA (0, 3 ml, 4.05 mmol, 39 eq.). The reaction mixture was stirred at 25° for 1 hour before diluting with saturated aq. NaHCO ₃ solution (5 ml), and extracted using DCM (3x3 ml). The combined organic layer was washed with brine (5 ml), dried over Na2SO4, filtered and evaporated. The crude residue was purified by preparative TLC (petroleum ether: EtOAc=1:1) to give diethyl-2((((2S,3R,4R,5R))-5-(6-amino-2-chloro-9H-purine -9-yl)-3-ethynyl-4-hydroxytetrahydrofuran-2-yl)methoxy)-2-benzylmalonate (19 mg, 13% yield in 3 steps) as yellow gum.

Stage 10.

To a solution of diethyl-2-(((2S,3R,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-4-hydroxytetrahydrofuran-2-yl) methoxy)-2-benzyl malonate (19 mg, 34.05 µmol, 1 eq.) in THF (0.2 ml) was added LiOH-H2O (7.14 mg, 170.26 µmol, 5 eq.) in H2O (70 µl) at 25°C. The reaction mixture was stirred for 5.5 hours, then diluted with H2O (5 ml), and then acidified to pH 2-3 with 1 N aq. HCl. The mixture was extracted using EtOAc (3x5 ml). The combined organic layer was washed with brine (15 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated. The crude residue was dissolved in a mixture of H2O (3 ml) and MeCN (2 ml) and then lyophilized to give the title compound as a white solid.

1H NMR (400 MHz, CD3OD) δ ppm. 8.30 (s, 1H) 7.27 (br.d, J=5.27 Hz, 2H) 7.17-7.24 (m, 1H) 7.15 (br.d, J=6.78 Hz, 2H) 5.87-5.96 (m, 1H) 4.94 (br.s, 1H) 4.68 (br.s, 1H) 3.96-4.11 (m, 2H) 3, 34-3.40 (m, 2H) 2.57 (d, J=2.51 Hz, 1H) 2.32 (s, 1H); LC/MS [M+H] = 502.0.

- 54 046381

Example 3. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3,4-dihydroxy-3-(prop-1yne -1 -yl)tetrahydrofuran-2-yl)methoxy)-2-benzylmalonic acid

Ac ₂ Oh,

Example 3

Stage 1.

To a solution of (3aR,5R,6aS)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-2,2-dimethyldihydrofuro[2,3d][1,3]dioxol-6(3aH)-one (10 g , 23.44 mmol, 1 equiv.) (prop-1-ynyl)magnesium bromide (0.5 M, 93.77 ml, 2 equiv.) was added to THF (100 ml) at 20°C under N ₂ atmosphere. The mixture was stirred at 40°C for 2 hours before diluting with saturated aq. NH ₄ Cl solution (250 ml). The aqueous phase was extracted using EtOAc (3x200 ml). The combined organic layer was washed with brine (200 ml), dried over Na ₂ SO ₄ , filtered and evaporated to give crude (3aR,5R,6R,6aR)-5(((tert-butyldiphenylsilyl)oxy)methyl)-2,2 -dimethyl-6-(prop-1-yn-1-yl)tetrahydrofuro[2,3^][1,3]dioxol-6-ol (11.81 g) as yellow gum.

Stage 2.

To a solution of crude (3aR,5R,6R,6aR)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-2,2-dimethyl6-(prop-1-in-1-yl)tetrahydrofuro[2,3^ ][1,3]dioxol-6-ol (12.2 g, 26.14 mmol, 1 equiv.) in pyridine (120 ml) was added 4-DMAP (3.51 g, 28.76 mmol) at 20°C , 1.1 eq.) and Ac ₂ O (4.90 ml, 52.29 mmol, 2 eq.). The mixture was stirred at 20°C for 16 hours, then diluted with H2O (200 ml) and extracted using EtOAc (3x100 ml). The combined organic layer was washed with brine (250 ml), dried over Na ₂ SO ₄ and filtered. The filtrate was evaporated to give crude (3aR,5R,6R,6aR)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-2,2-dimethyl-6-(prop-1-yn-1-yl)tetrahydrofuro[ 2,3^][1,3]dioxol-6-yl acetate (15 g) as yellow gum.

Stage 3.

To a solution of crude (3aR,5R,6R,6aR)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-2,2-dimethyl6-(prop-1-in-1-yl)tetrahydrofuro[2,3^ ][1,3]dioxol-6-ylacetate (15 g, 29.49 mmol, 1 eq.) in THF (300 ml) at 0°C under N2 was added a mixture of TBAF (1 M, 44.23 ml, 1 .5 eq.) and AcOH (1.26 ml, 22.12 mmol, 0.75 eq.). The mixture was stirred at 20°C for 7 hours before diluting with saturated aq. NH ₄ Cl solution (300 ml) and extracted using EtOAc (3x200 ml). The combined organic layer was washed with brine (300 ml), dried over Na ₂ SO ₄ , filtered and evaporated. The crude product was purified by silica gel flash column chromatography (petroleum ether: EtOAc=1:0-1:1) to give (3aR,5R,6R,6aR)-5-(hydroxymethyl)-2,2-dimethyl-6 -(prop-1-yn1-yl)tetrahydrofuro[2,3^][1,3]dioxol-6-ylacetate (5.78 g, 72.5% yield) as a white solid.

Stage 4.

To a solution of (3aR,5R,6R,6aR)-5-(hydroxymethyl)-2,2-dimethyl-6-(prop-1-in-1-yl)tetrahydrofuro[2,3d][1,3]dioxol- 6-yl acetate (5.78 g, 21.39 mmol, 1 eq.) in dichloroethane (60 ml) at 15°C under N2 was added Rh ₂ (OAc) ₄ (945.21 mg, 2.14 mmol, 0 .1 eq.) and diethyldiazomalonate (7.96 g, 42.77 mmol, 2 eq.). The mixture was stirred at 40°C for 7 hours before evaporation to dryness. The crude product was purified by silica gel flash column chromatography (0-25% EtOAc in petroleum ether) to give diethyl 2-(((3aR,5R,6R,6aR)-6-acetoxy-2,2-dimethyl-6 -(prop-1-yn-1yl)tetrahydrofuro[2,3^][1,3]-dioxol-5-yl)methoxy)malonate in the form of yellow gum.

Stage 5.

To a solution of diethyl-2-(((3aR,5R,6R,6aR)-6-acetoxy-2,2-dimethyl-6-(prop-1-in-1-yl)tetrahydrofuro [2,3^][1 Cs ₂ CO ₃ (11.07 g, 33 .98 mmol, 2 equiv.) and BnBr (3.03 ml, 25.49 mmol, 1.5 equiv.). The mixture was stirred at 20°C for 2 hours, then diluted with H ₂ O (300 ml) and extracted using EtOAc (3x100

- 55 046381 ml). The combined organic layer was washed with brine (200 ml), dried over Na ₂ SO ₄ , filtered and evaporated. The crude product was purified by silica gel flash column chromatography (petroleum ether: EtOAc=1:0-3:1) to give 2-(((3aR,5R,6R,6aR)-6acetoxy-2,2-dimethyl-6-( prop-1-yn-1-yl)tetrahydrofuro[2,3D][1,3]dioxol-5-yl)methoxy)-2-benzylmalonate (7.67 g, 87% yield) as yellow gum.

Stage 6.

To a solution of diethyl-2-(((3aR,5R,6R,6aR)-6-acetoxy-2,2-dimethyl-6-(nrop-1-in-1-yl)tetrahydrofuro[2,3D][1, 3]dioxol-5-yl)methoxy)-2-benzylmalonate (7.67 g, 14.79 mmol, 1 eq.) in TFA (80 ml) at 20°C was added H2O (6.97 ml, 387.05 mmol, 26 eq.). The mixture was stirred at 20°C for 8 hours before quenching with saturated aq. NaHCO ₃ to pH 7 and separated using EtOAc (3x100 ml). The combined organic layer was washed with brine (200 ml), dried over Na2SO4 and filtered. The filtrate was evaporated to dryness, yielding crude diethyl-2-benzyl-2-(((2S,3S,4R)3,4,5-trihydroxy-3-(prop-1-yn-1-yl)tetrahydrofuran-2- yl)methoxy)malonate (5.95 g) as a yellow gum.

Stage 7.

To a solution of crude diethyl-2-benzyl-2-(((2S,3S,4R)-3,4,5-trihydroxy-3-(prop-1-yn-1yl)tetrahydrofuran-2-yl)methoxy)malonate ( 5.95 g, 13.63 mmol, 1 eq.) in pyridine (60 ml) at 20°C was added 4-DMAP (5.00 g, 40.90 mmol, 3 eq.) and Ac ₂ O (6, 38 ml, 68.16 mmol, 5 eq.). The mixture was stirred at 20°C for 16 hours, then diluted with H ₂ O (300 ml) and extracted using EtOAc (3x150 ml). The combined organic layer was washed with brine (300 ml), dried over Na ₂ SO ₄ , filtered and evaporated. The crude product was purified by silica gel flash column chromatography (petroleum ether: EtOAc=1:0-3:1) to form diethyl-2-benzyl-2(((2R,3R,4R)-3,4,5-triacetoxy- 3-(prop-1-yn-1-yl)tetrahydrofuran-2-yl)methoxy)malonate (5.76 g, 66% yield) as yellow gum.

Stage 8.

To a solution of crude diethyl-2-benzyl-2-(((2R,3R,4R)-3,4,5-triacetoxy-3-(prop-1-yn-1yl)tetrahydrofuran-2-yl)methoxy)malonate ( 1 g, 1.78 mmol, 1 eq.) in MeCN (10 ml) at 20°C was added N,O-bis-(trimethylsilyl)acetamide (BSA) (1.32 ml, 5.33 mmol, 3 eq. ) and 2-chloroadenine (301.43 mg, 1.78 mmol, 1 eq.). The mixture was stirred at 65°C for 30 min, then cooled to 0°C and then TMSOTf (642 μl, 3.56 mmol, 2 eq.) was added dropwise. The mixture was stirred at 0°C for 10 min and then at 65°C for 2 h before quenching with saturated aq. NaHCO ₃ solution (100 ml) and extracted using EtOAc (2x60 ml). The combined organic layer was washed with brine (100 ml), dried over Na ₂ SO ₄ , filtered and evaporated. The crude product was purified by silica gel flash column chromatography (0-33% EtOAc in petroleum ether) to form diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5 -(6-amino-2-chloro-9H-purin-9yl)-3-(prop-1-yn-1-yl)tetrahydrofuran-2-yl)methoxy)malonate (218 mg, 18% yield) as yellow gum

Stage 9.

To a solution of diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-amino-2-chloro-9H-purin-9-yl)-3( prop-1-yn-1-yl)tetrahydrofuran-2-yl)methoxy)malonate (218 mg, 324.37 µmol, 1 eq.) in THF (2 ml) was added LiOH-H ₂ O (136.12 mg, 3.24 mmol, 10 eq.) in H2O (2 ml) at 20°C. The mixture was heated at 45°C for 2 hours, then diluted with H2O (10 ml) and extracted using EtOAc (10 ml). The organic layer was removed and the aqueous phase was acidified with 2 N aqueous HCl to pH 2-3. The aqueous phase was then extracted using EtOAc (3x20 ml). The combined organic layer was washed with brine (30 ml), dried over Na ₂ SO ₄ , filtered and evaporated. The crude product was purified by preparative HPLC to give the title compound (39.8 mg, 23% yield) as a white solid.

1H NMR (DMSO-de, 400 MHz) δ ppm. 12.70-14.12 (m, 2H), 8.37 (s, 1H), 7.80 (br. s, 2H), 7.19 (br. d, J=7.03 Hz, 2H) , 7.00-7.11 (m, 3H), 5.88-6.03 (m, 2H), 5.81 (d, J=7.53 Hz, 1H), 4.78 (br.s , 1H), 4.12 (dd, J=4.52, 3.01 Hz, 1H), 3.95 (br. dd, J=9.91, 4.89 Hz, 1H), 3.82 ( br.d, J=8.53 Hz, 1H), 3.25 (s, 2H), 1.81 (s, 3H); LC/MS [M+H] = 532.0.

- 56 046381

Example 4. Synthesis of 2-U(VR,3S,4R,5R)-5-(Y)-aMHiio-2-x.iop-9l 1-11uri11-9-i.1')-3-(cycl.. lo11po11i..teti11i..l')-3,4dihydroxytetrahydrofuran-2-yl)methoxy)-2-benzylmalonic acid

Stage 1.

To a solution of ethynitcyctopropane (4.96 g, 75.02 mmol, 6.22 mt, 2 eq.) in THF (80 mt) at -78°C under N2, add n-BuLi (2.5 M, 30 .01 mt, 2 eq.). Stir the solution at -78°C for 0.5 h, after which a solution of (3aR,5R,6aS)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-2,2-dimethyldihydrofuro[2, 3-d][1,3]-dioxot-6(3aH)-one (16.0 g, 37.51 mmol, 1 eq.) in THF (60 mt). Then allow the solution to warm up to 20°C and stir for 1 hour, then cool to 0°C and quench with water (120 mt). The mixture is extracted using EtOAc (2x120 mt). The combined organic mixture is washed with saline solution (200 mt), dried over Na ₂ SO ₄ , filtered and evaporated. The crude product was purified by combi-phthash chromatography with sitikagetem (0-15% ethyl acetate in petroleum ether) to give (3aR,5R,6R,6aR)-5-(((tert-bugyldiphenylsilyl)oxy)methyl)-6-(cyclopropylethynyl )-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxot-6-ota (15.8 g, 86% yield) as syrup.

Stage 2.

To a solution of crude (3aR,5R,6R,6aR)-5-(((tert-butytdiphenylsilyl)oxy)methyl)-6(πictopropylethynyl)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol -6-ol (15.8 g, 32.07 mmol, 1 eq.) in pyridine (160 mt) at 20°C add 4-DMAP (4.70 g, 38.48 mmol, 1.2 eq.) and Ac ₂ O (9.01 mt, 96.21 mmot, 3 eq.). Stir the solution for 3 hours, then dilute with water (200 mt) and extract with ethyl acetate (2x200 mt). The combined organic residue is washed with a saline solution (400 mt), dried using Na ₂ SO ₄ , filtered and evaporated. The crude residue is purified by combi-phthash chromatography with sitikat (0-15% ethyl acetate in petroleum ether) to give (3aR,5R,6R,6aR)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-6- (cyclopropylethynyl)-2,2dimethyltetrahydrofuro[2,3-d][1,3]dioxol-6-ylacetate (14.7 g, 86% yield) as a clear syrup.

Stage 3.

To a solution of crude (3aR,5R,6R,6aR)-5-(((tert-butytdiphenylsilyl)oxy)methyl)-6(πictopropylethynyl)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol -6-yl acetate (14.7 g, 27.49 mmol, 1 eq.) in THF (150 mt) at 0°C add a solution of TBAF (1 M, 41.24 mt, 1.5 eq.) and AcOH ( 1.18 mt, 20.62 mmot, 0.75 eq.). The solution was stirred at 20°C for 16 hours, then diluted with water (300 mt) and extracted using ethyl acetate (2x200 mt). The combined organic sludge is washed with water (400 mt), honeycomb solution (400 mt), dried using Na ₂ SO ₄ , filtered and evaporated. The crude residue is purified by combi-phthash chromatography with sitikaget (20-60% ethyl acetate in petroleum ether) to give (3aR,5R,6R,6aR)-6-(cyclopropylethynyl)-5(hydroxymethyl)-2,2-dimethyltetrahydrofuro [2,3-d][1,3]dioxol-6-ylacetate (8.15 g, 100% yield) as a beta solid.

Stage 4.

To a solution of (3aR,5R,6R,6aR)-6-(cyclopropylethynyl)-5-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-6-ylacetate (8.15 g, 27.50 mmH, 1 eq.) in dichloroethane (80 mt) at 20°C under N2 atmosphere, add Rh ₂ (OAc) ₄ (1.00 g, 2.26 mmH, 0.08 eq.) and solution diethyl diazomatonate (10.24 g, 55.01 mmot, 2 eq.) in dichtoethane (20 mt). Stir the zetan solution for 16 hours before evaporating it. The crude residue is purified by combi-phthash chromatography with sitikaget (15-50% ethyl acetate in petroleum ether) to form diethyl-2-(((3aR,5R,6R,6aR)-6-acetoxy-6(πictopropylethynyl)-2 ,2-dimethyl-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl)methoxy)malonate (9.52 g, 76% yield) as a yellow paste.

Stage 5.

To a solution of crude diethyl-2-(((3aR,5R,6R,6aR)-6-acetoxy-6-(cyclopropylethynyl)-2,2dimethyl-tetrahydrofuro[2,3-d][1,3]dioxol-5- yl)methoxy)malonate (4.50 g, 9.90 mmol, 1 eq.) in DMF

- 57 046381 (50 ml) Cs ₂ CO ₃ (9.68 g, 29.71 mmol, 3 eq.) and benzyl bromide (1.76 ml, 14.85 mmol, 1.5 eq.) were added at 20°C. . The suspension was stirred for 16 hours, then diluted with water (80 ml) and extracted using ethyl acetate (3x80 ml). The combined organic layer was washed with water (200 ml), brine (200 ml), dried with Na ₂ SO ₄ , filtered and evaporated. The crude product was purified by combi-flash silica gel chromatography (15-50% ethyl acetate in petroleum ether) to form diethyl-2-(((3aR,5R,6R,6aR)-6-acetoxy-6-(cyclopropylethynyl)- 2,2dimethyltetrahydrofuro[2,3^][1,3]dioxol-5-yl)methoxy)-2-benzylmalonate (4.10 g, 76% yield) as a colorless syrup.

Stage 6.

To a solution of diethyl-2-(((3aR,5R,6R,6aR)-6-acetoxy-6-(cyclopropylethynyl)-2,2-dimethylmetrahydrofuro[2,3^][1,3]dioxol-5-yl) methoxy)-2-benzyl malonate (4.10 g, 7.53 mmol, 1 eq.) in DCM (50 ml) was added with H2O (10 ml) and TFA (50 ml) at 0°C. The solution was stirred at 20°C for 2 hours before quenching with saturated aq. NaHCO ₃ solution (80 ml) to pH ~7. The reaction mixture was extracted using DCM (100 ml). The organic layer was washed with brine (10 ml), dried using Na ₂ SO ₄ , filtered and evaporated to give crude diethyl 2-(((2R,3S,4R)-3acetoxy-3-(cyclopropylethynyl)-4,5- Dihydroxytetrahydrofuran-2-yl)methoxy)-2-benzylmalonate (3.80 g) as yellow gum.

Stage 7.

To a solution of crude diethyl 2-(((2R,3S,4R)-3-acetoxy-3-(cyclopropylethynyl)-4,5dihydroxytetrahydrofuran-2-yl)methoxy)-2-benzylmalonate (3.80 g, 7.53 mmol, 1 eq.) in pyridine (40 ml) at 20°C was added 4-DMAP (2.76 g, 22.60 mmol, 3 eq.) and Ac ₂ O (5.64 ml, 60.25 mmol, 8 eq.). The solution was stirred for 16 hours, then diluted with water (80 ml) and extracted using ethyl acetate (2x80 ml). The combined organic layer was washed with water (150 ml), brine (150 ml), dried using Na2SO4, filtered and evaporated. The crude product was purified by combi-flash silica gel chromatography (10-50% ethyl acetate in petroleum ether) to give diethyl-2-benzyl-2-(((2R,3R,4R)-3,4,5-triacetoxy- 3-(cyclopropylethynyl)tetrahydrofuran-2yl)methoxy)malonate (2.91 g, 66% yield) as yellow gum.

Stage 8.

To a solution of diethyl 2-benzyl-2-(((2R,3R,4R)-3,4,5-triacetoxy-3-(cyclopropylethynyl)tetrahydrofuran-2-yl)methoxy)malonate (980 mg, 1.66 mmol , 1 equiv.) in MeCN (24 ml) at 25°C was added 2chloroadenine (338.80 mg, 2.00 mmol, 1.2 equiv.) and BSA (987.71 μl, 4.00 mmol, 2.4 eq.). The suspension was stirred at 65°C for 0.5 h until it became clear. The resulting solution was cooled to 0°C followed by the addition of TMSOTf (444.06 mg, 2.00 mmol, 361.03 μL, 1.2 eq.) dropwise. The reaction mixture was stirred at 40°C for 4 hours, after which it was left to cool to room temperature. The reaction mixture was diluted with water (20 ml) and extracted using ethyl acetate (3x20 ml). The combined organic layer was washed with brine (50 ml), dried with Na2SO4, filtered and evaporated. The crude product was purified by combi-flash silica gel chromatography (30-80% ethyl acetate in petroleum ether) to give diethyl-2benzyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6 -amino-2-chloro-9H-purin-9-yl)-3-(cyclopropylethynyl)tetrahydrofuran-2-yl)methoxy)malonate (270 mg, 23% yield) as yellow gum.

Stage 9.

To a solution of diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-amino-2-chloro-9H-purin-9-yl)-3( cyclopropylethynyl)tetrahydrofuran-2-yl)methoxy)malonate (270 mg, 386.75 µmol, 1 eq.) in THF (8 ml) was added aq. LiOH solution (1 M, 5.80 ml, 15 eq.). The mixture was stirred at 20°C for 16 hours and then treated with 1 N HCl to adjust the pH to 5. The mixture was evaporated. The crude residue was purified by preparative HPLC to give the title compound (23 mg, 11% yield) as a white solid.

1H NMR (400 MHz, CD3OD) δ ppm. 8.04 (s, 1H) 7.14-7.27 (m, 2H) 7.01-7.08 (m, 3H) 5.92 (d, J=6.63 Hz, 1H) 4.70 -4.83 (m, 1H) 4.24 (t, J=3.50 Hz, 1H) 4.02 (t, J=3.31 Hz, 2H) 3.31-3.45 (m, 2H ) 1.25-1.33 (m, 1H) 0.72-0.79 (m, 2H) 0.63-0.71 (m, 2H); LC/MS [M+H] = 559.0.

- 58 046381

Example 5. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-nurin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2- yl)methoxy)-2-((2-chloropyridin-4-yl)methyl)malonic acid

Proceeding as described in Example 1 above, by replacing BnBr with 2-chloro-4-(chloromethyl)pyridine, the title compound was obtained as a white solid.

¹ H NMR (CD3OD, 400 MHz) δ ppm. 8.40 (s, 1H), 8.00 (d, J=5.13 Hz, 1H), 7.36 (s, 1H), 7.23 (d, J=5.13 Hz, 1H), 6.01 (d, J=7.63 Hz, 1H), 5.08 (d, J=7.63 Hz, 1H), 4.39 (dd, J=4.88, 2.75 Hz, 1H ), 4.16 (dd, J=10.07, 5.19 Hz, 1H), 4.05 (dd, J=10.01, 2.63 Hz, 1H), 3.47 (s, 2H) , 3.05 (s, 1H); LC/MS [M+H] = 553.1.

Example 6. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3ethynyl-3,4 -dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

Example 6

Stage 1.

To a mixture of (3aR,5R,6R,6aR)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-6-ethynyl-2,2-dimethyltetrahydrofuro[2,3^][1,3]dioxol-6- yl acetate (27.4 g, 55.39 mmol, 1 eq.) in THF (250 ml) at 0°C add AcOH (2.38 ml, 41.54 mmol, 0.75 eq.) in TBAF (1 M , 83.09 ml, 1.5 eq.). The mixture was stirred at 15°C for 15 hours before partitioning it between water (800 ml) and EtOAc (300 ml). The aqueous phase was further extracted using EtOAc (3x200 ml). The combined organic layer was washed with brine (300 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure to leave a residue. The residue was purified by column chromatography on SiO ₂ (1133% EtOAc in petroleum ether) to give (3aR,5R,6R,6aR)-6-ethynyl-5-(hydroxymethyl)-2,2dimethyltetrahydrofuro-[2,3^][ 1,3]dioxol-6-ylacetate (15.2 g, 91% yield) as a light yellow solid.

Stage 2.

To a solution of (3aR,5R,6R,6aR)-6-ethynyl-5-(hydroxymethyl)-2,2-dimethyltetrahydrofuro-[2,3d][1,3]dioxol-6-yl acetate (15.2 g, 59 .32 mmol, 1 eq.) in dichloroethane (150 ml) at 0°C was added Rh ₂ (OAc) ₄ (1.31 g, 2.97 mmol, 0.05 eq.) and diethyldiazomalonate (13.25 g, 71.18 mmol, 1.2 eq.) in dichloroethane (30 ml). The mixture was stirred at 15°C under N ₂ for 15 hours, after which additional diethyl diazomalonate (6 g) in dichloroethane (15 ml) was added. The mixture was further stirred at 15°C for 2 hours, after which it was evaporated under reduced pressure. The crude residue was purified by silica gel column chromatography (11-33% EtOAc in petroleum ether) with

- 59 046381 by the formation of diethyl-2-(((3aK,5K,6K,6aK)-6-acetoxy-6-ethynyl-2,2-dimethyltetragvdrofuro-[2,3d][1,3]dioxol-5-yl) methoxy)malonate (15 g, 61% yield) as a white solid.

Stage 3.

To a mixture of diethyl-2-(((3aR,5R,6R,6aR)-6-acetoxy-6-ethynyl-2,2-dimethyltetrahydrofuro-[2,3d][1,3]dioxol-5-yl)methoxy) malonate (14 g, 33.78 mmol, 1 eq.) in DMF (140 ml) at 25°C was added Cs ₂ CO ₃ (22.01 g, 67.57 mmol, 2 eq.) and BnBr (6.02 ml, 50.68 mmol, 1.5 eq.). The mixture was stirred at 25°C for 3 hours, then filtered and the filter cake washed with EtOAc (50 ml). The mixture was diluted with water (400 ml) and extracted with EtOAc (3x150 ml). The combined organic layer was washed with brine (200 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (0-33% EtOAc in petroleum ether) to give diethyl 2-(((3aR,5R,6R,6aR)-6-acetoxy-6-ethynyl-2,2-dimethyltetrahydrofuro[ 2,3C][1,3]dioxol-5-yl)methoxy)-2-benzyl malonate (13.3 g, 78% yield) as a white solid.

Stage 4.

To a solution of diethyl-2-(((3aR,5R,6R,6aR)-6-acetoxy-6-ethynyl-2,2-dimethyltetrahydrofuro[2,3d][1,3]dioxol-5-yl)methoxy)- 2-benzylmalonate (13.20 g, 26.16 mmol, 1 eq.) in DCM (100 ml) and H2O (20 ml, 1.11 mol, 42 eq.) added TFA (100 ml, 1.35 mol, 52 eq. .). The mixture was stirred at 15°C for 12 hours, then water (200 ml) was added. The aqueous phase was extracted using DCM (2x100 ml). The combined organic extract was washed with sat. aq. NaHCO3 solution (2x100 ml), brine (100 ml), dried over anhydrous Na2SO4, filtered and evaporated under reduced pressure. The residue was purified by flash column chromatography (SiO ₂ , petroleum ether:ethyl acetate=10:1 to 1:3) to give diethyl-2-benzyl-2-(((2R,3S,4R)-3-ethynyl- 3,4,5trihydroxytetrahydrofuran-2-yl)methoxy) malonate (7.8 g, 71% yield) as a colorless oil.

Stage 5.

To a mixture of diethyl 2-benzyl-2-(((2R,3S,4R)-3-ethynyl-3,4,5-trihydroxytetrahydrofuran-2-yl)methoxy)malonate (7.8 g, 18.46 mmol, 1 eq.) in pyridine (70 ml) at 15°C was added 4-DMAP (6.77 g, 55.39 mmol, 3 eq.) and Ac ₂ O (17.29 ml, 184.65 mmol, 10 eq. .). The mixture was stirred for 15 hours, then water (400 ml) was added. The mixture was extracted using EtOAc (3x200 ml). The combined organic layer was washed with 1 N aq. HCl solution (2x200 ml), saturated with water, NaHCO ₃ solution (300 ml), brine (300 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The residue was purified by silica gel flash column chromatography (petroleum ether: Ethyl acetate=1:0 to 2:1) to obtain the desired product (7 g). This product was triturated with EtOH (10 ml) and filtered to give diethyl 2-benzyl-2-(((2R,3R,4R)-3,4,5-triacetoxy3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (3 .79 g, yield 37%) as a white solid. The filtrate was evaporated under reduced pressure to obtain additional product with minor impurities (3 g).

Stage 6.

To a solution of diethyl 2-benzyl-2-(((2R,3R,4R)-3,4,5-triacetoxy-3-ethynyltetrahydrofuran-2yl)methoxy)malonate (116 mg, 0.21 mmol, 1.0 eq. .) uracil (28 mg, 0.25 mmol, 1.2 equiv) was added to MeCN (3 ml) at 25°C and then N,O-bis-(trimethylsilyl)acetamide (BSA) (124 µl, 0. 51 mmol, 2.4 eq.). The resulting suspension was heated at 65°C for 30 minutes until it became transparent. The reaction mixture was cooled to 0°C and then TMSOTf (46 μl, 0.25 mmol, 1.2 eq.) was added dropwise. The reaction mixture was left to warm up on its own and then heated at 65°C for 3 hours since all the starting material had been consumed. The reaction was quenched with cold saturated aq. NaHCO ₃ solution (3 ml), then diluted with EtOAc (15 ml). The organic layer was separated, washed with H ₂ O (2x10 ml), brine, dried (MgSO ₄ ), filtered and evaporated. The crude residue was purified by silica gel flash column chromatography (0-75% EtOAc in hexanes) to give diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5- (2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (104 mg, 82% yield).

Stage 7.

To a solution of diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl) -3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (100 mg, 0.166 µmol, 1 eq.) in a mixture of THF (1 ml) and MeOH (2 ml) was added aq. LiOH solution (1 M, 3 ml). The mixture was stirred at 40°C for 24 hours, after which volatile organic compounds were removed under reduced pressure. The residue was diluted with water (2 ml) and treated with 1 N HCl to adjust the pH to 4. The mixture was extracted using EtOAc (3x10 ml). The combined organic layer was washed with brine, dried (MgSO4), filtered and evaporated to give the title compound (67 mg) as a white solid.

¹ H NMR (CD3OD, 300 MHz) δ 8.09 (br. s, 1H), 7.86 (d, J=8 Hz, 1H), 7.33-7.16 (m, 5H), 6, 01 (d, J=7 Hz, 1H), 5.07 (d, J=8 Hz, 1H), 4.45 (d, J=7 Hz, 1H), 4.22 (br. s, 1H) , 4.11-3.94 (m, 2H), 3.55-3.31 (m, 2H), 2.95 (s, 1H); LC/MS [M+H] = 461.0.

- 60 046381

Example 7. Synthesis of 2-(((2R.3S.4R.5R)-5-(6-aMUHO-2-xjop-9l 1-11U ^; rin-9-yl)-3-ethynyl-3.4-dihydroxytetrahydrofuran-2 -yl)methoxy)-2-(pyridin-4-ylmethyl)malonic acid

Example 7

Stage 1.

To a mixture of diethyl-2-(((3aR,5R,6R,6aR)-6-acetoxy-6-ethynyl-2,2-dimethyltetrahydrofuro-[2,3d][1,3]dioxol-5-yl)methoxy) malonate (1.2 g, 2.90 mmol, 1 eq.) in DMF (20 ml) at 20°C was added Cs ₂ CO ₃ (6.60 g, 20.27 mmol, 7 eq.) and 4-( chloromethyl)pyridine hydrochloride (1.90 g, 11.58 mmol, 4 eq.). The mixture was stirred for 2 hours, then filtered and the filter cake washed with EtOAc (20 ml). The filtrate was diluted with water (60 ml) and extracted using EtOAc (3x50 ml). The combined extract was washed with water (2x50 ml), saturated aq. NH ₄ Cl solution (50 ml), brine (50 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The residue was purified by column chromatography on SiO ₂ (14-33% EtOAc in petroleum ether) to give diethyl 2-(((3aR,5R,6R,6aR)-6-acetoxy-6-ethynyl-2,2-dimethyltetrahydrofuro [2,3-d][1,3]dioxol5-yl)methoxy)-2-(pyridin-4-ylmethyl)malonate (900 mg, 61% yield) as a yellow oil.

Stage 2.

To a mixture of diethyl-2-(((3aR,5R,6R,6aR)-6-acetoxy-6-ethynyl-2,2-dimethyltetrahydrofuro[2,3d][1,3]dioxol-5-yl)methoxy)- TFA (5 ml, 67.53 mol, 37.93 eq.). The mixture was stirred at 20°C for 12 hours and then evaporated under reduced pressure. The crude residue was azeotroped with DCM (3x10 ml) under reduced pressure to give crude diethyl 2-(((2R,3S,4R)-3-ethynyl-3,4,5-trihydroxytetrahydrofuran-2-yl)methoxy)- 2-(pyridin-4ylmethyl)malonate (1.1 g) as a brown oil.

Stage 3.

To a mixture of crude diethyl 2-(((2R,3S,4R)-3-ethynyl-3,4,5-trihydroxytetrahydrofuran-2yl)methoxy)-2-(pyridin-4-ylmethyl)malonate (1.1 g, 2.60 mmol, 1 equiv.) in pyridine (8 ml) at 20°C, 4-DMAP (952.17 mg, 7.79 mmol, 3 equiv.) and Ac ₂ O (2.43 ml, 25, 98 mmol, 10 eq.). The mixture was stirred for 12 hours before partitioning it between water (30 ml) and EtOAc (20 ml). The aqueous phase was further extracted using EtOAc (2x20 ml). The combined extract was washed with water (20 ml), 0.5 N aq. lCl solution (2x10 ml), saline (20 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The residue was purified by flash column chromatography on SiO2 (25-50% EtOAc in petroleum ether) to give diethyl-2-(pyridin4-ylmethyl)-2-(((2R,3R,4R)-3,4,5- triacetoxy-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (640 mg, 45% yield) as brown syrup.

Stage 4.

To a mixture of diethyl 2-(pyridin-4-ylmethyl)-2-(((2R,3R,4R)-3,4,5-triacetoxy-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (40 mg, 72 .79 µmol, 1 equiv.) and 2-chloro-adenine (13.58 mg, 80.07 µmol, 1.1 equiv.) in MeCN (1.5 ml) was added BSA (44.98 µl, 181.98 µmol, 2.5 eq.) at 25°C in an N2 atmosphere. The mixture was stirred at 65°C for 0.5 h, then cooled to 0°C, after which TMSOTf (26.31 μl, 145.58 μmol, 2 eq.) was added dropwise. The mixture was stirred at 0°C for 0.5 h and then at 65°C for 2 h. The reaction mixture was cooled to room temperature and quenched with saturated aq. NaHCO ₃ solution (6 ml). The mixture was extracted using EtOAc (3x8 ml). The combined organic layer was washed with brine (5 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The residue was purified by preparative TLC (EtOAc) to give diethyl 2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-amino-2-chloro-9H-purine-9- yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)-2-(pyridin-4-ylmethyl)malonate (13 mg, 26% yield) as yellow gum.

Stage 5.

To a solution of diethyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-amino-2-chloro-9H-purin-9-yl)-3ethynyltetrahydrofuran-2-yl) methoxy)-2-(pyridin-4-ylmethyl)malonate (50 mg, 75.87 µmol, 1 eq.) in THF (1 ml) was added 1 N aq. LiOH solution (1 ml). The mixture was stirred at 50°C for 1 hour, then cooled to room temperature and adjusted to pH 6-7 with 2 N aq. HCl solution. The mixture was evaporated under reduced pressure and the residue was purified using preparative HPLC

- 61 046381 phase to obtain the title compound (34 mg) as a white solid.

1H NMR (CD3OD, 300 MHz) δ 8.50 (br.s, 1H), 8.32 (d, J=4 Hz, 2H), 7.50 (d, J=5 Hz, 2H), 6, 01 (d, J=7 Hz, 1H), 4.80 (d, J=6 Hz, 1H), 4.38 (k, J=3 Hz, 1H), 4.10-3.95 (m, 2H), 3.45 (br.s, 2H), 3.06 (s, 1H); LC/MS [M+H] = 519.0.

Example 8. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2- yl)methoxy)-2-(furan-3-ylmethyl)malonic acid

Example 8

Stage 1.

To a mixture of PPh ₃ (4.28 g, 16.31 mmol, 1.6 eq.) and CBr ₄ (4.06 g, 12.23 mmol, 1.2 eq.) in DCM (20 ml) at 0° C under N ₂ atmosphere, furan-3-ylmethanol (1 g, 10.19 mmol, 1 eq.) was added dropwise to DCM (5 ml). The mixture was stirred at 20°C for 2 hours before quenching with saturated aq. NaHCO ₃ solution (30 ml), then extracted using EtOAc (2x15 ml). The combined organic layer was washed with brine (30 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure to obtain crude 3-(bromomethyl)furan (2.9 g) as a yellow gum, which was used in the next step directly without additional cleaning.

Stage 2.

To a solution of diethyl 2-(((3aR,5R,6R,6aR)-6-acetoxy-6-ethynyl-2,2-dimethyltetrahydrofuro[2,3d][1,3]dioxol-5-yl)methoxy)malonate (1 g, 2.41 mmol, 1 eq.) in DMF (5 ml) at 20°C was added Cs ₂ CO ₃ (2.36 g, 7.24 mmol, 3 eq.) and crude 3-(bromomethyl) furan (2.9 g) in DMF (6 ml). The mixture was stirred for 2 hours before partitioning it between water (30 ml) and EtOAc (30 ml). The aqueous phase was extracted using EtOAc (3x15 ml). The combined organic extract was washed with water (20 ml), saturated aq. NH ₄ Cl solution (2x20 ml), saline (20 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The residue was purified by column chromatography on SiO ₂ (10-25% EtOAc in petroleum ether) to give diethyl 2(((3aR,5R,6R,6aR)-6-acetoxy-6-ethynyl-2,2-dimethyltetrahydrofuro[ 2,3-d][1,3]dioxol-5-yl)methoxy)-2(furan-3-ylmethyl)malonate (375 mg, 31% yield) as a yellow oil.

Stage 3.

To a mixture of diethyl-2-(((3aR,5R,6R,6aR)-6-acetoxy-6-ethynyl-2,2-dimethyltetrahydrofuro[2,3d][1,3]dioxol-5-yl)methoxy)- 2-(furan-3-ylmethyl)malonate (375 mg, 758.36 µmol, 1 eq.) in DCM (2 ml) and H2O (0.4 ml, 22.20 mmol, 29 eq.) added TFA (2 ml, 27.01 mmol, 36 eq.). The mixture was stirred at 20°C for 12 hours, then evaporated under reduced pressure to obtain crude diethyl 2-(((2R,3S,4R)-3-ethynyl-3,4,5-trihydroxytetrahydrofuran-2-yl)methoxy )-2-(furan-3-ylmethyl)malonate (420 mg) as an oil, which was used in the next step without further purification.

Stage 4.

To a mixture of crude diethyl 2-(((2R,3S,4R)-3-ethynyl-3,4,5-trihydroxytetrahydrofuran-2yl)methoxy)-2-(furan-3-ylmethyl)malonate (420 mg, 1. 02 mmol, 1 eq.) in pyridine (4 ml) at 20°C, Ac ₂ O (954 μl, 10.18 mmol, 10 eq.) and 4-DMAP (373 mg, 3.06 mmol, 3 eq.) were added. ). The mixture was stirred for 12 hours before partitioning it between water (15 ml) and EtOAc (10 ml). The aqueous phase was extracted using EtOAc (2x20 ml). The combined organic extract was washed with water (10 ml), 0.5N aq. HCl solution (2x5 ml), saline (10 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated. The residue was purified by preparative TLC (petroleum ether: EtOAc=3:1) to give diethyl-2-(furan-3-ylmethyl)-2-(((2R,3R,4R)-3,4,5-triacetoxy- 3ethynyltetrahydrofuran-2-yl)methoxy)malonate (95 mg, 17% yield) as a yellow oil.

Stage 5.

To a mixture of diethyl 2-(furan-3-ylmethyl)-2-(((2R,3R,4R)-3,4,5-triacetoxy-3-ethynyltetrahydrofuran-2yl)methoxy)malonate (50 mg, 92.85 µmol, 1 equiv.) and 2-chloro-adenine (17.32 mg, 102.14 µmol, 1.1 equiv.) in MeCN (1.2 ml) was added BSA (57.38 µl, 232.13 µmol, 2.5 eq.) at 25°C in an N2 atmosphere. The mixture was stirred at 65°C for 0.5 h, then cooled to 0°C, after which TMSOTf (33.56 μl, 185.70 μmol, 2 eq.) was added dropwise. The mixture was stirred at 0°C for 0.5 h and then at 65°C for

- 62 046381 for 2 hours, after which it was cooled to room temperature and quenched with saturated aq. NaHCO3 solution (2 ml). The mixture was extracted using EtOAc (3x2 ml). The combined organic layer was washed with brine (2 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The residue was purified by preparative TLC (petroleum ether: EtOAc=1:1) to give diethyl 2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-amino-2-chloro -9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)-2-(furan-3-ylmethyl)malonate (17 mg, 29% yield) as yellow gum.

Stage 6.

To a solution of diethyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-amino-2-chloro-9H-purin-9-yl)-3ethynyltetrahydrofuran-2-yl) methoxy)-2-(furan-3-ylmethyl)malonate (33 mg, 50.92 µmol, 1 eq.) in THF (1 ml) was added 1 N aq. LiOH solution (1 ml). The mixture was stirred at 20°C for 3 hours, then extracted using EtOAc (2 ml). The organic layer was removed. The pH of the aqueous phase was adjusted to 2-3 using 2 N aq. HCl before extraction using EtOAc (4x5 ml). The combined organic extract was washed with brine (3 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The residue was dissolved in a mixture of MeCN (1 ml) and H2O (1 ml) and then dried by lyophilization to obtain the title compound (10.0 mg, 37% yield) as a white solid.

1H NMR (400 MHz, DMSOD6) δ ppm. 8.48 (s, 1H) 7.81 (br.s, 2H) 7.35 (s, 2H) 6.29 (s, 1H) 6.22 (br.s, 1H) 6.02 (br. d, J=6.50 Hz, 1H) 5.83 (d, J=7.50 Hz, 1H) 4.75-4.90 (m, 1H) 4.16 (dd, J=4.75, 2.75 Hz, 1H) 3.92 (br. dd, J=10.07, 5.07 Hz, 1H) 3.77 (br. d, J=8.00 Hz, 1H) 3.48 (s , 1H) 3.08 (s, 2H); LC/MS [M+H] = 507.9.

Example 9. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2- yl)methoxy)-2-(4-(2-oxotetrahydropyrimidin-1 (2H-yl)benzyl)malonic acid

Stage 1.

To a solution of diethyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-N,N'-(bis-(tert-butoxycarbonyl)amino)2-chloro-9H- purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (7.26 g, 4.92 µmol, 1 eq.) in DMF (80 ml) at 25°C and added K2CO3 (13.60 g, 98.40 mmol, 20 equiv.). The reaction mixture was stirred for 0.5 h and then 1-(bromomethyl)-4-nitrobenzene (15.94 g, 73.80 mmol, 15 eq.) was added. The mixture was stirred for 24 hours, then diluted with H ₂ O (300 ml) and extracted using EtOAc (3x60 ml). The combined organic layer was washed with brine (100 ml), dried over Na ₂ SO ₄ , filtered and evaporated. The crude residue was purified by silica gel flash column chromatography (petroleum ether: EtOAc=10:1-2:1) to give diethyl-2(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6 -((tert-butoxycarbonyl)amino)-2-chloro-9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)-2-(4-nitrobenzyl)malonate (2.36 g) was prepared from in the form of brown kamma di.

Stage 2.

To a solution of diethyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-((tert-butoxycarbonyl)amino)-2-chloro9H-purin-9-yl)-3 -ethynyltetrahydrofuran-2-yl)methoxy)-2-(4-nitrobenzyl)malonate (2.26 g, 2.81 mmol, 1 eq.) in EtOH (23 ml) was added with Fe (786 mg) at 0°C , 14.07 mmol, 5 eq.) and NH ₄ Cl (151 mg, 2.81 mmol, 1 eq.) in H2O (8.5 ml). The reaction mixture was stirred at 50°C for 4 hours, then filtered and the filtrate was evaporated. The crude residue was purified by silica gel flash column chromatography (petroleum ether: EtOAc=1:0-1:1) to give diethyl-2-(4-aminobenzyl)-2(((2R,3R,4R,5R)- 3,4-diacetoxy-5-(6-((tert-butoxycarbonyl)-amino)-2-chloro-9H-purin-9-yl)-3-ethyltetrahydrofuran-2-yl)methoxy)malonate (280 mg) in in the form of yellow foam.

Stage 3.

To a solution of diethyl-2-(4-aminobenzyl)-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-((tertbutoxycarbonyl)amino)-2-chloro-9H- purin-9-yl)-3-ethyltetrahydrofuran-2-yl)methoxy)malonate (280 mg, 362.14 µmol, 1 eq.) in DCM (3 ml) at 0°C and added 1-chloro-3-isocyanatopropane (86.59 mg, 724.28 µmol, 2 eq.). The reaction mixture was stirred at 25°C for 16 h before evaporation. The crude residue was purified by preparative TLC (petroleum ether: EtOAc=1:0-1:1) with

- 63 046381 by the formation of gHOTun-2-(4-(3-(3-xTOpnponun)ypeugo)6eH3un)-2-(((2R,3R,4R,5R)-3,4-gua^OKCu-5-(6 ((tert-butoxycarbonyl)amino)-2-chloro-9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (120 mg, 33% yield) as a foam.

Stage 4.

To a solution of diethyl-2-(4-(3-(3-chloropropyl)ureido)benzyl)-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6((tert- butoxycarbonyl)amino)-2-chloro-9H-pyrin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (120 mg, 134.42 µmol, 1 eq.) in THF (1.2 ml) at 0°C, NaH (11 mg, 268.84 µmol, 60% in mineral oil, 2 eq.) was added. The reaction mixture was stirred at 25°C for 2 hours, then quenched with H2O (0.2 ml) at 0°C. After this, the reaction mixture was stirred at 25°C for 16 hours. The reaction mixture was acidified to pH 3-4 using 1 N aq. HCl solution and then extracted using EtOAc (3x5 ml). The combined organic layer was washed with brine (15 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated to form crude 2-(((2R,3S,4R,5R)-5-(6((tert-butoxycarbonyl)amino )-2-chloro-9H-pyrin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrophyran-2yl)methoxy)-2-(4-(2-oxo-tetrahydropyrimidin-1(2H)-yl)benzyl )malonic acid (77 mg, 73% yield) as a white solid.

Stage 5.

To a solution of crude 2-(((2R,3S,4R,5R)-5-(6-((tert-butoxycarbonyl)amino)-2-chloro-9H-pyrin9-yl)-3-ethynyl-3,4- dihydroxytetrahydropyran-2-yl)methoxy)-2-(4-(2-oxo-tetrahydropyrimidin-1(2H)yl)benzyl)malonic acid (76 mg, 106.13 µmol, 1 eq.) in DCM (0.5 ml) at 0°C, TFA (0.25 ml, 3.38 mmol, 32 eq.) was added. The reaction mixture was stirred at 25°C for 2 h before evaporation. The residue was re-dissolved with saturated aq. NaHCO ₃ solution (5 ml) and extracted using EtOAc (3x5 ml). The combined organic layer was washed with brine (15 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated. The crude residue was purified by preparative reverse phase HPLC to give the title compound (8.6 mg, 12% yield) as a white solid.

^1H NMR (CD3OD, 300 MHz) δ 8.28 (s, 1H), 7.11 (d, J=8.52 Hz, 2H), 7.02 (d, J=8.52 Hz, 2H) , 5.99 (d, J=7.44 Hz, 1H), 4.79 (d, J=7.41 Hz, 1H), 4.29 (t, J=2.76 Hz, 1H), 4 .01-3.91 (m, 2H), 3.54-3.41 (m, 4H), 3.363.32 (m, 2H), 3.05 (s, 1H), 2.03-1.94 (m, 2H); LC/MS [M+H] = 616.2.

Example 10. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-pyrin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl) methoxy)-2-(4-(2-oxopiperidin-1-yl)benzyl)malonic acid

Example 10

Stage 1.

To a solution of crude diethyl-2-(4-aminobenzyl)-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-((tertbutoxycarbonyl)amino)-2-chloro-9H -pyrin-9-yl)-3-ethyltetrahydrofuran-2-yl)methoxy)malonate (140 mg, 160 µmol, 1 eq.) was added to DCM (2 ml) at 25°C with TEA (107 mg, 1.06 mmol , 147 µl, 6.59 eq.), followed by 5-chloropentanoyl chloride (24.9 µl, 192 µmol, 1.2 eq.). The mixture was stirred for 1 hour before partitioning it between DCM (20 ml) and H2O (20 ml). The organic phase was washed with H2O (10 ml), dried over Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The residue was purified by preparative TLC (petroleum ether: EtOAc=1:1) to give diethyl 2-(4-(5chloropentanamido)benzyl)-2-(((2R,3R,4R,5R)-3,4-diacetoxy -5-(6-((tert-butoxycarbonyl)amino)-2-chloro9H-pyrin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (130 mg, 69% yield) as a yellow foam.

Stage 2.

To a solution of diethyl-2-(4-(5-chloropentanamido)benzyl)-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-((tertbutoxycarbonyl)amino)-2 -chlor-9H-pyrin-9-yl)-3-ethynyltempahydrofuran-2-yl)methoxy)malonate (104 mg, 105 µmol, 1 equiv.) in THF (2 ml) at 25°C was added NaH (25.2 mg, 630 µmol, 60% in mineral oil, 6 eq.). The mixture was stirred for 4 hours and then quenched with H ₂ O (1 ml). The reaction mixture was stirred at 20°C for 14 hours before partitioning it between EtOAc (10 ml) and water (20 ml). The aqueous phase was acidified to pH 5-6 with 2 N aq. HCl solution before partitioning it between EtOAc (20 ml) and brine (10 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure with

- 64 046381 by the formation of crude 2-(((2R,3 S,4R,5R)-5 -(6-((tert-butoxycarbonyl)amino)-2-chloro-9H-purin-9-yl)-3 ethynyl- 3,4-dihydroxytetrahydrofuran-2-yl)methoxy)-2-(4-(2-oxopiperidin-1 -yl)benzyl)malonic acid (58 mg) as a colorless gum.

Stage 3.

To a mixture of crude 2-(((2R,3S,4R,5R)-5-(6-((tert-butoxycarbonyl)amino)-2-chloro-9H-purin-9yl)-3-ethynyl-3,4- dihydroxytetrahydrofuran-2-yl)methoxy)-2-(4-(2-oxopiperidin-1-yl)benzyl)malonic acid (58 mg, 81 µmol, 1 eq.) in DCM (500 µl) was added TFA (400 µl, 5.40 mmol, 67 eq.). The mixture was stirred at 20°C for 2 hours, then quenched using 2 N aq. LiOH solution (500 µl). The mixture was partitioned between EtOAc (10 ml) and water (10 ml). The aqueous phase was adjusted to pH 5-6 with 2M aq. HCl solution. The aqueous phase was partitioned between EtOAc (2x20 ml) and brine (5 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The crude product was purified by preparative HPLC and lyophilized to give the title compound (6.9 mg, 14% yield) as a white solid.

¹ H NMR (400 MHz, DMSO-dg) δ ppm. 8.32 (s, 1H) 7.33 (d, J=8.53 Hz, 2H) 6.98 (d, J=8.28 Hz, 2H) 5.98 (d, J=7.53 Hz , 1H) 4.79 (m, 1H) 4.28 (t, J=2.76 Hz, 1H) 4.04 (br. s, 2H) 3.39-3.54 (m, 4H) 3, 05 (s, 1H) 2.43 (m, 2H) 1.88 (br.t, J=2.89 Hz, 4H); LC/MS [M+H] = 615.3.

Example 11. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2- yl)methoxy)-2-(4-(1-(methoxymethyl)-2-oxo-1,2-dihydropyridin-3-yl)benzyl)malonic acid

Stage 1.

K ₂ CO ₃ (4.47 g, 32.33 mmol, 2.5 eq.). The suspension was stirred for 0.5 h and then MOM-Cl (2.79 mL, 36.76 mmol, 2.84 eq.) was added dropwise. The mixture was stirred at 25°C for 15 hours, then diluted with water (40 ml) and extracted using ethyl acetate (2x30 ml). The combined organic layer was washed with brine (50 ml), dried with Na ₂ SO ₄ , filtered and evaporated. The crude residue was purified by combi-flash silica gel chromatography (20-60% ethyl acetate in petroleum ether) to give 3-bromo-1-(methoxymethyl)pyridin-2(III)-one (1.22 g, 43% yield ) in the form of a clear oil.

Stage 2.

To a solution of 3-bromo-1-(methoxymethyl)pyridin-2(III)-one (1.38 g, 6.33 mmol, 1 equiv.) and (4-(4,4,5,5tetramethyl-1,3 ,2-dioxaborolan-2-yl)phenyl)methanol (1.06 g, 6.96 mmol, 1.1 eq.) in dioxane (12 ml) was added K ₂ CO ₃ (2.62 g, 18.99 mmol , 3 eq.), Pd(dppf)Cl ₂ (463 mg, 632.89 µmol, 0.1 eq.) and H2O (4 ml). The mixture was degassed using N ₂ for 10 minutes and then heated at 80°C for 16 hours under N ₂ atmosphere. The reaction mixture was diluted with water (10 ml) and extracted using ethyl acetate (3x10 ml). The combined organic layer was washed with brine (25 ml), dried with Na ₂ SO ₄ , filtered and evaporated. The crude residue was purified by combi-flash silica gel chromatography (50-100% ethyl acetate in petroleum ether) to give 3-(4(hydroxymethyl)phenyl)-1-(methoxymethyl)pyridin-2(III)-one (1.30 g , yield 84%) in the form of yellow gum.

Stage 3.

To a solution of PPh ₃ (8.34 g, 31.80 mmol, 6 eq.) in DCM (50 ml) at -25°C was added CBr ₄ (10.55 g, 31.80 mmol, 6 eq.). The yellow suspension was stirred at -25°C for 1 hour, followed by the dropwise addition of a solution of 3-(4-(hydroxymethyl)phenyl)-1-(meximethyl)pyridin-2(1H)-one (1.30 g, 5. 30 mmol, 1 eq.) in DCM (10 ml). The yellow suspension was stirred at -25°C for 0.5 h, then diluted with MTBE (180 ml). The precipitate was filtered off and the filtrate was evaporated to give the crude product (2.8 g) as yellow gum. The crude residue was purified by combi-flash silica gel chromatography (30-100% ethyl acetate in petroleum ether) to give 3-(4-(bromomethyl)phenyl)-1-(methoxymethyl)pyrigin-2(1H)-one (760 mg, yield 46%) as a yellow solid.

Stage 4.

To a solution of diethyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-N,N'-(bis-(tert-butoxycarbonyl)amino)2-chloro-9H- purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (100 mg, 130.18 µmol, 1 eq.)

- 65 046381 in DMF (1.5 ml) at 20°C and added K ₂ CO ₃ (53.97 mg, 390.54 µmol, 3 eq.). The suspension was stirred for 0.5 h and then 3-(4-(bromomethyl)phenyl)-1-(methoxymethyl)pyridin-2(III)-one (44.13 mg, 143.20 µmol, 1.1 eq.). The suspension was stirred at 20°C for 16 hours, then diluted with water (2 ml) and extracted using ethyl acetate (3x2 ml). The combined organic layer was dried using Na ₂ SO ₄ , filtered and evaporated. The crude residue was purified by preparative TLC (petroleum ether:ethyl acetate=1:1), to give diethyl-2-(((2R,3R,4R,5R)-3,4diacetoxy-5-(6-(N,N' -(bis-(tert-butoxycarbonyl)amino)-2-chloro-9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)-2-(4-(1-(methoxymethyl)-2- oxo-1,2-dihydropyridin-3-yl)benzyl)malonate (71 mg, 55% yield) as a clear syrup.

Stage 5.

To a solution of diethyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-(N,N'-(bis-(tert-butoxycarbonyl)amino)-2-chloro- 9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)-2-(4-(1-(methoxymethyl)-2oxo-1,2-dihydropyridin-3-yl)benzyl)malonate (68 mg , 68.31 µmol, 1 equiv.) TFA (0.3 ml, 4.05 mmol, 59 equiv.) was added to DCM (1.7 ml) at 0°C.The mixture was stirred at 20°C for 2 hours before quenching with saturated aq NaHCO ₃ to adjust pH to 9. The mixture was extracted using ethyl acetate (3 x 8 ml). The combined organic layer was evaporated to give the crude product (98 mg) as yellow gum. The crude residue was purified by preparative TLC (ethyl acetate) to form diethyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-amino-2-chloro-9H-nurin-9-yl)-3ethynyltetrahydrofuran-2 -yl)methoxy)-2-(4-(1-(methoxymethyl)-2-oxo-1,2-dihydropyridin-3-yl)benzyl)malonate (21 mg, 38% yield) as a colorless syrup.

Stage 6.

To a solution of diethyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-amino-2-chloro-9H-purin-9-yl)-3ethynyltetrahydrofuran-2-yl) methoxy)-2-(4-(1-(methoxymethyl)-2-oxo-1,2-dihydropyridin-3-yl)benzyl)malonate (20 mg, 25.15 µmol, 1 eq.) in THF (1 ml ) added 1 M aq. LiOH solution (503 µl, 20 eq.). The mixture was stirred at 18°C for 22 hours, then acidified to pH 2 with 1 N aq. HCl and evaporated. The crude residue was purified by preparative HPLC and the fraction was dried by lyophilization to give the title compound (2.1 mg, 13% yield) as a white solid.

¹ H NMR (400 MHz, DMSO) δ ppm. 8.45 (br.s, 1H), 7.81 (br.s, 2H), 7.67 (dd, J=6.75, 1.75 Hz, 1H), 7.51 (br.d, J=6.75 Hz, 1H), 7.36 (br. d, J=6.88 Hz, 2H), 7.20 (br. d, J=7.50 Hz, 2H), 6.33 ( t, J=6.75 Hz, 1H), 6.20 (br. s, 1H), 6.01 (br. d, J=6.88 Hz, 1H), 5.82 (d, J=7 .50 Hz, 1H), 5.28 (s, 2H), 4.65-4.89 (m, 1H), 4.06-4.23 (m, 1H) 3.88-4.06 (m , 1H), 3.67-3.86 (m, 1H), 3.40-3.52 (m, 3H), 3.18-3.30 (m, 2H); LC/MS [M+H] = 655.1.

Example 12. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2- yl)methoxy)-2-(4-(2-oxo-1,2-dihydropyridin-3-yl)benzyl)malonic acid

Stage 1.

To a solution of diethyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-N,N'-(bis-(tert-butoxycarbonyl)amino)2-chloro-9H- purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (12.59 g, 16.39 mmol, 1 eq.) and 1-(bromomethyl)-4-iodobenzene (48.67 g, 163 .90 mmol, 10 eq.) in DMF (120 ml) at 20°C was added K2CO3 (33.98 g, 245.85 mmol, 15 eq.) The solution was stirred for 16 hours, then diluted with water (200 ml) and extracted using ethyl acetate (3x200 ml). The combined organic layer was washed with water (400 ml), brine (400 ml), dried using Na ₂ SO ₄ , filtered and evaporated. The crude residue was purified by combi-flash silica gel chromatography (15-40% ethyl acetate in petroleum ether) to give diethyl 2-(((2H,3H,4H^)-3,4-diacetoxy-5-(6- ^Ni-(bis-(tert-butoxycarbonyl)amino)-2-chloro-9H-purin-9-ul)-3-ethynyltetrahydrofuran-2-yl)methoxy)-2-(4-iodobenzyl)malonate (2.94 g, yield 18%) as a yellow solid.

Stage 2.

To a solution of diethyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-N,N'-(bis-(tert-butoxycarbonyl)amino)2-chloro-9H- purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)-2-(4-iodobenzyl)malonate (1.10 g, 1.12 mmol, 1 eq.) and (2-oxo-1,2 -dihydropyridin-3-yl)boronic acid (310.53 mg, 2.24 mmol,

- 66 046381 eq.) in dioxane (12 ml) was added K ₂ CO ₃ (463.41 mg, 3.35 mmol, 3 eq.), Pd(dppf)Cl ₂ (81.78 mg, 111.77 µmol, 0.1 eq.) and H2O (4 ml). The mixture was degassed using N2 for 10 min and then stirred at 80°C for 1 h under N2. The dark mixture was diluted with water (10 ml) and extracted using ethyl acetate (3x10 ml). The combined organic layer was washed with brine (30 ml), dried with Na ₂ SO ₄ , filtered and evaporated. The crude residue was purified by combi-flash silica gel chromatography (40-100% ethyl acetate in petroleum ether) to give diethyl 2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6 -N,N'-(bis-(tert-butoxycarbonyl)amino)-2-chloro-9H-nurin9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)-2-(4-(2-oxo-1 ,2-dihydropyridin-3-yl)benzyl)malonate (220 mg, 21% yield) as yellow gum.

Stage 3.

To a solution of diethyl-2-(((2K,3K,4K,5K)-3,4-diacetoxy-5-(6-HNa-(bis-(tert-butoxycarbonyl)amino)2-chloro-9H-nurine- 9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)-2-(4-(2-oxo-1,2-dihydropyridin-3yl)benzyl)malonate (180 mg, 189.20 µmol, 1 eq.) TFA (0.6 mL, 8.10 mmol, 43 eq.) was added to DCM (2.4 mL). The yellow solution was stirred at 20°C for 2.5 hours before quenching with saturated aq. NaHCO ₃ solution (5 ml) and extracted using ethyl acetate (3x4 ml). The combined organic layer was evaporated to give the crude product (108 mg) as yellow gum. The crude residue was purified by preparative TLC (ethyl acetate) to give diethyl-2(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-amino-2-chloro-9H-purine-9- yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)-2-(4-(2-oxo-1,2-dihydropyridin-3-yl)benzyl)malonate (23 mg, 16% yield) as a yellow solid .

Stage 4.

To a solution of diethyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-amino-2-chloro-9H-purin-9-yl)-3ethynyltetrahydrofuran-2-yl) methoxy)-2-(4-(2-oxo-1,2-dihydropyridin-3-yl)benzyl)malonate (23 mg, 30.62 µmol, 1 eq.) in THF (2.5 ml) was added 1 M aq. LiOH solution (0.6 ml, 20 eq.). The reaction mixture was stirred at 20°C for 4 h, then acidified to pH 6 with 1 N aq. HCl solution and evaporated to give the crude product (32 mg) as yellow gum. The crude residue was purified by preparative HPLC (column: YMC-Triart Prep C18 150-40 mm-7 µm; mobile phase: [water (0.225%FA)-CAN]; B%: 15%-35%, 10 min). The product was dried by lyophilization to obtain the title compound (2.1 mg, 11% yield) as a white solid.

¹ H NMR (400 MHz, CD3OD) δ ppm. 8.24 (s, 1H), 7.46 (dd, J=6.88, 1.63 Hz, 1H), 7.28-7.34 (m, 5H), 6.39 (t, J= 6.75 Hz, 1H), 5.96 (d, J=7.38 Hz, 1H), 4.77-4.84 (m, 1H), 4.29 (t, J=2.88 Hz, 1H), 4.03 (d, J=2.75 Hz, 2H), 3.38-3.51 (m, 2H), 3.04 (s, 1H); LC/MS [M+H] = 611.0.

Example 13. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2- yl)methoxy)-2-((2-carboxy[1,1'-biphenyl]-4-yl)methyl)malonic acid

Stage 1.

To a mixture of diethyl 2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-N,N'-(bis-(tert-butoxycarbonyl)amino)-2chloro-9H-purine K2CO3 (53.90 mg, 390, 03 µmol, 3 eq.). The mixture was stirred at 40°C for 0.5 h followed by the addition of methyl 4-(bromomethyl)-[1,1'-biphenyl]-2-carboxylate (79.35 mg, 260.02 µmol, 2 eq.) , which was obtained according to the procedure described in the publication D. Stoermer et al. (J. of Med. Chem. 2012, 55, 5922-5932). The mixture was stirred at 40°C for 15.5 hours, then diluted with water (4 ml) and extracted using EtOAc (3x5 ml). The combined organic layer was washed with water (2x5 ml) and brine (5 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated. The crude residue was purified by preparative TLC (petroleum ether: EtOAc=1:1) to give diethyl 2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-N,N' -(bis-(tert-butoxycarbonyl)-amino)2-chloro-9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)-2-((2-(methoxycarbonyl)-[1,1 '-biphenyl]4-yl)methyl)malonate (70 mg, 53% yield) as a colorless syrup.

- 67 046381

Stage 2.

To a solution of diethyl-2-('('('2K.,3K.,4K.,5K.)-3,4-diacetoxy-5-('6-NI-('bis-(tert-butoxycarbonyl)amino) -2-chloro-9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)-2-((2-(methoxycarbonyl)-[1,1'biphenyl]-4-yl)methyl)malonate (70 mg, 70.53 µmol, 1 eq.) to DCM (2 ml) was added TFA (0.5 ml, 6.75 mmol, 96 eq.). The mixture was stirred at 20°C for 2 hours before quenching with saturated aq. NaHCO ₃ solution to pH 7-8 and extracted using EtOAc (3x10 ml). The combined organic layer was washed with water (2x8 ml) and brine (8 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated. The crude residue was purified by preparative TLC (petroleum ether: EtOAc=1:1) to give diethyl 2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-amino-2- chlor9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)-2-((2-(methoxycarbonyl)-[1,1'-biphenyl]-4yl)methyl)malonate (40 mg, yield 71 %) in the form of a colorless syrup.

Stage 3.

To a solution of diethyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-amino-2-chloro-9H-purin-9-yl)-3ethynyltetrahydrofuran-2-yl) methoxy)-2-((2-(methoxycarbonyl)-[1,1'-biphenyl]-4-yl)methyl)malonate (30 mg, 37.87 µmol, 1 eq.) in THF (1 ml) was added 1 M aq. LiOH solution (568 µl, 15 eq.). The mixture was stirred at 25°C for 20 hours, then diluted with water (1 ml) and extracted using EtOAc (3x2 ml). The organic layer was removed. The pH of the aqueous phase was adjusted to 2 using 2 N aq. HCl with the formation of a precipitate. The precipitate was collected by filtration to obtain the desired product (28 mg) as the first batch. The aqueous phase was further extracted using EtOAc (4x2 ml). The combined organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and evaporated to give a second batch (10 mg) as a white solid. The two batches were combined and purified by preparative HPLC, and the fraction was lyophilized to give the title compound (8.0 mg, 33% yield) as a white solid.

¹ H NMR (400 MHz, CD3OD) δ ppm. 8.21 (s, 1H), 7.66 (s, 1H), 7.46 (br. d, J=7.6 Hz, 1H), 7.24-7.32 (m, 3H), 7 .12 (dd, J=7.1, 2.3 Hz, 2H), 7.07 (d, J=7.9 Hz, 1H), 5.99 (d, J=7.4 Hz, 1H) , 4.84 (br.s, 1H), 4.33 (br.s, 1H), 4.02-4.16 (m, 2H), 3.43-3.60 (m, 2H), 3 .03 (s, 1H); LC/MS [M+H] = 638.2.

Example 14. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-phenyl-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran- 2-yl)methoxy)malonic acid θ-Β(ΟΗ) ₂ ³ d(OAc) ₂ Cs ₂ CO ₃ P(C ₆ H ₄ SO ₃ Na)3 _r T MeCN:H ₂ O (2:1) j

N^N^CI ΓΊ

Example 14

Stage 1.

To a solution of 2,6-dichloroadenine (0.8 g, 4.23 mmol, 1 equiv.) in H2O (10 ml) and MeCN (5 ml) was added phenylboronic acid (464.49 mg, 3.81 mmol, 0. 9 eq.), Cs ₂ CO ₃ (3.45 g, 10.58 mmol, 2.5 eq.), Pd(OAc) ₂ (47.51 mg, 211.64 µmol, 0.05 eq.) and trisodium; 3-bis-(3-sulfonatophenyl)phosphanylbenzenesulfonate (601.50 mg, 1.06 mmol, 0.25 eq.) at 20°C under N ₂ atmosphere. The mixture was stirred at 110°C for 2 hours and then allowed to cool and diluted with H ₂ O (50 ml). The reaction mixture was extracted using EtOAc (5x50 ml). The combined organic layer was washed with brine (50 ml), dried over Na ₂ SO ₄ , filtered and evaporated. The crude product was triturated with petroleum ether (9 ml) and EtOAc (3 ml) to give 2-chloro-6-phenyl-9H-purine (140 mg, 14% yield) as a yellow solid.

Stage 2.

Diethyl-2-benzyl-2-(((2R, 3R,4R)-3,4,5-riacetoxy-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (135 mg, 585.30 µmol, 1 eq.) and BSA (347 µl, 1.40 mmol, 2. 4 equiv.) at 15°C. The mixture was stirred at 65°C for 30 minutes, causing it to become clear. The mixture was cooled to 0°C and then TMSOTf (126.91 µl, 702.35 µmol, 1.2 eq.) was added dropwise. The mixture was stirred at 0°C for 10 min and then at 65°C for 3 h , after which it was cooled and quenched with saturated aq. NaHCO ₃ (40 ml). The reaction mixture was extracted using EtOAc (2x30 ml). The combined organic layer was washed with brine (40 ml), dried over Na ₂ SO ₄ , filtered and evaporated. The crude product was first purified by silica gel flash column chromatography (petroleum ether: EtOAc=1:0-1:1) and then by preparative TLC (petroleum ether: EtOAc=1:1) to give diethyl-2-benzyl- 2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(2-chloro-6-phenyl-9H-purin-9-yl)-3ethynyltetrahydrofuran-2-yl)methoxy)malonate ( 135 mg) as yellow gum.

Stage 3.

To a solution of diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(2-chloro-6-phenyl-9H-purin-9-yl)-3ethynyltetrahydrofuran- 2-yl)methoxy)malonate (135 mg, 187.73 µmol, 1 eq.) in THF (2 ml) was added

- 68 046381

LiOH-H2O (78.77 mg, 1.88 mmol, 10 eq.) in H2O (0.2 ml) at 20°C. The mixture was heated at 45°C for 2 hours, then diluted with H2O (10 ml) and extracted using EtOAc (2 ml). The organic layer was removed and the aqueous phase was acidified with 2 N aqueous HCl to pH 2-3. The aqueous phase was then extracted using EtOAc (3x20 ml). The combined organic layer was washed with brine (30 ml), dried over Na ₂ SO ₄ , filtered and evaporated. The crude product was purified by preparative HPLC to give the title compound (27.6 mg, 24% yield) as a white solid.

¹ H NMR (400 MHz, DMSO-06) δ ppm. 9.23 (s, 1H), 8.75-8.81 (m, 2H), 7.61-7.68 (m, 3H), 7.07-7.22 (m, 5H), 6. 03 (d, J=6.53 Hz, 1H), 4.62 (d, J=6.53 Hz, 1H), 4.20 (dd, J=6.90, 2.64 Hz, 1H), 3.72 (br.dd, J=9.91, 7.15 Hz, 2H), 3.55 (s, 1H), 3.03 (br.d, J=1.51 Hz, 2H); LC/MS [M+H] = 579.1.

Example 15. Synthesis of 2-allyl-2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran- 2-yl)methoxy)malonic acid.

Example 15

Stage 1.

To a solution of diethyl-2-(((3aR,5R,6R,6aR)-6-acetoxy-6-ethynyl-2,2-dimethyltetrahydrofuro-[2,3d][1,3]dioxol-5-yl)methoxy) malonate (600 mg, 1.45 mmol, 1 eq.) in DMF (1 ml) was added allyl bromide (263 mg, 2.17 mmol, 1.5 eq.) and Cs ₂ CO ₃ (943 mg, 2.90 mmol , 2 eq.). The mixture was stirred at 20°C for 2 hours, then diluted with water (15 ml) and extracted using EtOAc (4x10 ml). The combined organic phase was washed with water (10 ml), brine (10 ml), dried over Na2SO4, filtered and evaporated to give crude diethyl 2-(((3aR,5R,6R,6aR)-6-acetoxy-6-ethynyl -2,2dimethyltetrahydrofuro[2,3D][1,3]dioxol-5-yl)methoxy)-2-allylmalonate (685 mg) as a colorless gum.

Stage 2.

To a solution of diethyl-2-(((3aR,5R,6R,6aR)-6-acetoxy-6-ethynyl-2,2-dimethyltetrahydrofuro[2,3d][1,3]dioxol-5-yl)methoxy)- 2-allylmalonate (685 mg, 1.51 mmol, 1 eq.) in DCM (5 ml) was added TFA (5 ml, 67.53 mmol, 45 eq.) and H2O (1 ml, 55.51 mmol, 37 eq. .). The mixture was stirred at 20°C for 16 hours, then diluted with water (15 ml) and adjusted to pH 7-8 using solid NaHCO ₃ . The reaction mixture was extracted with a mixture of DCM and MeOH (4x12 ml; 10:1/v:v). The combined extract was washed with sat. aq. NaHCO ₃ (8 ml), brine (8 ml), dried using Na ₂ SO ₄ , filtered and evaporated under reduced pressure to obtain crude diethyl 2-(((2R,3S,4R)-3acetoxy-3-ethynyl -4,5-dihydroxytetrahydrofuran-2-yl)methoxy)-2-allyl malonate (580 mg) as yellow gum.

Stage 3.

To a mixture of crude diethyl 2-(((2R,3S,4R)-3-acetoxy-3-ethynyl-4,5-dihydroxytetrahydrofuran-2-yl)methoxy)-2-allylmalonate (580 mg, 1.40 mmol, 1 eq.) in pyridine (5 ml) was added Ac ₂ O (1.31 ml, 14.00 mmol, 10 eq.) and 4-DMAP (513 mg, 4.20 mmol, 3 eq.). The mixture was stirred at 20°C for 15 hours, then diluted with water (15 ml) and extracted using EtOAc (3x10 ml). The combined extract was washed with 0.5 N sat. aq. HCl solution (2x8 ml), NaHCO ₃ (2x8 ml), brine (8 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: EtOAc=5:1-2:1) to give (420 mg, 60% yield) as a colorless gum.

Stage 4.

To a mixture of diethyl 2-allyl-2-(((2R,3R,4R)-3,4,5-triacetoxy-3-ethynyltetrahydrofuran-2yl)methoxy)malonate (360 mg, 722.20 µmol, 1 eq.) and 2-chloro-9H-purine-6-amine (135 mg, 794.42 µmol, 1.1 equiv.) in MeCN (5 ml) was added BSA (446.28 µl, 1.81 mmol, 2.5 equiv. .) at 25°C in N2 atmosphere. The mixture was stirred at 65°C for 0.5 h. The reaction mixture was cooled to 0°C and then TMSOTf (261.00 μl, 1.44 mmol, 2 eq.) in MeCN (1 ml) was added dropwise. The mixture was stirred at 65°C for 3 hours, then allowed to cool and quenched with saturated aq. NaHCO ₃ solution (15 ml). The mixture was then extracted, the mixture was extracted using EtOAc (4x10 ml), washed with brine (8 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated. Unrefined product

- 69 046381 was purified by combi-flash silica gel chromatography (20-40% EtOAc in petroleum ether) to form diethyl-2-allyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy -5-(6-amino-2-chloro-9H-purin-9-yl)-3ethyltetrahydrofuran-2-yl)methoxy)malonate (190 mg) as a colorless gum.

Stage 5.

To a solution of diethyl-2-allyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-amino-2-chloro-9H-purin-9-yl)-3ethyltetrahydrofuran- 2-yl)methoxy)malonate (100 mg, 164.47 µmol, 1 eq.) in THF (0.5 ml) was added LiOH-H ₂ O (6.90 mg, 164.47 µmol, 1 eq.). The mixture was stirred at 50°C for 1 hour, then diluted with water (6 ml) and extracted using EtOAc (3x4 ml). The organic layer was removed. The pH of the aqueous phase was adjusted to 2 using 2 N aq. HCl solution. The aqueous phase was then extracted using EtOAc (4x6 ml). The combined organic layer was washed with brine (6 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated to give a solid. The solid was dissolved in a mixture of MeCN (1 ml) and water (1 ml) and then lyophilized directly to give the title compound (65.2 mg, 83% yield) as a white solid.

¹ H NMR (400 MHz, CD3OD) δ ppm. 8.83 (s, 1H) 6.05 (d, J=7.5 Hz, 1H) 5.84 (br. dd, J=17.2, 10.2 Hz, 1H) 5.15 (dd, J=17.2, 1.6 Hz, 1H) 4.99-5.05 (m, 2H) 4.27 (t, J=2.5 Hz, 1H) 4.00 (dd, J=10, 2, 2.6 Hz, 1H) 3.79 (dd, J=10.3, 2.8 Hz, 1H) 3.06 (s, 1H) 2.88 (d, J=7.3 Hz, 2H ); LC/MS [M+H] = 467.9.

Example 16. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl) methoxy)-2-ethylmalonic acid

but he

Example 16

Proceeding as described in Example 15 above, but replacing allyl bromide with EtBr, afforded the title compound as a white solid.

¹ H NMR (400 MHz, DMSO-06) δ ppm. 8.53-8.83 (m, 1H) 7.81 (br. s, 2H) 5.91-6.40 (m, 2H) 5.83 (d, J=7.88 Hz, 1H) 4 .91 (d, J=7.75 Hz, 1H) 4.14 (t, J=2.88 Hz, 1H) 3.75 (dd, J=10.26, 3.25 Hz, 1H) 3, 56 (s, 1H) 3.47-3.51 (m, 1H) 1.93-2.04 (m, 2H) 0.76 (t, J=7.38 Hz, 3H); LC/MS [M+H] = 455.9.

Example 17. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl) methoxy)-2-methylmalonic acid

n<3 'he

Example 17

Proceeding as described in Example 15 above, but replacing allyl bromide with methyl bromide, afforded the title compound as a white solid.

1H NMR (400 MHz, DMSO-4 ₆ ) δ ppm. 13.38 (br.s, 2H) 8.69 (s, 1H) 7.82 (br.s, 2H) 6.17 (br.s, 1H) 5.98 (br.d, J=7, 25 Hz, 1H) 5.82 (d, J=7.63 Hz, 1H) 4.85 (br.t, J=6.94 Hz, 1H) 4.15 (t, J=2.75 Hz, 1H) 3.83 (dd, J=10.13, 3.25 Hz, 1H) 3.53-3.65 (m, 2H) 1.52 (s, 3H); LC/MS [M+H] = 441.8.

Example 18. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(thiophen-2-yl)-9H-purin-9-yl)-3- ethynyl3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

o O y-OEt . g 9 &K“ -X a. ► HA ^{Ac0 OAc} >- H IT'ci Pd(OAc) ₂ Cs ₂ CO ₃ DBU, TMSOTf, MeCN P(C ₆ H ₄ SO ₃ Na) ₃ H 91 MeCN, H ₂ O VX О KOEt Ν^Λ _Μ °Λζ°γ ^{Ν Ν} ^Ι О AX Ac<5 'OAc aq. LiOH, THF I v У 0 Koh n^A _m o no OH Example 18

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Stage 1.

4,4,5,5-tetramethyl-2- (thiophen-2-yl)-1,3,2-dioxaborolane (800.37 mg, 3.81 mmol, 0.9 eq.), Pd(OAc)2 (47.51 mg, 211.64 µmol, 0 .05 eq.), Cs ₂ CO ₃ (3.45 g, 10.58 mmol, 2.5 eq.) and triphenylphosphine-3,3'-3-trisulfonic acid trisodium salt (601.50 mg, 1.06 mmol, 0.25 eq.). The mixture was stirred at 110°C for 3 hours, after which it was allowed to cool and partitioned between EtOAc (3x20 ml) and H2O (10 ml). The organic phase was washed with H2O (2x10 ml), dried over Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The crude product was triturated (petroleum ether: EtOAc=3:1) and left for 14 hours. The precipitate was collected by suction filtration and dried to give 2-chloro-6-(thiophen-2-yl)-9H-purine (190 mg, yield 19%) as a yellow solid.

Stage 2.

To a solution of 2-chloro-6-(thiophen-2-yl)-9H-purine (140 mg, 591.51 µmol, 1 eq.) and diethyl 2-benzyl-2(((2R,3R,4R)-3 ,4,5-triacetoxy-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (324.47 mg, 591.51 µmol, 1 equiv.) in MeCN (3 ml) at 0°C was added DBU (267 µl, 1 .77 mmol, 3 eq.). The mixture was stirred at 0°C for 10 min, then TMSOTf (481 μL, 2.66 mmol, 4.5 eq.) was added dropwise. The mixture was stirred at 0°C for 30 min and then at 65°C under N2 for 14 h. The reaction mixture was allowed to cool and partitioned between EtOAc (3x20 ml) and saturated aq. NaHCO ₃ (2x10 ml). The combined organic phase was washed with brine (2x20 ml), dried over Na ₂ SO ₄ , filtered and evaporated. The crude product was purified by preparative TLC (petroleum ether: EtOAc=2:1) to give diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(2chloro- 6-(thiophen-2-yl)-9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (150 mg, 31% yield) as a white solid.

Stage 3.

To a mixture of diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(2-chloro-6-(thiophen-2-yl)-9H-purin-9yl )-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (150 mg, 206.85 µmol, 1 eq.) in THF (2 ml) was added LiOH-H ₂ O (2 M, 2 ml, 19.34 eq. ). The mixture was stirred at 25°C for 2 hours before partitioning it between EtOAc (10 ml) and water (10 ml). The aqueous phase was brought to pH~ 2 with 2M aq. HCl solution. The aqueous phase was partitioned between EtOAc (40 ml) and brine (20 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The crude product was purified by preparative HPLC (column: YMC-Triart Prep C18 150-40 mm-7 µm; mobile phase: [water (0.225%FA)-ACN]; B%: 43% - 63%, 10 min) and lyophilized to give the title compound (31.6 mg, 26% yield) as a white solid.

¹ H NMR (400 MHz, DMSO-de) δ ppm. 8.87 (s, 1H) 8.60 (dd, J=3.76, 1.25 Hz, 1H) 8.02 (dd, J=5.02, 1.00 Hz, 1H) 7.36 ( dd, J=4.89, 3.89 Hz, 1H) 7.16-7.28 (m, 2H) 6.93-7.10 (m, 3H) 6.31 (br. s, 1H) 6 .11 (br.d, J=6.02 Hz, 1H) 5.99 (d, J=7.53 Hz, 1H) 4.88-4.97 (m, 1H) 4.23 (dd, J =4.27, 2.76 Hz, 1H) 3.99 (br. dd, J=10.42, 4.39 Hz, 1H) 3.84 (br. d, J=8.53 Hz, 1H) 3.56 (s, 1H) 3.26 (s, 2H); LC/MS [M+H] = 585.0.

Example 19. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl) methoxy)-2-(4-(2-oxo-1-propyl-1,2-dihydropyridin-3-yl)benzyl)malonic acid

Stage 1.

To a solution of 3-bromopyridin-2(III)-one (3 g, 17.24 mmol, 1 eq.) in acetone (100 ml) was added K ₂ CO ₃ (11.91 g, 86.21 mmol, 5 eq. ). The suspension was stirred at 20°C for 0.5 h and then 1 bromopropane (4.71 ml, 51.73 mmol, 3 eq.) and KI (859 mg, 5.17 mmol, 0.3 eq.) were added. The mixture was stirred at 20°C for 16 hours. An additional amount of 1-bromopropane (1.0 g) was added to the reaction mixture and the mixture was further stirred at 20°C for 3 hours. The reaction mixture was diluted with water (50 ml) and extracted with using EtOAc (3x40 ml). The combined organic layer was washed with brine (100 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated. The residue was purified by combi-flash chromatography (silica gel, 20-60% EtOAc in petroleum ether) to give 3-bromo-1-propylpyridin-2(III)-one (1.23 g, 33% yield) as a clear oils

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Stage 2.

To a solution of 3-bromo-1-propylpyridin-2(1H)-one (1.79 g, 8.28c mmol, 1 eq.) and (4-(hydroxymethyl)phenyl)boronic acid (1.38 g, 9. 11 mmol, 1.1 eq.) in dioxane (18 ml) was added K2CO ₃ (3.43 g, 24.84 mmol, 3 eq.), Pd(dppf)Cl ₂ (606 mg, 828.00 µmol, 0 .1 eq.) and H2O (6 ml). The mixture was degassed using N2 for 10 min and then stirred at 80°C for 16 h under N2. The reaction mixture was cooled and filtered. The filtrate was evaporated. The residue was purified by combi-flash chromatography (silica gel, 30-50% EtOAc in petroleum ether) to give 3-(4(hydroxymethyl)phenyl)-1-propylpyridin-2(III)-one (1.84 g, 91% yield ) in the form of a clear oil.

Stage 3.

To a solution of PPh ₃ (1.94 g, 7.40 mmol, 6 eq.) in DCM (15 ml) was added CBr ₄ (2.45 g, 7.40 mmol, 6 eq.) at -25° C. Yellow the solution was stirred at -25°C for 1 hour, followed by the dropwise addition of 3-(4-(hydroxymethyl)phenyl)-1-propylpyridin-2(III)-one (300 mg, 1.23 mmol, 1 eq.) in DCM (3 ml). The yellow suspension was stirred at -25° C. for 0.5 h until a yellow suspension was obtained. The reaction mixture was diluted with MTBE (50 ml) to obtain more precipitate. The precipitate was filtered off and the filtrate was evaporated. The residue was purified by combi-flash chromatography (silica gel column, 10-100% EtOAc in petroleum ether) to give 3-(4-(bromomethyl)phenyl)-1propylpyridin-2(III)-one (243 mg, 64% yield %) in the form of a clear oil.

Stage 4.

To a solution of diethyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-N,N'-(bis-(tert-butoxycarbonyl)amino)2-chloro-9H- nurin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (554 mg, 721.20 µmol, 1 eq.) in DMF (5 ml) was added K ₂ CO ₃ (299.03 mg, 2. 16 mmol, 3 equiv.). The mixture was stirred at 20°C for 0.5 h and then 3-(4-(bromomethyl)phenyl)-1-propylpyridin-2(III)-one (242.91 mg, 793.32 µmol, 1.1 eq.). The mixture was stirred at 20°C for 16 hours, then diluted with water (30 ml) and extracted using EtOAc (4x20 ml). The combined organic layer was washed with water (100 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated. The residue was purified by combi-flash chromatography (silica gel, 20-30% EtOAc in petroleum ether) to give diethyl-2(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-N,N '-(bis-(tert-butoxycarbonyl)amino)-2-chloro-9H-nurin-9-yl)-3ethynyltetrahydrofuran-2-yl)methoxy)-2-(4-(2-oxo-1-propyl-1 ,2-dihydropyridin-3-yl)benzyl)malonate (209 mg) as a colorless gum.

Stage 5.

To a solution of diethyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-N,N'-(bis-(tert-butoxycarbonyl)amino)2-chloro-9H- purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)-2-(4-(2-oxo-1-propyl-1,2-dihydropyridin-3-yl)benzyl)malonate (209 mg, 210 .38 µmol, 1 equiv.) TFA (0.7 ml, 9.45 mmol, 44.94 equiv.) was added to DCM (2 ml) at 0°C. The solution was stirred at 20°C for 2 hours, then quenched with saturated aq. NaHCO ₃ solution (5 ml) and extracted using EtOAc (4x10 ml). The combined organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and evaporated. The residue was purified by combi-flash chromatography (silica gel, 30-70% EtOAc in petroleum ether) to give diethyl 2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-amino -2-chloro-9H-nurin-9-yl)-3-ethynyltetrahydrofuran-2yl)methoxy)-2-(4-(2-oxo-1-propyl-1,2-dihydropyridin-3-yl)benzyl)malonate (91 mg, 55% yield) as a white solid.

Stage 6.

To a solution of diethyl 2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-amino-2-chloro-9H-nurin-9-yl)-3ethynyltetrahydrofuran-2-yl)methoxy )-2-(4-(2-oxo-1-propyl-1,2-dihydropyridin-3-yl)benzyl)malonate (91 mg, 114.72 µmol, 1 eq.) in THF (1 ml) was added 1 N aq. LiOH solution (1 ml). The mixture was stirred at 20°C for 2.5 hours, then diluted with water (5 ml) and the resulting solution washed with EtOAc (2x10 ml). The organic extract was removed. The aqueous phase was acidified to pH 2 with 2 N aq. HCl and then extracted using EtOAc (4x8 ml). The combined organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and evaporated. The residue was dissolved in a mixture of MeOH (5 ml) and water (20 ml) and dried by lyophilization to obtain the title compound (51 mg, 67% yield) as a white solid.

1H NMR (400 MHz, CD3OD) δ ppm. 8.08 (s, 1H), 7.53 (dd, J=6.80, 2.0 Hz, 1H), 7.28-7.39 (m, 5H), 6.34 (t, J= 6.9 Hz, 1H), 5.96 (d, J=7.6 Hz, 1H), 4.77 (d, J=7.6 Hz, 1H), 4.28 (s, 1H), 4 .07-4.15 (m, 1H), 4.01 (dd, J=10.3, 2.8 Hz, 1H), 3.92 (t, J=7.4 Hz, 2H), 3, 39-3.58 (m, 2H), 3.06 (s, 1H), 1.65-1.80 (m, 2H), 0.94 (t, J=7.4 Hz, 3H); LC/MS [M+H] = 653.1.

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Example 20. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purun-9-ul)-3-ethunyl-3,4dihydroxytetrahydrofuran-2-yl) methoxy)-2-(4-(1-ethyl-2-oxo-1,2-dihydropyridin-3-yl)benzyl)malonic acid

Example 20

Proceeding as described in Example 19 above, but replacing propyl bromide with ethyl bromide, afforded the title compound as a white solid.

1H NMR (400 MHz, CD3OD) δ ppm. 8.15 (s, 1H), 7.53 (dd, J=6.6, 1.3 Hz, 1H), 7.25-7.38 (m, 5H), 6.32 (t, J= 6.9 Hz, 1H), 5.96 (d, J=7.6 Hz, 1H), 4.78 (d, J=7.5 Hz, 1H), 4.30 (s, 1H), 4 .09-4.15 (m, 1H), 3.95-4.04 (m, 3H), 3.39-3.58 (m, 2H), 3.06 (s, 1H), 1.28 -1.32 (m, 3H); LC/MS [M+H] = 639.1.

Example 21. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2- yl)methoxy)-2-(4-(1-(2-hydroxyethyl)-2-oxo-1,2-dihydropyridin-3-yl)benzyl)malonic acid

Stage 1.

To a mixture of 3-bromopyridin-2(III)-one (3 g, 17.24 mmol, 1 eq.) in acetone (100 ml) at 20°C was added KI (859 mg, 5.17 mmol, 0.3 eq. .), K ₂ CO ₃ (5.96 g, 43.10 mmol, 2.5 eq.) and 2-bromoethanol (4.90 ml, 68.97 mmol, 4 eq.). The mixture was stirred for 4 hours, then filtered and the filter cake washed with EtOAc (100 ml). The filtrate was evaporated under reduced pressure to obtain a residue. The residue was purified by silica gel flash column chromatography (petroleum ether: EtOAc=5:1-0:1) to give 3-bromo-1-(2-hydroxyethyl)pyridin-2(III)-one (2.2 g , yield 59%) in the form of yellow gum.

Stage 2.

Imidazole (1 .72 g, 25.22 mmol, 2.5 eq.) and TBDPSCl (5.18 ml, 20.18 mmol, 2 eq.). The mixture was stirred for 2 hours, then diluted with H2O (60 ml) and extracted using EtOAc (3x30 ml). The combined extract was washed with sat. aq. NaHCO ₄ solution (2x30 ml), saline (30 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The residue was purified by silica gel flash column chromatography (petroleum ether: EtOAc=1:0-6:1) to give 3-bromo-1-(2-((tert-butyldiphenylsilyl)oxy)ethyl)pyridine-2(III )-she (3.75 g, 79% yield) as a light yellow oil.

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Stages 3-8.

Proceeding as described in Example 19 above, but replacing 3-(4-(bromomethyl)phenyl)-1-propylpyridin2(1H)-one with 3-bromo-1-(2-((tert-butyldiphenyl)oxy)ethyl) Pyridin-2(1H)-one gives the title compound as a white solid.

¹ H NMR (400 MHz, CD3OD) δ ppm. 8.33 (s, 1H), 7.50-7.57 (m, 1H), 7.41-7.47 (m, 1H), 7.39 (d, J=8.13 Hz, 2H) , 7.29 (br.d, J=8.13 Hz, 2H), 6.35 (t, J=6.88 Hz, 1H), 5.98 (d, J=7.13 Hz, 1H) , 4.71 (d, J=7.00 Hz, 1H), 4.30 (br. s, 1H), 3.93-4.16 (m, 4H), 3.83 (m, 2H), 3.36-3.50 (m, 2H), 3.05 (s, 1H); LC/MS [M+H] = 655.1.

Example 22. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2- yl)methoxy)-2-(4-(2-methoxypyridin-3-yl)benzyl)malonic acid

Stage 1.

To a solution of diethyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-N,N'-(bis-(tert-butoxycarbonyl)amino)2-chloro-9H- purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)-2-(4-iodobenzyl)malonate (900 mg, 914.47 mmol, 1 eq.) and (2-methoxy-3-pyridyl)boronic acid (168 mg, 1.10 mmol, 1.2 eq.) in dioxane (9 ml) at 25°C added K ₂ CO ₃ (379 mg, 2.74 mmol, 3 eq.), Pd(dppf)Cl ₂ (67 mg, 91.45 µmol, 0.1 eq.) and H2O (3 ml). The mixture was degassed using N2 for some time and then heated to 80°C for 16 hours, after which it was diluted with water (10 ml) and extracted with ethyl acetate (2x10 ml). The combined organic layer was dried using Na ₂ SO ₄ , filtered and evaporated. The crude product was purified by combi-flash chromatography (silica gel, 10-50% EtOAc in petroleum ether) to give diethyl 2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6 -N,N'-(bis-(tertbutoxycarbonyl)amino)-2-chloro-9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)-2-(4-(2-methoxypyridin-3 -yl)benzyl)malonate (117 mg, 13% yield) as yellow gum.

Stage 2.

To a solution of diethyl 2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-N,N'-(bis-(tert-butoxycarbonyl)amino)-2-chloro9H-purine -9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)-2-(4-(2-methoxypyridin-3-yl)benzyl)malonate (90 mg, 93.23 µmol, 1 eq.) in DCM ( 3 mL) TFA (0.4 mL, 5.40 mmol, 58 eq.) was added. The solution was stirred at 20°C for 2 hours before quenching with saturated aq. NaHCO ₃ solution (4 ml) and extracted using EtOAc (3x4 ml). The combined organic layer was evaporated to give crude diethyl 2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-amino-2-chloro-9H-purin-9-yl)-3 -ethynyltetrahydrofuran2-yl)methoxy)-2-(4-(2-methoxypyridin-3-yl)benzyl)malonate (42 mg) as yellow gum.

Stage 3.

To a solution of crude diethyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-amino-2-chloro-9H-purin-9yl)-3-ethynyltetrahydrofuran-2-yl )methoxy)-2-(4-(2-methoxypyridin-3-yl)benzyl)malonate (42 mg, 54.89 µmol, 1 eq.) in THF (2.5 ml) was added 1 M aq. LiOH solution (0.8 ml, 15 eq.). The reaction mixture was stirred at 20°C for 4 h, then acidified to pH 6 with 1 N aq. HCl and evaporated. The crude product was purified by preparative HPLC and the fraction was dried by lyophilization to give the title compound (6 mg, 17% yield) as a white solid.

¹ H NMR (400 MHz, CD3OD) δ ppm. 8.10 (s, 1H), 8.01 (dd, J=4.94, 1.69 Hz, 1H), 7.48 (dd, J=7.25, 1.63 Hz, 1H), 7 ,24-7.35 (m, 4H), 6.93 (dd, J=7.32, 5.07 Hz, 1H), 5.97 (d, J=7.50 Hz, 1H), 4, 89-4.96 (m, 1H), 4.29 (br.s, 1H), 4.05 (br.d, J=5.00 Hz, 2H), 3.76 (s, 3H), 3 .51 (br.d, J=14.76 Hz, 1H), 3.42 (br.d, J=14.51 Hz, 1H), 3.01 (s, 1H); LC/MS [M+H] = 625.1.

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Example 23. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl) methoxy)-2-(3-(trifluoromethoxy)benzyl)malonic acid

Proceeding as described in the example above, but replacing benzyl bromide with 1-(bromomethyl)-4 (trifluoromethoxy)benzene, afforded the title compound as a white solid.

^1H NMR (CD3OD, 300 MHz) δ 8.43 (s, 1H), 7.09-7.25 (m, 3H), 6.95-6.98 (d, J=8.1 Hz, 1H ), 6.01-6.04 (d, J=7.32 Hz, 1H), 5.00-5.03 (d, J=7.41 Hz, 1H), 4.35-4.37 ( t, J=3.33 Hz, 1H), 4.05-4.15 (m, 2H), 3.38-3.53 (k, J=15 Hz, 2H), 2.99 (s, 1H ); LC/MS [M+H] = 602.0.

Example 24. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl) methoxy)-2-(thiophen-3-ylmethyl)malonic acid

Proceeding as described in Example 1 above, but replacing benzyl bromide with 3-(bromomethyl)thiophene, afforded the title compound as a white solid.

1H NMR (CD3OD, 300 MHz) δ 8.39 (s, 1H), 7.09-7.17 (m, 2H), 6.98-7.00 (d, J=5.04.0 Hz, 1H), 6.01-6.04 (d, J=7.47 Hz, 1H), 5.00-5.02 (d, J=7.29 Hz, 1H), 4.32-4.34 (t, J=2.76 Hz, 1H), 4.01-4.11 (m, 2H), 3.41-3.54 (k, J=15 Hz, J=9.03 Hz, 2H) , 2.98 (s, 1H); LC/MS [M+H] = 524.0.

Example 25. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl) methoxy)-2-(prop-2-yn-1 -yl)malonic acid

Proceeding as described in Example 1 above, but replacing benzyl bromide with propargyl bromide, afforded the title compound as a white solid.

1H NMR (CD3OD, 300 MHz) δ 8.96 (s, 1H), 6.07-6.09 (d, J=7.53 Hz, 1H), 5.01-5.04 (d, J= 7.53 Hz, 1H), 4.29-4.30 (m, 1H), 3.92-4.05 (m, 2H), 3.01-3.15 (m, 2H), 2.99 (s, 1H), 2.28-2.30 (t, J=2.58 Hz, 1H); LC/MS [M+H] = 467.

Example 26. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl) methoxy)malonic acid.

Stage 1.

Into a solution of diethyl 2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-N,N'-(bis-(tert-butoxycarbonyl)amino)2-chloro-9H- purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (200 mg, 0.26 mmol) in CH ₂ Cl ₂ (1 ml) under argon at 0°C was added TFA (0.5 ml ). The mixture was stirred for 5 min, allowed to warm and stirred for 1 h. Additional TFA (0.4 mL) was added to the reaction mixture and stirred for an additional 1.5 h before evaporation. The residue was azeotroped with DCM (5x5 ml) under reduced pressure to give crude diethyl-2(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-amino-2-chloro-9H- purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate, which was used in the next step without further purification.

Stage 2.

To a solution of crude diethyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-amino-2-chloro-9H-purin-9yl)-3-ethynyltetrahydrofuran-2-yl )methoxy)malonate (0.26 mmol) from the previous step, powdered lithium hydroxide monohydrate (86 mg, 2.08 mmol) was added to a mixture of MeOH (8.5 ml) and water (1.5 ml).

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The mixture was stirred for 16 hours, after which volatile organic compounds were removed under reduced pressure. The residue was diluted with additional water (11 ml) and extracted using EtOA (12 ml). The organic layer was removed. The aqueous phase was acidified to pH ~ 2.5 with 1 N aq. HCl solution and extracted using EtOAc (3x12 ml). The combined organic layer was dried (Na ₂ SO ₄ ), filtered and evaporated to give the title compound (45.5 mg) as a light brown solid.

^1H NMR (CD3OD, 300 MHz): δ 8.94 (s, 1H), 6.08 (d, J=7.52 Hz, 1H), 5.05 (d, J=7.52 Hz, 1H ), 4.65 (s, 1H), 4.29 (t, J=2.40 Hz, 1H), 4.06 (dd, J=10.7, 2.5 Hz, 1H), 3.93 (dd, J=10.64, 2.50 Hz, 1H), 3.12 (s, 1H); LC/MS [M+H] = 428.

Example 27. Synthesis of 2-(((2R,3S,4R,5R)-3-((1H-pyrazol-3-yl)ethynyl)-5-(6-amino-2-chloro-9H-purin-9yl) -3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

Example 27

Stage 1.

To a mixture of 3-iodo-III-pyrazole (407 mg, 2.1 mmol), PdCl ₂ (PPh ₃ ) ₂ (82 mg, 0.12 mmol), CuI (22 mg, 0.12 mmol) and Et ₃ N (10 ml) in THF (10 ml) under an argon atmosphere, diethyl 2-(((2R,3R,4R,5R)-3,4diacetoxy-5-(6-N,N'-(bis-(tert-butoxycarbonyl) )amino)-2-chloro-9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (1 g, 1.2 mmol). The resulting mixture was stirred at 60°C overnight, after which it was allowed to cool to room temperature and volatile organic compounds were removed under reduced pressure. The resulting crude residue was purified by silica gel flash column chromatography (60-100% EtOAc in hexane) to give diethyl 2-(((2R,3R,4R,5R)-3((1H-pyrazol-3-yl)ethynyl) -3,4-diacetoxy-5-(6-N,N'-(bis-(tert-butoxycarbonyl)amino)-2-chloro-9H-purin-9-yl)tetrahydrofuran-2-yl)methoxy)malonate in form of a solid.

Stage 2.

To a solution of diethyl-2-(((2R,3R,4R,5R)-3-((1H-pyrazol-3-yl)ethynyl)-3,4-diacetoxy-5-(6-N,N'-( bis(tert-butoxycarbonyl)amino)-2-chloro-9H-purin-9-yl)tetrahydrofuran-2-yl)methoxy)malonate (100 mg, 0.12 mmol) in DCM (3 ml) added TFA (1 ml ). The resulting mixture was stirred at 25°C for 4 hours, then evaporated to form crude diethyl 2-(((2R,3R,4R,5R)-3-((1H-pyrazol-3yl)ethynyl)-3,4- diacetoxy-5-(6-amino-2-chloro-9H-purin-9-yl)tetrahydrofuran-2-yl)methoxy)malonate, which was used in the next step without further purification.

Stage 3.

To a solution of crude diethyl-2-(((2R,3R,4R,5R)-3-((1H-pyrazol-3-yl)ethynyl)-3,4-diacetoxy-5(6-amino-2-chloro- 9H-purin-9-yl)tetrahydrofuran-2-yl)methoxy)malonate in a mixture of THF (5 ml) and H2O (2 ml) was added LiOH-H ₂ O (50 mg, 1.2 mmol). The resulting mixture was stirred at 25°C for 24 hours, then cooled to 0°C and acidified to pH 6.5 with 1 N aq. HCl. The reaction mixture was evaporated. The crude residue was purified by preparative reverse phase HPLC and lyophilized to give the title compound as a white solid.

1H NMR (CD3OD, 300 MHz) δ 8.96 (s, 1H), 7.56-7.76 (m, 1H), 6.50-6.57 (m, 1H), 6.12-6, 14 (d, J=7.14 Hz, 1H), 5.13-5.15 (d, J=6.78 Hz, 1H), 4.63-4.70 (m, 1H), 4.38 (s, 1H), 3.99-4.13 (m, 2H); LC/MS [M+H] = 495.

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Examples 28 and 29. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-((1-benzyl-1Hpyrazol- 3-yl)ethynyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid and 2(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purine- 9-yl)-3-((1-benzyl-1H-pyrazol-5-yl)ethynyl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

Stage 1.

To a solution of diethyl-2-(((2R,3R,4R,5R)-3-((1H-pyrazol-3-yl)ethynyl)-3,4-diacetoxy-5-(6-N,N'-( bis(tert-butoxycarbonyl)amino)-2-chloro-9H-purin-9-yl)tetrahydrofuran-2-yl)methoxy)malonate (100 mg, 0.12 mmol) in anhydrous DMF (2 ml) under argon at Oven-dried Cs ₂ CO ₃ (78 mg, 0.24 mmol) was added at 0°C. The mixture was stirred at room temperature for 20 min followed by the addition of benzyl bromide (29 μL, 0.24 mmol). The resulting mixture was stirred at room temperature for 2 hours, then diluted with EtOAc (15 ml) and H2O (5 ml). The organic layer was separated, washed with H2O (20 ml), brine, dried over Na ₂ SO ₄ and evaporated. The crude residue was purified by silica gel flash column chromatography (0-50% EtOAc in hexanes) to give diethyl 2-(((2R,3R,4R,5R)-3,4-diacetoxy-3-((1- benzyl-1H-pyrazol-3-yl)ethynyl)-5-(6-N,N'-(bis(tert-butoxycarbonyl)amino)-2-chloro-9H-purin-9-yl)tetrahydrofuran-2-yl )methoxy)malonate and diethyl2-(((2R,3R,4R,5R)-3,4-diacetoxy-3-((1-benzyl-1H-pyrazol-5-yl)ethynyl)-5-(6-N ,N'-(bis-(tert-butoxycarbonyl)amino)-2-chloro-9H-purin-9-yl)tetrahydrofuran-2-yl)methoxy)malonate.

Stages 2, 3.

Proceeding as described in Example 27 above, but replacing diethyl 2-(((2R,3R,4R,5R)-3-((1H-pyrazol-3yl)ethynyl)-3,4-diacetoxy-5-(6 -N,N'-(bis-(tert-butoxycarbonyl)amino)-2-chloro-9H-purin-9-yl)tetrahydrofuran-2-yl)methoxy)malonate on diethyl 2-(((2R,3R, 4R,5R)-3,4-diacetoxy-3-((1-benzyl-1H-pyrazol3-yl)ethynyl)-5-(6-N,N'-(bis-(tert-butoxycarbonyl)amino)-2 -chloro-9H-purin-9-yl)tetrahydrofuran-2yl)methoxy)malonate, the title compound (Example 28) acid was obtained as a white solid.

^1H NMR (CD3OD, 300 MHz) δ 8.95 (s, 1H), 7.685 (s, 1H), 7.22-7.38 (m, 5H), 6.50 (s, 1H), 6, 10-6.13 (d, J=7.05 Hz, 1H), 5.35 (s, 2H), 5.16-5.18 (d, J=7.35 Hz, 1H), 4.68 -4.76 (m, 1H), 4.38 (s, 1H), 3.96-4.16 (dd, J=9.84 Hz, J=17 Hz, 2H); LC/MS [M+H] = 585.

Stages 4, 5.

Proceeding as described in Example 27 above, but replacing diethyl 2-(((2R,3R,4R,5R)-3-((1H-pyrazol-3yl)ethynyl)-3,4-diacetoxy-5-(6 -N,N'-(bis-(tert-butoxycarbonyl)amino)-2-chloro-9H-purin-9-yl)tetrahydrofuran-2-yl)methoxy)malonate on diethyl 2-(((2R,3R, 4R,5R)-3,4-diacetoxy-3-((1-benzyl-1H-pyrazol5-yl)ethynyl)-5-(6-N,N'-(bis-(tert-butoxycarbonyl)amino)-2 -chloro-9H-purin-9-yl)tetrahydrofuran-2yl)methoxy)malonate gave the title compound (Example 29) as a white solid.

^1H NMR (CD3OD, 300 MHz) δ 8.88 (s, 1H), 7.53 (s, 1H), 7.23-7.35 (m, 5H), 6.59 (s, 1H), 6.10-6.13 (d, J=6.09 Hz, 1H), 5.46 (s, 2H), 5.15-5.17 (d, J=6.9 Hz, 1H), 4 .35-4.41 (m, 2H), 3.71-4.01 (dd, J=10.71, J=33, 2H); LC/MS [M+H] = 585.

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Examples 30 and 31. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(5-chloro-7-(isopropylamino)-3Himidazo[4,5-b]pyridin-3-yl )-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid and 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(dimethylamino )-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

Example 30 Example 31

Stage 1.

To a solution of diethyl 2-benzyl-2-(((2R,3R,4R)-3,4,5-triacetoxy-3-ethynyltetrahydrofuran-2yl)methoxy)malonate (500 mg, 0.91 mmol) in MeCN (6 ml) at 25°C, add 5,7-dichloro-Chimidazo^^-^pyridine (223 mg, 1.18 mmol) and then N,O-bis-(trimethylsilyl)acetamide (BSA) (535 µl, 2.19 mmol). The resulting suspension was heated at 85°C for 15 minutes until it became clear. The reaction mixture was allowed to cool to room temperature followed by the addition of TMSOTf (262 mg, 1.18 mmol) dropwise. The reaction mixture was then refluxed at 85°C for 3 hours since all the starting material had been consumed. The reaction was quenched with cold saturated aq. NaHCO ₃ solution and diluted with EtOAc (15 ml). The organic layer was separated, washed with H2O (20 ml), brine, dried over Na ₂ SO ₄ and evaporated. The crude residue was purified by silica gel flash column chromatography (0-50% EtOAc in hexanes) to give diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5- (5,7-dichloro-3H-imidazo[4,5-b]pyridin-3-yl)3-ethynyltetrahydrofuran-2-yl)methoxy)malonate in the form of foam.

Stage 2.

In a sealed test tube containing diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(5,7dichloro-3H-imidazo[4,5-b]pyridinium- 3-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (80 mg, 0.12 mmol) in anhydrous DMF (1 ml) was added isopropylamine (0.5 ml, 5.9 mmol) and Et ₃ N ( 1 ml, 7.1 mmol). The reaction mixture was heated at 75°C for 72 hours, after which it was allowed to cool and diluted with EtOAc (15 ml) and H ₂ O (5 ml). The organic layer was separated, washed with H ₂ O (20 ml), brine, dried over Na ₂ SO ₄ and evaporated. The residue was purified by silica gel flash column chromatography (0-50% EtOAc in hexanes) to give diethyl-2-benzyl-2(((2R,3S,4R,5R)-5-(5-chloro-7-(isopropylamino) -3H-imidazo[4,5-b]pyridin-3-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonate and diethyl-2-benzyl-2-(((2R,3S ,4R,5R)-5-(5-chloro-7(dimethylamino)-3H-imidazo[4,5-b]pyridin-3-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy ) malonate in the form of foam.

Stage 3.

To a solution of diethyl-2-benzyl-2-(((2R,3S,4R,5R)-5-(5-chloro-7-(isopropylamino)-3H-imidazo[4,5b]pyridin-3-yl)- _LiOH - _H2O (10 mg, 0 .24 mmol). The resulting mixture was stirred at 25°C for 24 hours, then cooled to 0°C and acidified to pH 6.5 with 1 N aq. HCl. The crude residue was purified by preparative reverse phase HPLC and lyophilized to give 2-benzyl-2-(((2R,3S,4R,5R)-5-(5-chloro-7-(isopropylamino)-3H-imidazo[ 4,5b]pyridin-3-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid as a white solid.

^1H NMR (CD3OD, 300 MHz) δ 8.25 (s, 1H), 7.25-7.28 (m, 2H), 7.05 (m, 3H), 6.43 (s, 1H), 6.06-6.08 (d,

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J=7.17 Hz, 1H), 4.95-4.98 (d, J=7.05 Hz, 1H), 4.32 (s, 1H), 4.05-4.11 (m, 2H ), 3.89-3.93 (m, 1H), 3.31-3.39 (m, 2H), 2.99 (s, 1H), 1.30-1.33 (m, 6H); LC/MS [M+H] = 560.

Stage 4.

To a solution of diethyl-2-benzyl-2-(((2R,3 S,4R,5R)-5-(5-chloro-7-(dimethylamino)-3H-imidazo[4,5b]pyridin-3-yl) -3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonate (21 mg, 0.035 mmol) was added to _a mixture of THF (3 ml) and H ₂ O (1 ml) 0.71 mmol). The resulting mixture was stirred at 25°C for 24 hours, then cooled to 0°C and acidified to pH 6.5 with 1 N aq. HCl. The reaction mixture was evaporated. The crude residue was purified by preparative reverse phase HPLC and lyophilized to give the title compound as a white solid.

^1H NMR (CD3OD, 300 MHz) δ 8.68 (s, 1H), 7.21-7.24 (m, 2H), 6.99-7.03 (m, 3H), 6.49 (s , 1H), 6.15-6.17 (d, J=7.08 Hz, 1H), 4.99-5.02 (d, J=7.17 Hz, 1H), 4.35-4, 37 (t, J=3.12 Hz, 1H), 4.07-4.08 (m, 2H), 3.36-3.50 (m, 8H), 2.99 (s, 1H); LC/MS [M+H] = 546.

Example 32. Synthesis of 2-(((2R,3S,4R,5R)-5-(2-chloro-6-((2-hydroxyethyl)amino)-9H-purin-9-yl)-3ethynyl-3,4 -dihydroxytetrahydrofuran-2-yl)methoxy)-2-(thiophen-3-ylmethyl)malonic acid

Example 32

Stages 1-3.

Proceeding as described in Example 15 above, but replacing allyl bromide with 3-(bromomethyl)thiophene, diethyl-2-(thiophen-3-ylmethyl)-2-(((2R,3R,4R)-3,4,5 -triacetoxy-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate in the form of a solid.

Stage 4.

N,O-bis(trimethylsilyl)acetamide ( BSA) (571 µl, 2.34 mmol). The resulting suspension was heated at 85°C for 15 minutes until it became clear. The reaction mixture was allowed to cool to room temperature followed by the dropwise addition of diethyl 2-(thiophen-3-ylmethyl)2-(((2R,3R,4R)-3,4,5-triacetoxy-3-ethynyltetrahydrofuran-2-yl )methoxy)malonate (540 mg, 0.97 mmol) and TMSOTf (228 μl, 1.26 mmol). The reaction mixture was then refluxed at 85°C for 2.5 hours since all the starting material had been consumed. The reaction was quenched with cold saturated aq. NaHCO ₃ solution and diluted with EtOAc (15 ml). The organic layer was separated, washed with H2O (20 ml), brine, dried over Na ₂ SO ₄ and evaporated. The crude residue was purified by silica gel flash column chromatography (0-50% EtOAc in hexanes) to give diethyl 2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(2,6- dichloro-9H-purin-9-yl)-3-ethynyltetrahydrofuran2-yl)methoxy)-2-(thiophen-3-ylmethyl)malonate in the form of foam.

Stage 5.

To a solution of diethyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(2,6-dichloro-9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl) methoxy)-2-(thiophen-3-ylmethyl)malonate (100 mg, 0.146 mmol) in 1,4-dioxane (2 ml) TEA (20 µl, 0.146 mmol) was added followed by ethanolamine (13 µl, 0.219 mmol) . The resulting mixture was stirred at 25°C for 2 hours and then diluted with EtOAc (15 ml) and H2O (5 ml). The organic layer was separated, washed with H ₂ O (20 ml), brine, dried over Na ₂ SO ₄ and evaporated to give crude diethyl 2-(((2R,3R,4R,5R)-3,4-diacetoxy-5- (2-chloro-6-((2-hydroxyethyl)amino)-9H-purin-9-yl)3-ethynyltetrahydrofuran-2-yl)methoxy)-2-(thiophen-3-ylmethyl)malonate, which was used in the following stage without additional purification.

Stage 6.

To a solution of crude diethyl 2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(2-chloro-6-((2-hydroxyethyl)amino)

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9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)-2-(thiophen-3-ylmethyl)malonate in a mixture of THF (4 ml) and H2O (1 ml), LiOH H ₂ O (80 mg, 1.91 mmol). The resulting mixture was stirred at 25°C for 24 hours, then cooled to 0°C and acidified to pH 6.5 with 1 N aq. HCl. The reaction mixture was evaporated. The crude residue was purified by preparative reverse phase HPLC and lyophilized to give the title compound as a white solid.

^1H NMR (CD3OD, 300 MHz) δ 8.32 (s, 1H), 7.09-7.16 (m, 2H), 6.97-6.99 (m, 1H), 6.00-6 .03 (d, J=7.41 Hz, 1H), 4.99-5.02 (d, J=7.38 Hz, 1H), 4.32-4.34 (t, J=3.03 Hz, 1H), 4.00-4.11 (m, 2I), 3.65-3.79 (m, 4H), 3.40-3.53 (k, J=15.42 Hz, J= 5.79 Hz, 2H), 2.98 (s, 1H); LC/MS [M+H] = 568.0.

Example 33. Synthesis of 2-(((2R,3S,4R,5R)-5-(2-chloro-6-(isopropylamino)-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2- yl)methoxy)-2-(thiophen-3-ylmethyl)malonic acid

Proceeding as described in Example 32 above, but replacing ethanolamine with i-PrNH ₂ gave the title compound as a white solid.

^1H NMR (CD3OD, 300 MHz) δ 8.28 (s, 1H), 7.08-7.16 (m, 2H), 6.97-6.99 (m, 1H), 5.99-6 .02 (d, J=7.38 Hz, 1H), 5.00-5.02 (d, J=7.35 Hz, 1H), 4.31-4.43 (m, 2H), 4, 00-4.12 (m, 2H), 3.40-3.53 (k, J=15.63 Hz, J=5.4 Hz, 2H), 2.98 (s, 1H), 1.27 -1.32 (m, 6H); LC/MS [M+H] = 566.0.

Example 34. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-((3-hydroxypropyl)amino)-9H-purin-9yl)-3-ethynyl -3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

aq. LiOH

MeOH, THF

Example 34

Stage 1.

To a solution of 2,6-dichloro-9H-purine (690 mg, 3.65 mmol) in dry CH ₃ CN (15 ml) was added N,O-bis-(Ίrimethylsilyl)acetamide (0.28 ml, 1 .12 mmol). The mixture was heated to 95°C under argon for 5 min and then cooled to ambient temperature. To this mixture was added diethyl 2benzyl-2-(((2R,3R,4R)-3,4,5-triaceΊΌxy-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (2 g, 3.65 mmol) and then TMSOTf (0.09 ml, 0.494 mmol). The resulting mixture was heated at 95°C for 2.5 hours, then cooled to ambient temperature and diluted with water (60 ml) and EtOAc (60 ml). The organic phase was washed successively with equal volumes of saturated NaHCO ₃ solution and brine. The aqueous phase was further extracted using EtOAc (2x30 ml). The combined organic phase was dried (MgSO ₄ ), filtered and concentrated. The crude residue was purified by silica gel flash column chromatography (5-60% EtOAc in hexanes) to give diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3,4diacetoxy-5-(2,6 -dichloro-9H-purin-9-yl)tetrahydrofuran-2-yl)meΊΌxy)malonate (1.61 g) as an almost white solid.

Stage 2.

To a solution of diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(2,6-dichloro-9H-purin-9yl)tetrahydrofuran-2-yl)methoxy )malonate (102 mg, 0.15 mmol) in dry dioxane (1 ml), triethylamine (0.02 ml, 0.15 mmol) and 3-aminopropanol (16 mg, 0.212 mmol) were added. The resulting mixture was stirred for 1.5 h, then diluted with water (15 ml) and DCM (15 ml) and the organic phase was collected. The organic layer was washed with brine (15 ml). The aqueous phase was further extracted using EtOAc (2x10 ml). The combined organic layer was dried over MgSO ₄ , filtered and evaporated to give crude diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(2-chloro-6(( 3-hydroxypropyl)amino)-9H-purin-9-yl)tetrahydrofuran-2-yl)methoxy)malonate in the form of a clear viscous oil.

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Stage 3.

To a solution of crude diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(2-chloro-6-((3hydroxypropyl)amino)-9H-purine-9 -yl)tetrahydrofuran-2-yl)methoxy)malonate (0.15 mmol) in H2O (0.2 ml), MeOH (1 ml) and THF (0.28 ml) was added powdered LiOH monohydrate (43 mg, 1. 05 mmol)). The mixture was stirred for 4 hours and then sonicated for 30 minutes. Additional LiOH monohydrate (7 mg) was added and sonication continued for 1 h, then VOCs were removed under reduced pressure and the residue was diluted with water (10 ml) and EtOAc (10 ml). The mixture was cooled to 0°C and acidified to pH ~ 3 with 1 N aq. HCl. The organic phase was collected and the aqueous phase was further extracted using EtOAc (2x10 ml). The combined EtOAc phases were dried over MgSO ₄ , filtered and evaporated. The crude residue was purified by preparative reverse phase HPLC to give the title compound as an off-white solid.

^1H NMR (CD3OD, 300 MHz) δ 8.19 (br.s, 1H), 7.21-7.3 (m, 2H), 7.00-7.10 (m, 3H), 6.00 (d, J=7.36 Hz, 1H), 4.98 (d, J=7.36 Hz, 1H), 4.33 (t, J=3.18 Hz, 1H), 4.02-4 .15 (m, 2H), 3.68 (t, J=6.18 Hz, 2H), 3.59-3.72 (m, 2H), 3.47 (d, J=14.95 Hz, 1H), 3.38 (d, J=14.95 Hz, 1H), 2.99 (s, 1H), 1.84-1.95 (m, 2H); LC/MS [M+H] = 576.0.

Example 35. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(((R)-2-hydroxypropyl)amino)-9Hpurin-9-yl) -3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

Example 35

Proceeding as described in Example 34 above, but replacing propanolamine with N)-1-aminopropan-2-ol, followed by hydrolysis of the ester using LiOH, afforded the title compound as a white solid.

^1H NMR (CD3OD, 300 MHz) δ 8.19 (br.s, 1H), 7.22-7.30 (m, 2H), 7.02-7.10 (m, 3H), 6.01 (d, J=7.38 Hz, 1H), 4.98 (d, J=7.38 Hz, 1H), 4.33 (t, J=3.17 Hz, 1H), 3.99-4 .13 (m, 3H), 3.58-3.69 (m, 1H), 3.43 (kt, J=14.64 Hz, 2H), 3.41-3.55 (m, 1H), 2.99 (s, 1H), 1.24 (d, J=6.30 Hz, 3H); LC/MS [M+H] = 576.0.

Example 36. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(((S)-2-hydroxypropyl)amino)-9Hpurin-9-yl) -3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

Example 36

Proceeding as described in Example 34 above, but replacing propanolamine with N)-1-aminopropan-2-ol, followed by hydrolysis of the ester using LiOH, afforded the title compound as a white solid.

1H NMR (CD3OD, 300 MHz) δ 8.19 (br.s, 1H), 7.23-7.29 (m, 2H), 7.03-7.10 (m, 3H), 6.00 ( d, J=7.35 Hz, 1H), 4.96 (d, J=7.35 Hz, 1H), 4.32 (t, J=3.30 Hz, 1H), 3.98-4, 13 (m, 3H), 3.59-3.68 (m, 1h), 3.35-3.55 (m, 3H), 2.99 (s, 1H), 1.25 (d, J= 6.30 Hz, 3H); LC/MS [M+H] = 576.0.

Example 37. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(6-(bis-(2-hydroxyethyl)amino)-2-chloro-9H-purin9-yl)-3 -ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

Example 37

Proceeding as described in Example 34 above, but replacing propanolamine with diethanolamine followed by hydrolysis of the ester using LiOH, afforded the title compound as a white solid.

1H NMR (CD3OD, 300 MHz) δ 8.16 (s, 1H), 7.22-7.29 (m, 2H), 6.99-7.09 (m, 3H), 6.02 (d, J=7.33 Hz, 1H), 4.97 (d, J=7.33 Hz, 1H), 4.00-4.37 (m, 2H), 4.29-4.34 (m, 2H ), 4.03-4.13 (m, 3H), 3.84-3.91 (m, 4H), 3.46 (d, J=14.92 Hz, 1H), 3.37 (d, J=14.92 Hz, 1H), 2.98 (s, 1H); LC/MS [M+H] = 606.0.

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Example 38. Synthesis of 2-6eii3uj-2-(X(yR,3S,4R,5R')-5-(2-x.iop-6-((2-MeTOKCujTu.i')aMuiio)-9l l-iiypuii -9-u.i)3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

Proceeding as described in Example 34 above, but replacing propanolamine with 2-methoxyethylamine followed by hydrolysis of the ester using LiOH, afforded the title compound as a white solid.

1H NMR (CD3OD, 300 MHz) δ 8.17 (br. s, 1H), 7.22-7.30 (m, 2H), 7.01-7.09 (m, 3H), 6.01 ( d, J=7.33 Hz, 1H), 4.98 (d, J=7.33 Hz, 1H), 4.33 (t, J=3.20 Hz, 1H), 4.03-4, 13 (m, 2H), 3.69-3.79 (m, 2H), 3.62 (t, J=5.13 Hz, 2H), 3.47 (d, J=14.89 Hz, 1H ), 3.40 (s, 3H), 3.38 (d, J=14.89 Hz, 1H), 2.99 (s, 1H); LC/MS [M+H] = 576.0.

Example 39. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-((2-methoxyethyl)-(methyl)amino)-9Hpurin-9-yl) -3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

Proceeding as described in Example 34 above, but replacing propanolamine with (2-methoxyethyl)methylamine followed by hydrolysis of the ester using LiOl, afforded the title compound as a white solid.

1H NMR (CD3OD, 300 MHz) δ 8.18 (br.s, 1H), 7.22-7.29 (m, 2H), 6.99-7.09 (m, 3H), 6.02 ( d, J=7.21 Hz, 1H), 4.98 (d, J=7.21 Hz, 1H), 4.32 (t, J=3.41 Hz, 1H), 4.03-4, 14 (m, 2H), 3.68 (t, J=5.42 Hz, 2H), 3.34-3.49 (m, 7H), 3.36 (s, 3H), 2.99 (s , 1H); LC/MS [M+H] = 590.0.

Example 40. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(((1-hydroxycyclobutyl)methyl)amino)9H-purin-9-yl) -3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

Proceeding as described in Example 34 above, but replacing propanolamine with 1(aminomethyl)cyclobutanol followed by hydrolysis of the ester using LiOl, afforded the title compound as a white solid.

1H NMR (CD3OD, 300 MHz) δ 8.18 (br.s, 1H), 7.23-7.29 (m, 2H), 7.01-7.09 (m, 3H), 6.01 ( d, J=7.36 Hz, 1H), 4.98 (d, J=7.36 Hz, 1H), 4.32 (t, J=3.21 Hz, 1H), 4.03-4, 11 (m, 2H), 3.73-3.79 (m, 2H), 3.36-3.50 (m, 2H), 2.99 (s, 1H), 2.03-2.21 ( m, 4H), 1.59-1.85 (m, 2H); LC/MS [M+H] = 602.0.

Example 41. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(3-hydroxyazetidin-1-yl)-9H-purin-9yl)-3- ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

Proceeding as described in Example 34 above, but replacing propanolamine with azetidin-3-ol followed by hydrolysis of the ester using LiOl, afforded the title compound as a white solid.

1H NMR (CD3OD, 300 MHz) δ 8.29 (br.s, 1H), 7.21-7.29 (m, 2H), 6.99-7.11 (m, 3H), 6.01 ( d, J=7.33 Hz, 1H), 5.01 (d, J=7.33 Hz, 1H), 4.57-4.82 (m, 3H), 4.33 (t, J=3 ,39 Hz, 1H), 4.14-4.27 (m, 2H), 4.07 (cd, J=4.04, 2.92 Hz, 2H), 3.30-3.50 (m, 2H), 2.98 (s, 1H); LC/MS [M+H] = 574.0.

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Example 42. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(2-(hydroxymethyl)azetidin-1-yl)-9Hpurin-9-yl) -3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

Proceeding as described in Example 34 above, but replacing propanolamine with azetidin-2-ylmethanol followed by hydrolysis of the ester using LiOH, afforded the title compound as a white solid.

1H NMR (CD3OD, 300 MHz) δ 8.17-8.29 (m, 1H), 7.22-7.28 (m, 2H), 7.01-7.12 (m, 3H), 5. 98-6.03 (m, 1H), 4.98 (d, J=7.30 Hz, 1H), 4.27-4.45 (m, 3H), 4.00-4.13 (m, 3H), 3.83-3.92 (m, 1H), 3.33-3.50 (m, 3H), 2.99 (s, 0.5H), 2.97 (s, 0.5H) , 2.49-2.63 (m, 1H), 2.31-2.64 (m, 1H); LC/MS [M+H] = 588.0.

Example 43. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-((1-(hydroxymethyl)cyclopropyl)amino)-9H-purin-9-yl )-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

O V ⁴ hI - ? 'Sh'· 'S i/ =-4-/

Example 43

Proceeding as described in Example 34 above, but replacing propanolamine with (1-aminocyclopropyl)methanol followed by hydrolysis of the ester using LiOH, afforded the title compound as a white solid.

1H NMR (CD3OD, 300 MHz) δ 8.18 (s, 1H), 7.22-7.32 (m, 2H), 7.02-7.11 (m, 3H), 6.01 (d, J=7.36 Hz, 1H), 4.96 (d, J=7.36 Hz, 1H), 4.32 (t, J=3.24 Hz, 1H), 4.02-4.14 ( m, 2H), 3.76 (br. s, 2H), 3.46 (d, J=15.04 Hz, 1H), 3.38 (d, J=15.04 Hz, 1H), 2, 99 (s, 1H), 0.88-1.03 (m, 4H); LC/MS [M+H] = 588.0.

Example 44. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(((1-hydroxycyclopropyl)methyl)amino)-9H-purin-9-yl )-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

Proceeding as described in Example 34 above, but replacing propanolamine with 1(aminomethyl)cyclopropanol followed by hydrolysis of the ester using LiOH, afforded the title compound as a white solid.

^1H NMR (CD ₃ OD, 300 MHz) δ 8.18 (s, 1H), 7.08-7.25 (m, 5H), 5.98-6.02 (d, J=7 Hz, 1H ), 4.93-4.97 (m, 2H), 4.30 (br.s, 1H), 3.98-4.10 (m, 2H), 3.71 (br.s, 2H), 3.39-3.51 (m, 2H), 3.00-3.13 (s, 1H), 0.71-0.80 (m, 4H); LC/MS [M+H] = 588.2.

Example 45. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-((cyclobutylmethyl)amino)-9H-purin-9yl)-3-ethynyl-3 ,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

Proceeding as described in Example 34 above, but replacing propanolamine with cyclobutylmethylamine followed by hydrolysis of the ester using LiOH, afforded the title compound as a white solid.

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1H NMR (CD3OD, 300 MHz) δ 8.19 (s, 1H), 7.24-7.27 (m, 2H), 7.05-7.07 (m, 3H), 5.98-6, 01 (d, J=8 Hz, 1H), 4.93-4.95 (d, J=7 Hz, 1 Hz), 4.31-4.33 (br. s, 1H), 4.01- 4.10 (m, 2H), 3.58 (s, 2H), 3.39-3.48 (m, 2H), 3.00 (s, 1H), 2.66-2.71 (m, 1H), 2.12-2.15 (m, 2H) 1.84-1.97 (m, 4H); LC/MS [M+H] = 586.2.

Example 46. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(3-(hydroxymethyl)azetidin-1-yl)-9Hpurin-9-yl) -3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

Proceeding as described in Example 34 above, but replacing propanolamine with azetidin-3-ylmethanol followed by hydrolysis of the ester using LiOH, afforded the title compound as a white solid.

1H NMR (CD3OD, 300 MHz) δ 8.09 (s, 1H), 7.05-7.25 (m, 5H), 6.00-6.02 (d, J=7 Hz, 1H), 4 .93-4.97 (m, 1H), 4.33 (br.s, 1H), 3.96-4.11 (m, 2H), 3.78 (br.s, 2H), 3.36 -3.40 (m, 6H), 2.99 (br.s, 2H); LC/MS [M+H] = 588.2.

Example 47. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(3-hydroxy-3-methylazetidin-1-yl)-9Hpurin-9-yl )-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

Proceeding as described in Example 34 above, but replacing propanolamine with 3-methylazetidin-3-ol followed by hydrolysis of the ester using LiOH, afforded the title compound as a white solid.

1H NMR (CD3OD, 300 MHz) δ 8.30 (s, 1H), 7.08-7.25 (m, 5H), 6.00-6.03 (d, J=7 Hz, 1H), 4 .98-5.01 (m, 1H), 4.33 (br.s, 4H), 4.01-4.09 (m, 2H), 3.73 (br.s, 1H), 3.35 -3.47 (m, 2H), 2.98 (s, 1H), 1.57 (s, 3H); LC/MS [M+H] = 588.2.

Example 48. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-((3-hydroxycyclobutyl)-(methyl)amino)9H-purin-9-yl )-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

Proceeding as described in Example 34 above, but replacing propanolamine with 3-(methylamino)cyclobutanol followed by hydrolysis of the ester using LiOH, afforded the title compound as a white solid.

1H NMR (CD3OD, 300 MHz) δ 8.26 (s, 1H), 6.96-7.28 (m, 5H), 6.02-6.04 (d, J=7 Hz, 1H), 5 .27 (br.s, 1H), 4.98-5.02 (m, 1H), 4.33 (br.s, 2H), 3.95-4.15 (m, 3H), 3.36 -3.51 (m, 4H), 2.99 (s, 1H), 2.67-2.69 (m, 2H), 2.22-2.25 (m, 2H); LC/MS [M+H] = 602.2.

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Examples 49 and 50. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2- yl)methoxy)-3-ethoxy-3-oxo-2-(3-(trifluoromethyl)benzyl)propanoic acid and 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro -9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)-2(3-(trifluoromethyl)benzyl)malonic acid

as described in example 8 above, but replacing furan with (3-trifluoromethyl)benzene, we obtained

Acting, the title compounds appear as a white solid by purification by preparative reverse phase HPLC.

2-(((2R,3S,4R,5R)-5-(6-Amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2yl)methoxy)-3 -ethoxy-3-oxo-2-(3-(trifluoromethyl)benzyl)propanoic acid:

1H NMR (CD3OD, 300 MHz) δ 8.33 (br.s, 1H), 7.51-7.54 (d, J=8 Hz, 2H), 7.38-7.40 (d, J= 6 Hz, 1H), 7.21-7.26 (t, J=7 Hz, 1H), 6.00-6.04 (m, 1H), 4.98-5.02 (dt, J=4 ,7, 52 Hz, 1H), 4.36 (br. s, 1H), 4.02-4.22 (m, 4H), 3.45-3.58 (m, 2H), 3.02- 3.12 (d, J=29 Hz, 1H), 1.18-1.24 (m, 3H); LC/MS [M+H] = 614.2.

2-(((2R,3S,4R,5R)-5-(6-Amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2yl)methoxy) 2- (3-(trifluoromethyl)benzyl)malonic acid:

1H NMR (CD3OD, 300 MHz) δ 8.30 (s, 1H), 7.52-7.54 (d, J=9 Hz, 2H), 7.37-7.39 (d, J=7 Hz , 1H), 7.237.25 (t, J=7 Hz, 1H), 6.01-6.03 (d, J=7 Hz, 1H), 4.97-5.00 (d, J=7 Hz , 1H), 4.37 (br.s, 1H), 4.12-4.14 (m, 2H), 3.44-3.57 (m, 2H), 3.01 (s, 1H); LC/MS [M+H] = 586.2.

Example 51. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl) methoxy)-2-(3-chlorobenzyl)malonic acid

Proceeding as described in Example 8 above, but replacing furan with 3-chlorobenzene, the title compound was obtained as a white solid by purification by preparative reverse phase HPLC.

1H NMR (CD3OD, 300 MHz) δ 8.42 (s, 1H), 7.27 (br. s, 1H), 7.14-7.15 (d, J=6 Hz, 1H), 7.02 -7.06 (m, 2H), 6.02-6.05 (d, J=8 Hz, 1H), 5.03-5.06 (d, J=7 Hz, 1H), 4.35- 4.39 (m, 2H), 3.39-3.49 (m, 2H), 3.01 (s, 1H), 2.48-2.54 (t, J=8 Hz, 1H) 2, 22-2.32 (m, 1H); LC/MS [M+H] = 552.1.

Example 52. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl) methoxy)-2-(3-methoxybenzyl)malonic acid

Proceeding as described in Example 8 above, but replacing furan with 3-methoxybenzene, the title compound was obtained as a white solid by purification by preparative reverse phase HPLC.

1H NMR (CD ₃ OD, 300 MHz) δ 8.40 (s, 1H), 6.96-7.02 (t, J=8 Hz, 1H), 6.82 (br.s, 2H), 6 ,59-6.62 (m, 1H), 6.02-6.04 (d, J=7 Hz, 1H), 5.01-5.04 (d, J=8 Hz, 1H), 4, 35-4.39 (m, 2H), 3.54 (s, 3H), 3.45-3.50 (m, 1H), 2.97 (s, 1H), 2.48-2.54 ( t, J=8 Hz, 1H) 2.22-2.32 (m, 1H); LC/MS [M+H] = 548.1.

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Example 53. Synthesis of 2-([1,1'-biphenyl]-4-ylmethyl)-2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purine- 9yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

Proceeding as described in Example 8 above, but replacing furan with 3-biphenyl, the title compound was obtained as a white solid by purification by preparative reverse phase HPLC.

¹ H NMR (CD3OD, 300 MHz) δ 8.23 (s, 1H), 7.28-7.43 (m, 9H), 5.99-6.02 (d, J=7 Hz, 1H), 4.96-4.98 (d, J=7 Hz, 1H), 4.35 (s, 1H), 4.08-4.15 (m, 2H), 3.41-3.57 (m, 2H), 3.04 (s, 1H): LC/MS [M+H]=594.2.

Example 54. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl) methoxy)-2-((2-carboxy-[1,1'-biphenyl]-4-yl)methyl)malonic acid

Proceeding as described in example 8

above, but replacing furan with 3-methyl[1,1'-biphenyl]-2 carboxylate, afforded the title compound as a white solid by purification by preparative reverse phase HPLC.

^1H NMR (CD3OD, 300 MHz) δ 8.27 (s, 1H), 7.71-7.73 (d, J=7 Hz, 1H), 7.29-7.48 (m, 4H), 7.14-7.16 (d,

J=8 Hz, 1H), 7.08-7.10 (d, J=8 Hz, 2H), 5.99-6.01 (d, J=8 Hz, 1H), 4.88-4, 91 (m, 1H), 4.30 (br.s, 1H), 4.034.12 (m, 2H), 3.39-3.57 (m, 2H), 2.99 (s, 1H); LC/MS [M+H] = 638.2.

Example 55. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethyl-3,4dihydroxytetrahydrofuran-2-yl) methoxy)malonic acid

Example 55

Diethyl 2-(((2R,3R,4S,5R)-5-(N6,N6-bis-Boc-2-chloro-9H-purin-9-yl)-3-((tert-butoxycarbonyl)oxy)- 4-Fluorotetrahydrofuran-2-yl)methoxy)malonate (100 mg, 0.13 mmol) was dissolved in EtOAc (2 ml). The solution was purged with argon gas three times, followed by careful addition of palladium on carbon (20 mg, 10 wt%). The resulting suspension was purged with argon gas three times and then placed in an H2 atmosphere (1 atm in a cylinder). The reaction was carried out for 70 h at ambient temperature. The suspension was filtered through diatomaceous earth, washed using EtOAc (3x2 ml). The filtrate was evaporated to an oil, which was then dissolved in DCM (1 ml), after which TFA (100 μl) was added. The resulting solution was kept overnight and then evaporated. The pale yellow oily residue was dissolved in THF (1 ml) and cooled to 0°C. 4 M NaOH (100 μL) was added to this reaction mixture and the reaction mixture was allowed to warm to ambient temperature for 14 h before evaporation. The crude residue was purified by preparative reverse phase HPLC to give the title compound as a white solid.

^1H NMR (CD3OD, 300 MHz) δ 8.90 (s, 1H), 6.09-6.11 (d, J=7 Hz, 1H), 4.69-4.72 (d, J=8 Hz, 1H), 4.64 (br.s, 1H), 4.19 (s, 1H), 3.83 (br.s, 3H), 1.88-1.95 (m, 3H), 1 .06-1.11 (t, J=7 Hz, 3H); LC/MS [M+H] = 432.2.

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Example 56.

Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethyl-3,4hydroxytetrahydrofuran-2-yl)methoxy)- 2-Benzylmalonic acid

Pd/C, H ₂ EtOH, EtOAc,

OAc

nh ₂ Ν·Α.

<' J 1st N^ci _>

OAc BSA, TMSOTf MeCN

N

ΌΙ aq. LiOH

MeOH, THF

Example 56

N

ΌΙ

Stage 1.

A mixture of diethyl 2-benzyl-2-(((2R,3R,4R)-3,4,5-triacetoxy-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (1.0 mmol, 549 mg) and palladium on charcoal (100 mg, 10 wt%) in EtOH (5 ml) and EtOAc (5 ml) under H2, stirred for 24 h, then filtered through diatomaceous earth, washed with EtOAc (3x5 ml). The filtrate was evaporated and purified by silica gel flash column chromatography to give diethyl 2-benzyl-2-(((2R,3R,4R)-3,4,5-triacetoxy-3-ethyltetrahydrofuran-2yl)methoxy)malonate.

Stages 2, 3.

Proceeding as described in Example 7 above, but replacing diethyl 2-(pyridin-4-ylmethyl)-2-(((2R,3R,4R)3,4,5-triacetoxy-3-ethynyltetrahydrofuran-2-yl) methoxy)malonate to diethyl 2-benzyl-2-(((2R,3R,4R)3,4,5-riacetoxy-3-ethyltetrahydrofuran-2-yl)methoxy)malonate followed by hydrolysis of the ester to obtain the title compound as a white solid by purification by preparative reverse phase HPLC ^1H NMR (CD ₃ OD, 300 MHz) δ 8.46 (s, 1H), 7.18-7.20 (m, 2H), 7 .04-7.09 (m, 3H), 6.01-6.04 (d, J=8 Hz, 1H), 4.63-4.66 (d, J=8 Hz, 1H), 4, 21 (br. s, 1H), 3.76-3.96 (m, 2H), 3.39-3.52 (m, 2H), 1.76-1.83 (m, 2H), 0.981, 03 (t, J=7 Hz, 3H); LC/MS [M+H] = 522.2.

Example 57. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3,4-dihydroxy-3vinyltetrahydrofuran-2-yl) methoxy)-2-benzylmalonic acid

ΝΗLindlar catalyst, H ₂ EtOH, EtOAc

CI aq. LiOH

MeOH,THF

C.I.

Example 57

Stage 1.

A mixture of diethyl 2-benzyl-2-(((2R,3R,4R)-3,4,5-triacetoxy-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (525 mg, 0.96 mmol) and Lindlar catalyst (105 mg, 5 wt%) in EtOH (5 ml) and EtOAc (5 ml) under H2 atmosphere was stirred for 24 h, then the mixture was filtered through diatomaceous earth and washed with EtOAc (3x5 ml). The filtrate was evaporated and purified by silica gel flash column chromatography to give diethyl 2-benzyl-2-(((2R,3R,4R)-3,4,5-triacetoxy-3-vinyltetrahydrofuran-2yl)methoxy)malonate.

Stages 2, 3.

Proceeding as described in Example 7 above, but replacing diethyl 2-(pyridin-4-ylmethyl)-2-(((2R,3R,4R)3,4,5-triacetoxy-3-ethynyltetrahydrofuran-2-yl) methoxy)malonate to diethyl 2-benzyl-2-(((2R,3R,4R)3,4,5-riacetoxy-3-vinyltetrahydrofuran-2-yl)methoxy)malonate followed by hydrolysis of the ester to obtain the title compound as a white solid by purification using preparative reverse phase HPLC.

^1H NMR (CD ₃ OD, 300 MHz) δ 8.44 (s, 1H), 7.21-7.22 (m, 2H), 7.06-7.11 (m, 3H), 6.14 -6.23 (m, 1H), 6.08-

- 87 046381

6.10 (d, J=8 Hz, 1H), 5.55-5.61 (m, 1H), 5.25-5.29 (m, 1H), 4.81 (s, 1H), 4 .14 (br. s, 1H), 3.91-3.94 (m, 1H), 3.62-3.65 (d, J=9 Hz, 1H), 3.50-3.55 (d , J=15 Hz, 1H), 3.39 (s, 1H); LC/MS [M+H] = 520.1.

Example 58

Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(5-chloro-3H-imidazo[4,5-b]pyridin-3-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran -2-yl)methoxy)malonic acid

Example 58

Stage 1.

To a solution of an anomeric mixture of diethyl-2-benzyl-2-(((2R,3R,4R)-3,4,5-triacetoxy-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (175 mg, 0.319 mmol) in dry dichloroethane (3.5 ml) was added 5-chloro3H-imidazo[4,5-b]pyridine (65 mg, 0.422 mmol) followed by N,O-6uc(trimethylsilyl)acetamide (BSA) (0.28 ml, 1 .12 mmol) using a syringe. The mixture was heated at 95°C under argon for 1 hour, then cooled to ambient temperature before TMSOTf (0.09 mL, 0.494 mmol) was added via syringe. The resulting mixture was heated at 95°C for 5 hours, after which it was allowed to cool, diluted with water (30 ml) and extracted using EtOAc (30 ml). The organic phase was washed successively with equal volumes of saturated NaHCO ₃ solution and brine. The aqueous phases were further extracted using EtOAc (2x30 ml). The combined organic phase was dried (MgSO ₄ ), filtered and concentrated. The crude residue was purified by preparative TLC (55% EtOAc in hexanes) to give the less polar diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(5-chloro -3H-imidαzo[4,5b]pyridin-3-yl)-3-((trimethylsilyl)ethynyl)tetrahydrofuran-2-yl)methoxy)malonate (44 mg) as a viscous oil and the desired diethyl-2-benzyl -2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(5-chloro-3H-imidazo[4,5b]pyridin-3-yl)-3-ethynyltetrahydrofuran-2-yl )methoxy)malonate (22 mg) as a viscous oil.

Stage 2.

To a solution of diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(5-chloro-3H-imidazo[4,5b]pyridin-3-yl)- 3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (22 mg, 0.034 mmol) in THF (0.6 ml) was added to a solution of 1 N aq. LiOH (0.24 ml). Additional 1 N aq. LiOH solution (0.58 ml) was applied for 2 days with a combination of intermittent sonication and stirring. The reaction mixture was evaporated and diluted with water (10 ml) and EtOAc (10 ml). The reaction mixture was cooled to 0°C and acidified to pH ~ 3 with 1 N aq. HCl solution. The layers were separated and aq. the layer was further extracted using EtOAc (2x10 ml). The combined organic layer was dried over MgSO ₄ , filtered and evaporated. The crude residue was purified by preparative reverse phase HPLC to give the title compound as an off-white solid.

^1H NMR (CD3OD, 300 MHz): δ 8.63 (br.s, 1H), 8.01 (d, J=8.14 Hz, 1H), 7.34 (d, J=8.14 Hz , 1H), 7.297.23 (m, 2H), 7.07-6.99 (m, 3H), 6.21 (d, J=7.33 Hz, 1H), 5.04 (d, J= 7.33 Hz, 1H), 4.34 (t, J=3.02 Hz, 1H), 4.144.03 (m, 2H), 3.48 (d, J=12.53 Hz, 1H), 3 .37 (d, J=12.53 Hz, 1H), 2.99 (s, 1H); LC/MS [M+H] = 502.0.

- 88 046381

Example 59. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(6-(benzylamino)-2-chloro-9H-purin-9-yl)-3ethynyl-3,4- Dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

Example 59

Stage 1.

A suspension of sodium hydride (60%, mineral dispersion; 1.91 g, 47.7 mmol) in anhydrous THF (150 ml) was cooled to 0°C and treated with a second solution of (3aR,5R,6R,6aR)-5-(( (tertbutyldimethylsilyl)oxy)methyl)-6-ethynyl-2,2-dimethyltetrahydrofuro[2,3^][1,3]dioxol-6-ol (10 g, 30.4 mmol) in THF (50 ml) more than 15 min. After stirring for 15 min at 0°C, the mixture was warmed to room temperature and stirred for an additional 1.5 h. The mixture was then cooled to 0°C and MOMCl (6.81 mL, 80.7 mmol, 2.6 eq) was added dropwise .). After addition was complete, the cooling bath was removed and stirring was continued for 2 hours at room temperature. The reaction was quenched by slow addition of saturated aqueous ammonium chloride (50 ml), washed with water and extracted with ethyl acetate (100 ml). The organic phase was dried over sodium sulfate, filtered and evaporated. The crude product was purified by silica gel chromatography (30% ethyl acetate in hexanes) to give tert-butyl (((3aR,5R,6R,6aR)-6-ethynyl-6-(methoxymethoxy)-2,2-dimethyltetrahydrofuro[2 ,3d][1,3]dioxol-5-yl)methoxy)dimethylsilane (9.95 g, 88% yield) as a white solid.

Stage 2.

A solution of the above alcohol (9.8 g, 26.3 mmol) in anhydrous THF (100 ml) was cooled to 0°C and treated with a 1 N solution of tetrabutylammonium fluoride in THF (37 ml, 36.8 mmol, 1.4 eq. ) for 15 minutes. After addition was complete, the reaction mixture was warmed to room temperature and stirred for 3 hours. When the reaction was complete (3 hours), the volatile components were evaporated to obtain a viscous residual oil, which was dissolved in dichloromethane (5 ml), loaded directly onto a silica gel column ( ~300 cm ³ ) and purified by silica gel chromatography, eluting with hexane to 50% ethyl acetate in hexane to give ((3aR,5R,6R,6aR)-6-ethynyl-6-(methoxymethoxy)-2,2dimethyltetrahydrofuro[2, 3^][1,3]-dioxol-5-yl)methanol (6.18 g, 91% yield) as a white solid.

Stage 3.

A solution of the above alcohol (175 mg, 0.678 mmol) in anhydrous benzene (8 ml) and 1-ethyl 3-(prop1-en-1-yl)2-diazomalonate (188 mg, 0.949 mmol) was treated with rhodium(II) acetate (5 .8 mg, 0.013 mmol, 0.02 eq.) and heated to 60-65°C for 2 hours. Upon completion, the solution was evaporated, dissolved in dichloromethane (1.5 ml), loaded directly onto a silica gel column (~ 100 cm ³ ) and purified by silica gel chromatography, eluting (0-50% ethyl acetate in hexane), to give 1-ethyl-3-prop-1-en1-yl 2-(((3aR,5R,6R,6aR)- 6-ethynyl-6-(methoxymethoxy)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]-dioxol5-yl)methoxy)malonate (252 mg, 87%) (mixture of isomers) as pale yellow oil.

Stage 4.

Under a nitrogen atmosphere, a solution of malonate from the previous step (250 mg, 0.583 mmol) and benzyl bromide (0.42 ml, 3.5 mmol, 6 eq.) in anhydrous DMF (8 ml) was treated with cesium carbonate (760 mg, 2.33 mmol ) and stirred at room temperature for 4 hours. Once complete, the reaction was filtered through a celite pad, washed with water and extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered and evaporated. The residual oil was dissolved in dichloromethane (2 ml), loaded onto a silica gel column (~100 cm ³ ), eluting with 30% ethyl acetate in hexane, to give 1-ethyl 3-prop-1-en-1-yl 2-benzyl-2 -(((3aR,5R,6R,6aR)-6-ethynyl-6-(methoxymethoxy)-2,2

- 89 046381 dimethyltetrahydro(])uro[2.3-c1][1.3]dio1<sol-5-yl)methoxy)malonate (261 mg, 86%) (mixture of isomers) in the form of a pale yellow oil.

Stage 5.

Under a nitrogen atmosphere, cooled with water (14-17°C), a solution of the acetonide from the last step (500 mg, 0.964 mmol) in acetic acid (3.9 ml) was treated with acetic anhydride (0.965 ml, 10.3 mmol, 10.7 eq. .) and concentrated sulfuric acid (410 μl, 0.326 mmol, 0.34 eq.). The resulting solution was stirred for 4 hours, diluted with water and extracted with ethyl acetate. The combined organic solution was washed with sodium bicarbonate (aq, saturated; 100 ml), dried (Na ₂ SO ₄ ), filtered and evaporated in vacuo. The residual oil was dissolved in dichloromethane (2 ml) and purified on a Biotage flash chromatography system, eluting with hexane to 50% ethyl acetate in hexane. Diastereomeric mixture of 1-ethyl 3-prop-1-en-1-yl 2-benzyl-2-(((2R,3R,4R)-3,4,5-triacetoxy-3-ethynyltetrahydrofuran-2yl)methoxy)malonate ( 420 mg, 8%) (anomers separated by silica gel chromatography, 40G silica gel column) were isolated as a clear oil.

Stage 6.

A suspension of 2,6-dichloroadenine (143 mg, 0.76 mmol, 1.01 eq.) and N-O-bis-(trimethylsilyl)acetamide (0.24 ml, 0.97 mmol, 1.29 eq.) in anhydrous acetonitrile (5 ml) was treated with a second solution of 1-ethyl3-((E)-prop-1-en-1-yl)2-benzyl-2-(((2R,3R,4R)-3,4,5-triacetoxy -3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (143 mg, 0.75 mmol) in anhydrous acetonitrile (15 ml) followed by dropwise addition of trimethylsilyl trifluoromethanesulfonate (0.18 ml, 1.0 mmol, 1.33 eq. ). After addition was complete, the reaction mixture was heated to 50°C for 18 hours, then cooled to room temperature. (The reaction starts out pale yellow and turns clear amber after 4 hours.) Once complete, saturated aqueous sodium bicarbonate was added and the mixture was stirred for 10 minutes. The crude product was then extracted with ethyl acetate (3x30 ml) and the combined organic layer was dried (Na ₂ SO ₄ ), filtered and evaporated. The residue was dissolved in dichloromethane and purified using a Biotage flash chromatography system, eluting with hexanes to 50% ethyl acetate in hexanes to give 1-ethyl-3-prop-1-en-1-yl-2-benzyl-2-((( 2R,3R,4R,5R)-3,4-diacetoxy-5-(2,6-dichloro-9Hpurin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (as a mixture of isomers) in the form white solid (400 mg, 77% yield).

Stage 7.

A solution of 1-ethyl-3-prop-1-en-1-yl-2-benzyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(2,6-dichloro- 9Hpurin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (80 mg, 0.116 mmol) in anhydrous dioxane (2 ml) was cooled to 0°C and treated with DIPEA (30 μl, 0.174 mmol, 1 .5 eq.) and benzylamine (13 μl, 0.116 mmol, 1 eq.). After addition was complete, the solution was warmed to room temperature with continued stirring overnight (18 hours). The mixture was diluted with ethyl acetate (50 ml) and washed with water (20 ml). The organic layer was dried (Na ₂ SO ₄ ), filtered and evaporated. The crude product was purified using a Biotage flash chromatography system, eluting with 50% ethyl acetate in hexanes, to give 1-ethyl-3-prop-1-en-1-yl-2-benzyl-2-(((2R,3R,4R ,5R)-3,4-diacetoxy-5-(6(benzylamino)-2-chloro-9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (as a mixture of isomers ~1: 1) as a white solid (75 mg, 85% yield).

Stage 8.

Solution of 1 -ethyl-3 -prop-1 -en-1 -yl-2-benzyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5 -(6-(benzylamino)- 2chloro-9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate from the last step (70 mg, 0.092 mmol) in THF (1 ml) was treated with a solution of LiOH (31 mg, 1.35 mmol, 15 eq.; in 1 ml of water) and stirred overnight. The resulting solution was acidified using 2 N HCl to pH 3 and the resulting suspension was stirred for 10 minutes, then filtered, washed with cold water and dried. The title compound was isolated as a white solid (50 mg, 89% yield).

^1H NMR (400 MHz, CDCl3/CD3OD=5:1) δ 7.98 (s, 1H), 7.25-6.84 (m, 10H), 5.85 (d, J=6.4 Hz , 1H), 4.63 (s, 2H), 4.54 (d, J=6.4 Hz, 1H), 4.17 (t, J=3.2 Hz, 1H), 3.88 (cd , J=10.3, 3.3 Hz, 2H), 3.36-3.16 (m, 2H), 2.49 (s, 1H). HPLC: 9.97 min, 97.0%. ESI-MS (m/z): [M] ⁺ calculated for C29H ₂₆ ClN ₅ O ₈ , 608.15; found 608.1.

Example 60. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-((tetrahydro-2H-pyran-4-yl)amino)-9Hpurin-9- yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that tetrahydro-2H-pyran-4amine was used instead of benzylamine in Step 7.

- 90 046381 ¹ H NMR (400 MHz, CDCl3/CD3OD=5:1) δ 8.09 (s, 1H), 7.18-7.12 (m, 2H), 7.01 (dd, J=12 ,1, 7.2 Hz, 3H), 5.90 (d, J=6.3 Hz, 1H), 4.57 (d, J=6.3 Hz, 1H), 4.25 (t, J =3.1 Hz, 2H), 4.00-3.89 (m, 4H), 3.51 (td, J=11.6, 2.2 Hz, 2H), 3.42-3.330 (m, 2H), 2.52 (s, 1H), 1.94 (d, J=13.0 Hz, 2H), 1.57 (td, J=11.2, 3.5 Hz, 2H). ESI-MS (m/z): [M] ⁺ calculated for C27H ₂₈ ClN ₅ O ₉ , 602.16; found 602.5.

Example 61. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-((2-(diethylamino)ethyl)amino)-9Hpurin-9-yl)- 3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid ζ ^Ί * ^r ; ^J

IlC jH

Prkier 51

The title compound was prepared in a manner similar to that described in Example 59, except that in step 7, N, N-diethylethane-1,2-diamine was used instead of benzylamine.

1H NMR (400 MHz, DMSO- _d6 ): δ 9.01 (s, 1H), 8.75 (s, 1H), 8.49 (s, 1H), 7.23-7.03 (m, 5H), 6.16 (s, 1H), 5.96 (d, J=7.0 Hz, 1H), 5.84 (d, J=6.9 Hz, 1H), 4.59 (t, J=7.1 Hz, 1H), 4.13 (dd, J=6.7, 2.7 Hz, 1H), 3.83-3.55 (m, 4H), 3.53 (s, 1H ), 3.30-3.15 (m, 6H), 3.06-2.97 (m, 2H), 1.26-1.12 (m, 6H). ESI-MS (m/z): [M] ⁺ calculated for C ₂₈ H ₃₃ ClN ₆ O ₈ , 617.20; found 617.5.

Example 62. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(((R)-1-phenylethyl)amino)-9H-purin-9yl) -3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid *; '“З μ\я.-j... _l

K-.S·' - D G '-'A· ΐ i

T ^-- ' d_.__ but O» - Ί ΰι

Piasher ί2

The title compound was prepared in a manner similar to that described in Example 59, except that N)-1-phenylethan-1-amine was used instead of benzylamine in Step 7.

1H NMR (400 MHz, CDCl ₃ /CD ₃ OD=5:1) δ 8.25 (s, 1H), 7.44-7.27 (m, 4H), 7.23-6.94 (m, 6H), 5.91 (dd, J=6.2, 1.7 Hz, 1H), 5.46 (d, J=8.0 Hz, 1H), 4.58 (d, J=6.2 Hz, 1H), 4.25 (t, J=2.4 Hz, 1H), 3.98-3.83 (m, 2H), 3.32 (dd, J=6.3, 1.7 Hz , 2H), 2.48 (d, J=1.7 Hz, 1H), 1.59 (dd, J=6.9, 1.7 Hz, 3H). ESI-MS (m/z): [M] ⁺ calculated for C ₃₀ H ₂₈ ClN ₅ O ₈ , 622.16; found 622.1.

Example 63. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(((S)-1-phenylethyl)amino)-9H-purin-9yl) -3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid p·. ',ι k-·/ ' ; Г ν Ϊ .

“ BUT Oh ι

Ui

Example:3

The title compound was prepared in a manner similar to that described in Example 59, except that N)-1-phenylethan-1-amine was used instead of benzylamine in Step 7.

1H NMR (400 MHz, CDCl ₃ /CD ₃ OD=5:1) δ 8.11 (s, 1H), 7.40-7.28 (m, 4H), 7.23-6.93 (m, 6H), 5.90 (d, J=5.8 Hz, 1H), 5.45 (s, 1H), 4.52 (d, J=5.8 Hz, 1H), 4.35-4, 22 (m, 1H), 3.99 (t, J=2.4 Hz, 2H), 3.42-3.25 (m, 2H), 2.50 (s, 1H), 1.58 (d , J=6.9 Hz, 3H). ESI-MS (m/z): [M]+ calculated for C ₃₀ H ₂₈ ClN ₅ O ₈ , 622.16; found 622.2.

Example 64. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-((tetrahydrofuran-3-yl)amino)-9Hpurin-9-yl)-3 -ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

Prüggr ί4

The title compound was prepared in a manner similar to that described in Example 59, except that tetrahydrofuran-3-amine was used instead of benzylamine in step 7.

1H NMR (400 MHz, CDCl ₃ /CD ₃ OD=5:1) δ 8.10 (s, 1H), 7.19-6.92 (m, 5H), 5.90 (d, J=6, 3 Hz, 1H), 4.72 (s, 1H), 4.58 (d, J=6.3 Hz, 1H) 4.25 (t, J=3.1 Hz, 1H), 3.94 ( tr, J=7.0, 4.0, 3.3 Hz, 3H), 3.70 (dt, J=9.4, 3.6 Hz, 2H), 3.43-3.24 (m, 2H), 2.52 (s, 1H), 2.29 (dk, J=13.2, 7.6 Hz, 1H), 1.93 (s, 1H). ESI-MS (m/z): [M] ⁺ calculated for C ₂₆ H ₂₆ ClN ₅ O ₉ , 588.14; found 588.3.

- 91 046381

Example 65. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-((S)-3-hydroxypyrrolidin-1-yl)-9Hpurin-9-yl )-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that (b)-pyrrolidin-3-ol was used instead of benzylamine in step 7.

¹ H NMR (400 MHz, CDCl3/CD3OD=5:1) δ 8.10 (s, 1H), 7.22-6.92 (m, 5H), 5.92 (d, J=5.4 Hz , 1H), 4.554.48 (m, 2H), 4.29 (t, J=3.7 Hz, 1H), 4.25-3.85 (m, 4H), 3.80-3.65 ( m, 2H), 3.35-3.24 (m, 2H), 2.51 (s, 1H), 2.081.98 (m, 2H). ESI-MS (m/z): [M] ⁺ calculated for C26H ₂₆ ClN ₅ O ₉ , 588.14; found 588.2.

Example 66. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(ethyl(methyl)amino)-9H-purin-9-yl)-3ethynyl- 3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that N-methylethanamine was used instead of benzylamine in step 7.

¹ H NMR (400 MHz, CDCl ₃ /CD ₃ OD=5:1) δ 8.05 (s, 1H), 7.24-7.02 (m, 5H), 5.92 (d, J=4 .8 Hz, 1H), 4.46 (dd, J=4.8, 1.3 Hz, 1H), 4.32 (dd, J=4.9, 3.2 Hz, 1H), 4.05 (dd, J=8.5, 4.0 Hz, 2H), 3.44-3.30 (m, 2H), 3.10-2.85 (m, 7H), 2.53 (s, 1H ), 1.22 (t, J=7.1 Hz, 3H). ESI-MS (m/z): [M] ⁺ calculated for C^H^CMOs, 560.15; found 560.5.

Example 67. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-((2-fluorobenzyl)amino)-9H-purin-9-yl)3 - ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that (2-fluorophenyl)methanamine was used instead of benzylamine in Step 7.

¹ H NMR (400 MHz, CDCl3/CD3OD=5:1) δ 8.29 (s, 1H), 7.47-7.36 (m, 1H), 7.26-6.94 (m, 8H) , 5.94 (d, J=6.2 Hz, 1H), 4.82-4.76 (m, 2H), 4.60 (d, J=6.2 Hz, 1H), 4.35- 4.23 (m, 1H), 3.98-3.84 (m, 2H), 3.44-3.30 (m, 2H), 2.46 (s, 1H). ESI-MS (m/z): [M] ⁺ calculated for C29H ₂₅ ClFN ₅ O ₈ , 626.14; found 626.7.

Example 68

Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-((4-fluorobenzyl)amino)-9H-purin-9-yl)-3-ethynyl3, 4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that (4-fluorophenyl)methanamine was used instead of benzylamine in Step 7.

1H NMR (400 MHz, CDCl ₃ /CD ₃ OD=5:1) δ 8.49 (s, 1H), 7.37 (dd, J=8.4, 5.3 Hz, 2H), 7.24 -6.89 (m, 7H), 5.97 (d, J=6.3 Hz, 1H), 4.72 (s, 2H), 4.63 (d, J=6.3 Hz, 1H) , 4.7-4.28 (m, 1H), 4.06-3.87 (m, 2H), 3.46-3.26 (m, 2H), 2.54 (s, 1H). ESI-MS (m/z): [M] ⁺ calculated for C ₂₉ H ₂₅ ClFN ₅ O ₈ , 626.14; found 626.4.

- 92 046381

Example 69. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(((R)-2,3-dihydro-1H-inden-1yl)amino )-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid '=· ¹ . '

-'·g ί, A-. _v ^r 'l

....4— ^f ' ή .. J'j /—\ i yu ch ' Ch y

The title compound was prepared in a manner similar to that described in Example 59, except that in step 7, N)-2,3-dihydro-N-inden-1-amine was used instead of benzylamine.

1H NMR (400 MHz, CDCl ₃ /CD ₃ OD=5:1) δ 8.30 (s, 1H), 7.29-6.97 (m, 9H), 5.95 (d, J=6, 2 Hz, 1H), 5.81 (t, J=7.1 Hz, 1H), 4.59 (d, J=6.3 Hz, 1H), 4.25 (d, J=3.1 Hz , 1H), 3.94-3.73 (m, 2H), 3.40-3.31 (m, 2H), 3.07 (ddd, J=14.0, 8.7, 4.8 Hz , 1H), 2.89 (dt, J=15.8, 7.7 Hz, 1H), 2.69-2.58 (m, 1H), 2.45 (s, 1H), 2.00 ( dd, J=13.6, 7.1 Hz, 1H). ESI-MS (m/z): [M] ⁺ calculated for C ₃₁ H ₂₈ ClN ₅ O ₈ , 634.16; found 634.8.

Example 70. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-phenethylamine-9H-purin-9-yl)-3-ethynyl3,4-dihydroxytetrahydrofuran- 2-yl)methoxy)malonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that 2-phenylethan-1-amine was used instead of benzylamine in step 7.

1H NMR (400 MHz, CDCl3/CD3OD=5:1) δ 8.14 (s, 1H), 7.26-6.91 (m, 10H), 5.91 (d, J=6.2 Hz, 1H), 4.57 (d, J=6.2 Hz, 1H), 4.27 (t, J=3.2 Hz, 1H), 3.96 (t, J=4.0 Hz, 2H) , 3.78 (m, 2H), 3.45-3.29 (m, 2H), 2.93 (t, J=7.3 Hz, 2H), 2.52 (s, 1H). ESI-MS (m/z): [M] ⁺ calculated for C ₃₀ H ₂₈ ClN ₅ O ₈ , 622.16; found 622.5.

Example 71. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(methylamino)-9H-purin-9-yl)-3-ethynyl3,4- Dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

Ph =4 c

H.. ^J —'' --1-./1

I sjH 5.

npEbISp 1

The title compound was prepared in a manner similar to that described in Example 59, except that methylamine was used instead of benzylamine in step 7.

1H NMR (400 MHz, CD3OD) δ 8.18 (s, 1H), 7.29-7.20 (m, 2H), 7.04 (dd, J=5.1, 1.9 Hz, 3H) , 5.99 (d, J=7.4 Hz, 1H), 4.99 (d, J=7 Hz, 1H), 4.30 (t, J=3.3 Hz, 1H), 4.11 -4.01 (m, 2H), 3.49-3.34 (m, 2H), 3.06 (s, 3H), 2.98 (s, 1H). ESI-MS (m/z): [M] ⁺ calculated for C ₂₃ H ₂₂ ClN ₅ O ₈ , 532.12; found 532.1.

Example 72. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(((S)-2,3-dihydro-1H-inden-1-yl )amino)9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid gl- 9°·'..... ^:

To.

b D Τ'* -γ.'' ·) , >

ⁿ -·- ' I ή /h

Si 'A

Example 72

The title compound was prepared in a manner similar to that described in Example 59, except that in step 7, N)-2,3-dihydro-N-inden-1-amine was used instead of benzylamine.

1H NMR (400 MHz, CD3OD) δ 8.14 (s, 1H), 7.41-7.13 (m, 6H), 7.04 (t, J=3.5 Hz, 3H), 6.01 (d, J=7.4 Hz, 1H), 5.88-5.76 (m, 1H), 4.99 (d, J=7.4 Hz, 1H), 4.32 (t, J= 3.3 Hz, 1H), 4.06 (dd, J=5.5, 3.3 Hz, 2H), 3.53-3.34 (m, 2H), 3.13-3.05 (m , 1H), 3.00 (s, 1H), 2.99-2.87 (m, 1H), 2.67 (dd, J=12.5, 4.2 Hz, 1H), 2.02 ( dd, J=12.8, 7.7 Hz, 1H). ESI-MS (m/z): [M] ⁺ calculated for C ₃₁ H ₂₈ ClN ₅ O ₈ , 634.16; found 634.5.

Example 73. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(ethylamino)-9H-purin-9-yl)-3-ethynyl3,4- Dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

- ...^—·’ .. )1 '''nG: ΰΗ '1

Priper 73

The title compound was prepared in a manner similar to that described in Example 59, for

- 93 046381 except that in step 7, ethylamine was used instead of benzylamine.

^1H NMR (400 MHz, CD3OD) δ 8.16 (s, 1H), 7.40-7.19 (m, 2H), 7.04 (dd, J=5.1, 1.9 Hz, 3H ), 5.98 (d, J=7.4 Hz, 1H), 4.97 (d, J=7.4 Hz, 1H), 4.30 (t, J=3.3 Hz, 1H), 4.14-3.98 (m, 2H), 3.57 (d, J=7.8 Hz, 2H), 3.49-3.33 (m, 2H), 2.98 (s, 1H) , 1.28 (t, J=7.2 Hz, 3H). ESI-MS (m/z): [M] ⁺ calculated for C24H24ClN5O ₈ , 546.13; found 546.1.

Example 74. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(6-((S)-sec-butylamino)-2-chloro-9H-purin-9-yl)- 3ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that N-butan-2-amine was used instead of benzylamine in Step 7.

1H NMR (400 MHz, CD3OD) δ 8.13 (s, 1H), 7.32-7.15 (m, 2H), 7.14-6.98 (m, 3H), 5.98 (d, J=7.4 Hz, 1H), 4.97 (d, J=7.4 Hz, 1H), 4.31 (t, J=3.2 Hz, 1H), 4.24 (s, 1H) , 4.14-3.97 (m, 2H), 3.53-3.33 (m, 2H), 2.99 (s, 1H), 1.63 (k, J=7.2 Hz, 2H ), 1.27 (d, J=6.5 Hz, 3H), 0.97 (t, J=7.4 Hz, 3H). ESI-MS (m/z): [M] ⁺ calculated for C26H2sClN5Os, 574.16; found 574.1.

Example 75. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-((cyclopropylmethyl)-(methyl)amino)-9Hpurin-9-yl)-3 -ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that 1-cyclopropyl-N-methylmethanamine was used instead of benzylamine in step 7.

1H NMR (400 MHz, CD3OD) δ 8.15 (s, 1H), 7.36-7.18 (m, 2H), 7.03 (dd, J=5.2, 2.0 Hz, 3H) , 6.00 (d, J=7.3 Hz, 1H), 4.97 (d, J=7.3 Hz, 1H), 4.30 (dd, J=4.1, 2.8 Hz, 1H), 4.07 (cd, J=10.2, 3.5 Hz, 2H), 3.52-3.31 (m, 7H), 2.98 (s, 1H), 1.20-1 .12 (m, 1H), 0.54 (dd, J=8.2, 1.8 Hz, 2H), 0.45-0.30 (m, 2H). ESI-MS (m/z): [M] ⁺ calculated for C ₂₇ H ₂₈ ClN ₅ O ₈ , 586.16; found 586.9.

Example 76. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(6-((carboxymethyl)amino)-2-chloro-9H-purin-9yl)-3-ethynyl-3 ,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that 2-amino-K,K-dimethylacetamide was used instead of benzylamine in step 7.

1H NMR (400 MHz, CD3OD) δ 8.21 (s, 1H), 7.38-7.14 (m, 2H), 7.12-6.98 (m, 3H), 6.11 (d, J=7.4 Hz, 1H), 4.96 (d, J=7.3 Hz, 1H), 4.30 (k, J=4.7, 4.0 Hz, 2H), 4.09- 4.02 (m, 2H), 3.54-3.33 (m, 2H), 2.97 (s, 1H). ESI-MS (m/z): [M] ^- calculated for C ₂₄ H ₂₂ ClN ₅ O ₁₀ , 574.11; found 574.1.

Example 77. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-((2-chlorobenzyl)amino)-9H-purin-9-yl)3- ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that (2-chlorophenyl)methanamine was used instead of benzylamine in step 7.

1H NMR (400 MHz, CD3OD) δ 8.17 (s, 1H), 7.43 (m, 2H), 7.28 (m, 3H), 7.22 (m, 1H), 7.00 (m , 3H), 5.99 (d, J=7.4 Hz, 1H), 4.98 (d, J=7.4 Hz, 1H), 4.82 (m, 2H), 4.30 (s , 1H), 4.05 (s, 2H), 3.42 (m, 2H), 2.98 (s, 1H). LC-MS: m/z=597 (M-CO2H); m/z=292 (M-ribose fragment).

- 94 046381

Example 78. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-((pyridin-4-ylmethyl)amino)-9H-purin9-yl)-3 -ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that pyridin-4-ylmethanamine was used instead of benzylamine in step 7.

1H NMR (400 MHz, CD3OD) δ 9.08 (br.s, 1H), 8.73 (d, J=5.7 Hz, 2H), 8.51 (s, 1H), 7.68 (d , J=5.6 Hz, 2H), 7.22 (m, 2H), 7.06 (m, 3H), 5.82 (d, J=7.7 Hz, 1H), 4.93 (d , J=6.9 Hz, 1H), 4.87 (d, J=7.6 Hz, 1H), 4.20 (m, 1H), 4.03 (m, 2H), 3.86 (m , 2H), 3.62 (s, 1H), 3.27 (m, 2H). HPLC: Room temperature=5.74 min, 97.7%. LC-MS: m/z=610 (M+); m/z=261 (M-ribose fragment).

Example 79 Synthesis

2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-morpholino-9H-purin-9-yl)-3-ethynyl-3,4 dihydroxytetrahydrofuran-2-yl )methoxy)malonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that morpholine was used instead of benzylamine in step 7.

1H NMR (400 MHz, CD3OD) δ 8.20 (s, 1H), 7.22 (m, 2H), 7.01 (m, 3H), 6.00 (d, J=7.6 Hz, 1H ), 4.99 (d, J=7.6 Hz, 1H), 4.30 (m, 1H), 4.22 (m, 4H), 4.04 (m, 2H), 3.78 (m , 4H), 3.47 (m, 2H), 2.99 (s, 1H). HPLC: 8.16 min, 98.2%. LC-MS: m/z=588 (M+), 544 (M-CO2H), m/z=240 (M-ribose fragment).

Example 80. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-(αzepan-1-yl)-2-chloro-9H-purin-9-yl)-3-ethynyl-3, 4dihydroxytetrahydrofuran-2-yl)methoxy)-2-benzylmalonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that azepane was used instead of benzylamine in step 7.

1H NMR (400 MHz, CD3OD) δ 8.16 (s, 1H), 7.22 (m, 2H), 7.00 (m, 3H), 5.99 (d, J=7.6 Hz, 1H ), 4.98 (d, J=7.6 Hz, 1H), 4.29 (m, 3H), 4.07 (m, 2H), 3.85 (m, 2H), 3.40 (m , 2H), 2.97 (s, 1H), 1.85 (m, 4H), 1.59 (m, 4H). HPLC: 9.67 min, 98.1%. LC-MS: m/z=600 (M+), m/z=556 (M-CO ₂ H), m/z=252 (M-ribose fragment).

Example 81. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(cyclobutyl(methyl)amino)-9H-purin-9yl)-3-ethynyl- 3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that N-methyl ring butanamine was used instead of benzylamine in step 7.

1H NMR (400 MHz, CD3OD) δ 8.21 (s, 1H), 7.21 (dd, J=7.0, 2.6 Hz, 2H), 6.99 (m, 3H), 5.99 (d, J=7.3 Hz, 1H), 5.74 (br. s, 1H), 4.96 (d, J=7.2 Hz, 1H), 4.28 (dd, J=4, 1, 2.8 Hz, 1H), 4.06 (dd, J=10.2, 4.2 Hz, 1H), 4.01 (dd, J=10.2, 2.9 Hz, 1H), 3.45-3.31 (m, 5H), 2.96 (s, 1H), 2.35 (k, J=10.0 Hz, 2H), 2.23 (m, 2H), 1.87 -1.63 (m, 2H); HPLC: 9.45 min, 98.9%. LC-MS: m/z=587 (M+H), 543 (M-CO2H), m/z=238 (M-ribose fragment).

- 95 046381

Example 82. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(isopropyl(methyl)amino)-9H-purin-9yl)-3-ethynyl- 3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that X-methylpropan-2-amine was used instead of benzylamine in step 7.

1H NMR (400 MHz, CD3OD) δ 8.17 (s, 1H), 7.21 (dd, J=7.3, 2.2 Hz, 2H), 7.03-6.94 (m, 3H) , 5.98 (d, J=7.3 Hz, 1H), 4.97 (d, J=7.3 Hz, 1H), 4.28 (dd, J=4.0, 2.8 Hz, 1H), 4.04 (cd, J=10.2, 3.5 Hz, 2H), 3.45-3.31 (m, 6H), 2.96 (s, 1H), 1.25 (d , J=6.8 Hz, 6H); HPLC: 9.08 min, 99.9%. LC-MS: m/z=575 (M+H), 531 (M-CO2H), m/z=226 (M-ribose fragment).

Example 83. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(cyclopropylamino)-9H-purin-9-yl)-3ethynyl-3,4- Dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that cyclopropanamine was used instead of benzylamine in step 7.

1H NMR (400 MHz, CD3OD) δ 8.14 (s, 1H), 7.23 (dd, J=6.7, 2.8 Hz, 2H), 7.01 (m, 3H), 5.97 (d, J=7.4 Hz, 1H), 4.95 (d, J=7.4 Hz, 1H), 4.28 (t, J=3.3 Hz, 1H), 4.10-3 .97 (m, 2H), 3.49-3.30 (m, 2H), 2.99 (m, 1H), 2.96 (s, 1H), 0.92-0.78 (m, 2H ), 0.66-0.54 (m, 2H); HPLC: 7.83 min, 99.1%. LC-Mc: m/z=559 (M+H).

Example 84. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-((pyridin-3-ylmethyl)amino)-9H-purin9-yl)-3 -ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that pyridin-3-ylmethanamine was used instead of benzylamine in step 7.

1H NMR (400 MHz, CD3OD) δ 8.87 (s, 1H), 8.70 (d, J=5.6 Hz, 1H), 8.56 (d, J=8.1 Hz, 1H), 8.22 (s, 1H), 7.97 (dd, J=8.1, 5.7 Hz, 1H), 7.21 (dd, J=6.5, 3.0 Hz, 2H), 7 .07-6.90 (m, 3H), 5.97 (d, J=7.4 Hz, 1H), 4.96 (d, J=7.4 Hz, 1H), 4.92 (m, 2H), 4.29 (t, J=3.4 Hz, 1H), 4.04 (cd, J=10.2, 3.4 Hz, 2H), 3.38 (m, 2H), 2, 97 (s, 1H); HPLC: 5.82 min, 99.9%. LC-MS: m/z=261 (M-ribose fragment).

Example 85. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-((3-fluorobenzyl)amino)-9H-purin-9-yl)3- ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that (3-fluorophenyl)methanamine was used instead of benzylamine in step 7.

1H NMR (400 MHz, CD3OD) δ 8.19 (s, 1H), 7.32 (td, J=8.0, 5.9 Hz, 1H), 7.24-7.16 (m, 3H) , 7.12 (dd, J=9.8, 2.3 Hz, 1H), 7.06-6.78 (m, 4H), 5.98 (d, J=7.3 Hz, 1H), 4.94 (d, J=7.3 Hz, 1H), 4.74 (m, 2H), 4.29 (t, J=3.3 Hz, 1H), 4.03 (cd, J=10 ,3, 3.5 Hz, 2H), 3.47-3.26 (m, 2H), 2.97 (s, 1H); HPLC: 9.04 min, 99.5%. LC-MS: m/z=627 (M+H), 583 (M-CO ₂ H), m/z=278 (M-ribose fragment).

- 96 046381

Example 86. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-((3-chlorobenzyl)amino)-9H-purin-9-yl)3- ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that (3-chlorophenyl)methanamine was used instead of benzylamine in step 7.

1H NMR (400 MHz, CD3OD) δ 8.18 (s, 1H), 7.40 (s, 1H), 7.34-7.05 (m, 5H), 6.98 (m, 3H), 5 .97 (d, J=7.4 Hz, 1H), 4.95 (d, J=7.4 Hz, 1H), 4.72 (m, 2H), 4.29 (t, J=3, 3 Hz, 1H), 4.09-3.87 (m, 2H), 3.46-3.31 (m, 2H), 2.97 (s, 1H); HPLC: 9.45 min, 98.3%. LC-MS: m/z=643 (M+H), 598 (M-CO ₂ H), m/z=294 (M-ribose fragment).

Example 87. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-((4-chlorobenzyl)amino)-9H-purin-9-yl)3- ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that (4-chlorophenyl)methanamine was used instead of benzylamine in step 7.

^1H NMR (400 MHz, CD3OD) δ 8.17 (s, 1H), 7.36 (d, J=8.6 Hz, 2H), 7.31 (d, J=8.5 Hz, 1H) , 7.21 (dd, J=6.8, 2.8 Hz, 2H), 7.02-6.96 (m, 3H), 5.97 (d, J=7.3 Hz, 1H), 4.94 (d, J=7.3 Hz, 1H), 4.71 (m, 2H), 4.29 (t, J=3.3 Hz, 1H), 4.03 (cd, J=10 ,2, 3.4 Hz, 2H), 3.46-3.30 (m, 2H), 2.96 (s, 1H); HPLC: 9.50 min, 98.7%. LC-MS: m/z=643 (M+H), 598 (M-CO2H), m/z=294 (M-ribose fragment).

Example 88. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-(azetidin-1-yl)-2-chloro-9H-purin-9-yl)-3-ethynyl-3, 4dihydroxytetrahydrofuran-2-yl)methoxy)-2-benzylmalonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that azetidine was used instead of benzylamine in step 7.

1H NMR (400 MHz, CD3OD) δ 8.25 (s, 1H), 7.24-7.08 (m, 2H), 7.10-6.85 (m, 3H), 5.97 (d, J=7.4 Hz, 1H), 4.98 (d, J=7.3 Hz, 1H), 4.43 (m, 4H), 4.29 (dd, J=4.1, 2.8 Hz, 1H), 4.07 (dd, J=10.2, 4.2 Hz, 1H), 4.00 (dd, J=10.2, 2.9 Hz, 1H), 3.42 (d , J=15.0 Hz, 1H), 3.34 (d, J=12.3 Hz, 2H), 2.95 (s, 1H), 2.51 (k, J=7.7 Hz, 2H ); HPLC: 7.58 min, 99.5%. LC-MS: m/z=558 (M+H), 554 (M-CO2H), m/z=210 (M-ribose fragment).

Example 89. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(dimethylamino)-9H-purin-9-yl)-3ethynyl-3,4- Dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that dimethylamine was used instead of benzylamine in step 7.

1H NMR (400 MHz, CD3OD) δ 8.17 (s, 1H), 7.22 (dd, J=7.5, 2.0 Hz, 2H), 7.05-6.89 (m, 3H) , 5.98 (d, J=7.2 Hz, 1H), 4.96 (d, J=7.3 Hz, 1H), 4.28 (dd, J=4.0, 2.9 Hz, 1H), 4.04 (cd, J=10.2, 3.5 Hz, 2H), 3.55-3.31 (m, 8H), 2.95 (s, 1H); HPLC: 8.16 min, 99.9%. LC-MS: m/z=546 (M+H), 502 (M-CO2H), m/z=198 (Mribose fragment).

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Example 90. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(pyrrolidin-1-yl)-9H-purin-9-yl)-3ethynyl- 3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid!'G'- ^- , /- ¹ '>I;

Prier I

The title compound was prepared in a manner similar to that described in Example 59, except that pyrrolidine was used instead of benzylamine in step 7.

1H NMR (400 MHz, CD3OD) δ 8.21 (s, 1H), 7.21 (dd, J=7.3, 2.3 Hz, 2H), 7.03-6.76 (m, 3H) , 5.98 (d, J=7.3 Hz, 1H), 4.98 (d, J=7.3 Hz, 1H), 4.29 (dd, J=4.2, 2.8 Hz, 1H), 4.15-3.86 (m, 4H), 3.66 (m, 2H), 3.41 (d, J=15.0 Hz, 1H), 3.34 (d, J=15 .0 Hz, 1H), 2.95 (s, 1H), 2.02 (m, 4H); HPLC: 8.42 min, 95.6%. LC-MS: m/z=573 (M+H), 529 (M-CO ₂ H), m/z=224 (M-ribose fragment).

Example 91. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(cyclopentylamino)-9H-purin-9-yl)-3ethynyl-3,4- Dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

Prkgk I

The title compound was prepared in a manner similar to that described in Example 59, except that cyclopentylamine was used instead of benzylamine in step 7.

1H NMR (400 MHz, CD3OD) δ 8.09 (s, 1H), 7.23 (dd, J=6.7, 2.9 Hz, 2H), 7.01 (m, 3H), 5.96 (d, J=7.4 Hz, 1H), 4.94 (d, J=7.4 Hz, 1H), 4.47 (m, 1H), 4.28 (t, J=3.3 Hz , 1H), 4.04 (m, 2H), 3.43 (d, J=14.9 Hz, 1H), 3.34 (d, J=14.9 Hz, 1H), 2.96 (s , 1H), 2.19-1.98 (m, 2H), 1.78 (m, 2H), 1.72-1.64 (m, 2H), 1.62-1.52 (m, 2H ); HPLC: 9.06 min, 98.9%. LC-MS: m/z=587 (M+H), 543 (M-CO2H), m/z=238 (M-ribose fragment).

Example 92. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-((cyclopropylmethyl)amino)-9H-purin9-yl)-3-ethynyl-3 ,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that cyclopropylmethanamine was used instead of benzylamine in step 7.

1H NMR (400 MHz, CD ₃ OD) δ 8.14 (s, 1H), 7.18 (d, J=6.9 Hz, 2H), 7.12-6.68 (m, 3H), 5 .92 (d, J=6.1 Hz, 1H), 4.58 (d, J=6.1 Hz, 1H), 4.28 (t, J=3.2 Hz, 1H), 3.98 (cd, J=10.3, 3.2 Hz, 2H), 3.50-3.04 (m, 4H), 2.55 (s, 1H), 1.08 (m, 1H), 0. 52 (m, 2H), 0.26 (m, 2H); HPLC: 8.52 min, 97.7%. LC-MS: m/z=572 (M+H), 528 (MCO ₂ H), m/z=224 (M-ribose fragment).

Example 93. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(isopropylamino)-9H-purin-9-yl)-3ethynyl-3,4- Dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that propan-2-amine was used instead of benzylamine in step 7.

1H NMR (400 MHz, CD3OD) δ 8.05 (s, 1H), 7.23-7.12 (m, 2H), 7.09-6.78 (m, 3H), 5.91 (d, J=6.2 Hz, 1H), 4.59 (d, J=6.2 Hz, 1H), 4.37 (m, 1H), 4.28 (m, 1H), 3.42 (d, J=14.8 Hz, 1H), 3.35-3.22 (m, 3H), 2.56 (s, 1H), 1.24 (d, J=6.5 Hz, 6H); HPLC: 8.39 min, 98.9%. LC-MS: m/z=560 (M+H), 516 (M-CO2H), m/z=212 (Mribose fragment).

- 98 046381

Example 94. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-((2-hydroxy-2-methylnropyl)amino)-9Hpurin-9-yl) -3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that 1-amino-2-methylpropan-2-ol was used instead of benzylamine in Step 7.

Ή-NMR (400 MHz, CD3OD) δ 8.16 (s, 1H) 7.24-7.22 (m, 2H) 7.03-7.01 (m, 3H) 5.97 (d, J= 7.4 Hz, 1H) 4.95 (d, J=7.4 Hz, 1H) 4.28 (t, J=3.2 Hz, 1H) 4.05-4.03 (m, 2H) 3 .72-3.69 (m, 2H) 3.48-3.31 (m, 2H) 2.97 (s, 1H) 1.24 (s, 6H). ESI-MS (m/z): [M] ^- calculated for C ₂₆ H ₂₈ ClN ₅ O ₉ 589.98; found 588.4.

Example 95. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(((S)-1-hydroxypropan-2-yl)amino)-9Hpurine- 9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that N-2-aminopropan-1-ol was used instead of benzylamine in step 7.

Ή-NMR (400 MHz, CD3OD) δ 8.18 (s, 1H) 7.24-7.22 (m, 2H) 7.03-7.01 (m, 3H) 5.97 (d, J= 7.4 Hz, 1H) 4.93 (d, J=7.4 Hz, 1H) 4.42-4.35 (m, 1H) 4.32-4.26 (m, 1H) 4.04 ( d, J=3.2 Hz, 2H) 3.62-3.60 (m, 2H) 3.48-3.32 (m, 2H) 2.96 (s, 1H) 1.29 (d, J =6.7 Hz, 3H). ESI-MS (m/z): ^-calculated for C _; ,H;..Cl\,O.. 575.96; found 574.2.

Example 96. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(diethylamino)-9H-purin-9-yl)-3ethynyl-3,4- Dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that diethylamine was used instead of benzylamine in step 7.

Ή-NMR (400 MHz, CD3OD) δ 8.15 (s, 1H) 7.23-7.20 (m, 2H) 7.00-6.98 (m, 3H) 5.98 (d, J= 7.3 Hz, 1H) 4.97 (d, J=7.3 Hz, 1H) 4.29 (m, 1H) 4.15-3.83 (m, 6H) 3.43-3.33 ( m, 2H) 2.96 (s, 1H) 1.24 (t, J=7.0 Hz, 6H). ESI-MS (m/z): [M] ^- calculated for C ₂₆ H ₂₈ ClN ₅ O ₉ 573.98; found 572.3.

Example 97. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-((2-hydroxyethyl)amino)-9H-purin-9yl)-3-ethynyl -3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that 2-aminoethan-1-ol was used instead of benzylamine in step 7.

Ή-NMR (400 MHz, CD3OD) δ 8.20 (s, 1H) 7.24-7.22 (m, 2H) 7.04-7.02 (m, 3H) 5.97 (d, J= 7.4 Hz, 1H) 4.91 (d, J=7.3 Hz, 1H) 4.29 (t, J=3.4 Hz, 1H)) 4.06-3.98 (m, 2H) 3.76-3.73 (m, 2H) 3.66-3.63 (m, 2H) 3.43-3.33 (m, 2H) 2.96 (s, 1H). ESI-MS (m/z): [M] ^- calculated for C ₂₄ H ₂₄ ClN ₅ O ₉ 561.93; found 560.2.

Example 98. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(((R)-1-hydroxypropan-2-yl)amino)-9Hpurin- 9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

- 99 046381

The title compound was prepared in a manner similar to that described in Example 59, except that in step 7, N)-2-aminopropan-1-ol was used instead of benzylamine.

Ή-NMR (400 MHz, CD3OD) δ 8.12 (s, 1H) 7.24-7.22 (m, 2H) 7.03-7.01 (m, 3H) 5.96 (d, J= 7.4 Hz, 1H) 4.95 (d, J=7.4 Hz, 1H) 4.42-4.32 (m, 1H) 4.28 (t, J=3.3 Hz, 1H) 4 .08-4.00 (m, 2H) 3.66-3.58 (m, 2H) 3.45-3.33 (m, 2H) 2.97 (s, 1H) 1.27 (d, J =6.7 Hz, 3H). ESI-MS (m/z): ^-calculated for C ₂₅ H ₂₆ ClN ₅ O ₉ , 575.96; found 574.2.

Example 99. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(cyclopropyl(2-hydroxyethyl)amino)9H-purin-9-yl)-3 -ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that 2-(cyclopropylamino)ethan-1-ol was used instead of benzylamine in step 7.

Ή-NMR (400 MHz, CD3OD) δ 8.23 (s, 1H) 7.24-7.22 (m, 2H) 7.04-7.01 (m, 3H) 6.02 (d, J= 7.4 Hz, 1H) 4.96 (d, J=7.3 Hz, 1H) 4.30-4.28 (m, 1H) 4.10-4.04 (m, 4H) 3.78 ( t, J=5.9 Hz, 2H) 3.44-3.32 (m, 2H) 3.23-3.19 (m, 1H) 2.96 (s, 1H) 1.01-0.97 (m, 2H) 0.78-0.74 (m, 2H).

Example 100. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-((R)-3-hydroxypyrrolidin-1-yl)-9Hpurin-9-yl )-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that (C)-pyrrolidin-3-ol was used instead of benzylamine in step 7.

¹ H NMR (400 MHz, CD3OD) δ 8.24 (s, 1H), 7.23 (d, J=7.2 Hz, 2H), 7.06-6.96 (m, 3H), 6, 01 (d, J=7.3 Hz, 1H), 5.01 (s, 1H), 4.56 (d, J=26.1 Hz, 1H), 4.33-4.31 (m, 1H ), 4.20 (d, J=12.5 Hz, 1H), 4.10-4.00 (m, 3H), 3.78 (m, 2H), 3.50-3.35 (m, 2H), 2.97 (s, 1H), 2.14-2.05 (m, 2H).

Example 101. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-((S)-3-(hydroxymethyl)πyrrolidin-1yl)-9H-purine- 9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that N-pyrrolidin-3-ylmethanol was used instead of benzylamine in step 7.

Ή NMR (400 MHz, DMSOHO δ 8.37 (s, 1H), 8.34 (s, 1H), 7.15 (m, 2H), 7.01 (m, 3H), 6.16 (br. s, 1H), 5.96 (br. s, 1H), 5.81 (d, J=7.5 Hz, 1H), 4.82 (d, J=7.4 Hz, 1H), 4, 18-4.08 (m, 1H), 3.94 (m, 2H), 3.71 (m, 2H), 3.50 (m, 2H), 3.20 (s, 4H), 3.13 (s, 1H), 2.33 (m, 1H), 2.04 (m, 1H), 1.98-1.89 (m, 1H), 1.77 (m, 1H), 1.65 ( m, 1H); HPLC: 7.22 min, 97.7%.

Example 102. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-((R)-3-(hydroxymethyl)pyrrolidin-1yl)-9H-purine- 9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that in step 7, N)-pyrrolidin-3-ylmethanol was used instead of benzylamine.

Ή NMR (400 MHz, DMSOO δ 8.37 (s, 1H), 8.34 (s, 1H), 7.21-7.11 (m, 2H), 7.01 (m, 3H), 6, 16 (br.s, 1H), 5.96 (br.s, 1H), 5.81 (d, J=7.5 Hz, 1H), 4.82 (d, J=7.5 Hz, 1H ), 4.44 (m, 1H), 4.25-4.07 (m, 3H), 3.95 (m,

- 100 046381

2H), 3.87-3.64 (m, 3H), 3.51 (m, 2H), 3.20 (s, 1H), 2.37-2.27 (m, 1H), 2.04 (m, 1H), 1.98-1.86 (m, 1H), 1.76 (m, 1H), 1.66 (m, 1H); HPLC: Rt=7.20 min, 97.0%.

Example 103. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-((R)-2-(hydroxymethyl)nirrolidin-1yl)-9H-purine- 9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

Н·,^ у= ^Сг \

NS-/ / V' %-. _t । k: sya 3

When-isp short circuit

The title compound was prepared in a manner similar to that described in Example 59, except that N-pyrrolidin-2-ylmethanol was used instead of benzylamine in Step 7.

^1H NMR (400 MHz, CD3OD) δ 8.21 (s, 1H), 7.28-7.16 (m, 2H), 7.08-6.87 (m, 3H), 5.99 (d , J=7.3 Hz, 1H), 4.97 (d, J=7.3 Hz, 1H), 4.41 (m, 1H), 4.28 (m, 1H), 4.12-3 .93 (m, 4H), 3.72 (m, 2H), 3.42 (d, J=15.0 Hz, 1H), 3.33 (d, J=15.0 Hz, 2H), 2 .96 (s, 1H), 2.05 (m, 4H); HPLC: 7.73 min, 98.2%; ESI-MS: m/z=254 (Mribose fragment).

Example 104. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-((S)-2-(hydroxymethyl)pyrrolidin-1ul)-9H-purine 9-ul)-3-ethynyl-3,4-dihydroxytetrahydrofvran-2-yl)methoxy)malonic acid

F- _h ·~ ^: -·ζΗ ^1. G .

I it: dn 1

The title compound was prepared in a manner similar to that described in Example 59, except that N-pyrrolidin-2-ylmethanol was used instead of benzylamine in Step 7.

1H NMR (400 MHz, CD3OD) δ 8.22 (s, 1H), 7.22 (m, 2H), 7.07-6.88 (m, 3H), 5.99 (d, J=7, 3 Hz, 1H), 4.98 (d, J=7.3 Hz, 1H), 4.44 (m, 1H), 4.29 (dd, J=4.0, 2.9 Hz, 1H) , 4.07 (m, 2H), 4.05 (cd, J=10.2, 3.5 Hz, 2H), 3.77 (dd, J=11.0, 4.2 Hz, 1H), 3.66 (dd, J=11.1, 6.5 Hz, 1H), 3.42 (d, J=15.1 Hz, 1H), 3.33 (d, J=15.1 Hz, 1H ), 2.95 (s, 1H), 2.07 (m, 4H); HPLC: 7.77 min, 99.3%; ESI-MS: m/z=642 (M+ACN).

Example 105. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(cyclopronul(methyl)amino)-9H-purun9-yl)-3-ethynyl- 3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that N-methylcyclopropanamine was used instead of benzylamine in step 7.

1H NMR (400 MHz, CD3OD) δ 8.36 (s, 1H), 7.26-7.12 (m, 2H), 7.02 (m, 3H), 6.03 (d, J=7, 3 Hz, 1H), 4.97 (d, J=7.3 Hz, 1H), 4.30 (dd, J=3.9, 2.8 Hz, 1H), 4.19-3.97 ( m, 2H), 3.57-3.29 (m, 5H), 3.21 (m, 1H), 2.96 (s, 1H), 1.05-0.85 (m, 2H), 0 .83-0.62 (m, 2H); HPLC: Rt=8.41 min, 98.6%; ESI-MS: m/z=224 (M-ribose fragment).

Example 106. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-((cyclopropylmethyl)amino)-9H-purin9-yl)-3-ethynyl-3 ,4-dihydroxytetrahydrofuran-2-yl)methoxy)-3-(methylamino)-3-oxopropanoic acid

benzene

2,6-dichloroadenine BSA, TMSOTf, MeSM

I

Example 106

Stage 1.

Stirred solution [(3aR,5R,6R,6aR)-6-ethyl-6-(methoxymethoxy)-2,2-dimethyl-tetragugro2H-furo[2,3-d][1,3]dioxol-5-yl] methanol (480 mg, 1.87 mmol) and methyl 2-diazo-2

- 101 046381 (methylcarbamoyl) acetate (440 mg, 2.8 mmol), prepared according to the literature: European Journal of Organic Chemistry, 2014 (24), 5302-5311), in anhydrous benzene (20 ml) under a nitrogen atmosphere was treated with tetraacetate rhodium (16 mg, 0.04 mmol) and heated to 60°C for 4 hours. The resulting mixture was cooled to room temperature and evaporated. The resulting oil was dissolved in dichloromethane, loaded onto a silica gel column, eluting with 10-100% ethyl acetate in hexane to give methyl 2{[(3aR,5R,6R,6aR)-6-ethynyl-6-(methoxymethoxy)-2,2 -dimethyl-tetrahydro-2H-furo[2,3-d][1,3]dioxol-5yl]methoxy}-2-(methylcarbamoyl)acetate (455 mg, 63% yield) as a diastereoisomeric pair.

Stage 2.

Solution of methyl 2-{[(3aR,5R,6R,6aR)-6-ethynyl-6-(methoxymethoxy)-2,2-dimethyl-tetrahydro-2Hfuro[2,3^][1,3]dioxol-5 -yl]methoxy}-2-(methylcarbamoyl)acetate (450 mg, 116 mmol) and benzyl bromide (0.97 ml, 8.13 mmol) in anhydrous DMF (4 ml) were treated with cesium carbonate (757 mg, 2.32 mmol ). The reaction mixture was stirred at room temperature overnight. The reaction was quenched with water (10 ml), diluted with diethyl ether (70 ml) and washed twice with saturated aqueous sodium chloride (50 ml). The organic layer was dried over NaSO4 and evaporated to give a yellow oil. The residue was purified by a silica gel column and eluted with 10-70% ethyl acetate in hexane to give a diastereomeric mixture of methyl 2-benzyl-2-(((3aR,5R,6R,6aR)-6-ethynyl-6-(methoxymethoxy)-2, 2dimethyltetrahydrofuro[2,3^][1,3]dioxol-5-yl)methoxy)-3-(methylamino)-3-oxopropanoate (500 mg, 90% yield) as a clear oil.

Stage 3.

Under a nitrogen atmosphere, an ice-cooled, stirred solution of methyl 2-benzyl-2(((3aR,5R,6R,6aR)-6-ethynyl-6-(methoxymethoxy)-2,2-dimethyltetrahydrofuro[2,3-d][1 ,3]dioxol-5-yl)methoxy)-3-(methylamino)-3-oxopropanoate (500 mg, 1.05 mmol) in acetic acid (4 ml) was treated with acetic anhydride (1 ml, 11.15 mmol) and concentrated sulfuric acid (0.02, 0.35 mmol). The reaction solution was slowly warmed to room temperature. After 4 hours, the reaction mixture was diluted with water and extracted with ethyl acetate (80 ml each). The organic solution was washed with saturated sodium bicarbonate (100 ml), dried over Na ₂ SO ₄ and evaporated. The residual oil was dissolved in dichloromethane and purified by flash chromatography to give (3R,4R,5R)-5-(((2benzyl-1-methoxy-3-(methylamino)-1,3-dioxopropan-2-yl)oxy )methyl)-4-ethynyltetrahydrofuran-2,3,4triyltriacetate in the form of a mixture of anomers/diastereomers.

Stage 4.

A suspension of 2-6-dichloroadenine (48 mg, 0.25 mmol) and N,O-bis-(trimethylsilyl)acetamide (0.08 ml, 0.32 mmol) in anhydrous acetonitrile (7 ml) was treated with a second solution of (3R, 4R,5R)-5-(((2-benzyl1-methoxy-3-(methylamino)1,3-dioxopropan-2-yl)oxy)methyl)-4-ethynyltetrahydrofuran-2,3,4-triyltriacetate (130 mg , 0.25 mmol) in anhydrous acetonitrile (10 ml), followed by dropwise addition of trimethylsilyl trifluoromethanesulfonate (0.06 ml, 0.33 mmol). After addition was complete, the reaction mixture was heated to 50°C for 18 hours, cooled to room temperature and quenched with saturated sodium bicarbonate solution (80 ml). After stirring for 5 minutes, the solution was extracted with ethyl acetate (3x80 ml), dried over Na ₂ SO ₄ and evaporated. The residue was dissolved in dichloromethane and purified by flash chromatography to give (2R,3R,4R,5R)-2-(((2-benzyl-1-methoxy-3(methylamino)-1,3-dioxopropan-2-yl )oxy)methyl)-5-(2,6-dichloro-9H-purin-9-yl)-3ethynyltetrahydrofuran-3,4-diyldiacetate (135 mg, 83% yield) as a foamy solid (diastereomeric pair).

Stage 5.

In a nitrogen atmosphere, a solution of (2R,3R,4R,5R)-2-(((2-benzyl-1-methoxy-3-(methylamino)-1,3dioxopropan-2-yl)oxy)methyl)-5-(2 ,6-dichloro-9H-purin-9-yl)-3-ethynyltetrahydrofuran-3,4-diyldiacetate (70 mg, 0.11 mmol) in dioxane (2 ml) was cooled to 0°C and treated with diisopropylethylamine (0.03 ml, 0.16 mmol) and cyclopropylamine (0.01 ml, 0.13 mmol) and warmed to room temperature with stirring overnight. The reaction mixture was diluted with ethyl acetate (80 ml), washed with water (50 ml) and saturated aqueous sodium chloride (50 ml). The organic layer was dried over Na2SO4, then filtered and evaporated. The residue was dissolved in dichloromethane and purified by flash column chromatography to give (2R,3R,4R,5R)-2-(((2-benzyl-1-methoxy-3-(methylamino)1,3-dioxopropane-2- yl)oxy)methyl)-5-(2-chloro-6-((cyclopropylmethyl)amino)-9H-purin-9-yl)-3ethynyltetrahydrofuran-3,4-diyldiacetate (70 mg, 95% yield) as almost white solid (diastereomeric pair).

Stage 6.

A solution of (2R,3R,4R,5R)-2-(((2-benzyl-1-methoxy-3-(methylamino)-1,3-dioxopropan-2-yl)oxy)methyl)-5-(2- chloro-6-((cyclopropylmethyl)amino)-9H-purin-9-yl)-3-ethynyltetrahydrofuran-3,4-diyldiacetate (70 mg, 0.10 mmol) in THF (1 ml) was treated with LiOH (24 mg, 1.02 mmol) in water (1 ml) and stirred at room temperature overnight. The pH of the reaction was adjusted to 4-5 using cold 2 N HCl. After precipitation, the suspension was stirred for another 10 minutes. The solid was collected, washed with cold water and dried to give 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6((cyclopropylmethyl)amino)-9H-purine-9- yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)-3

- 102 046381 (methylamino)-3-oxopropanoic acid (45 mg, 75% yield) as an almost white solid (diastereomeric pair).

¹ H NMR (400 MHz, CDCl3/CD3OD=5:1) δ 8.01 (s, 0.45H), 7.48 (s, 0.55H), 7.22-7.03 (m, 5H) , 5.86 (dd, J=4.7, 3.0 Hz, 1H), 4.43 (dd, J=8.5, 4.6 Hz, 1H), 4.31-4.06 (m , 2H), 3.84-3.72 (m, 1H), 3.45-3.21 (m, 4H), 2.62 (d, J=40.9 Hz, 1H), 2.46 ( d, J=8.1 Hz, 3H), 1.05 (dk, J=8.0, 3.7 Hz, 1H), 0.51 (ddd, J=8.1, 4.0, 1, 6 Hz, 2H), 0.310.18 (m, 2H). ESI-MS (m/z): [M] ⁺ calculated for C27H ₂₉ ClN ₆ O ₇ , 585.18; found 585.9.

Example 107. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(6-(benzylamino)-2-chloro-9H-purine-9-ul)-3ethynyl-3,4- dihydroxytetrahydrofuran-2-yl)methoxy)-3-(methylamino)-3-oxopropanoic acid

The title compound was prepared in a manner similar to that described in Example 106, except that benzylamine was used instead of cyclopropylamine in step 5.

¹ H NMR (400 MHz, CDCl ₃ /CD ₃ OD=5:1) δ 8.03 (s, 0.46H), 7.52 (d, J=2.7 Hz, 0.54H), 7. 42-7.04 (m, 10H), 5.89 (t, J=4.3 Hz, 1H), 4.73 (m, 2H), 4.44 (dd, J=6.0, 4, 5 Hz, 1H), 4.33-4.15 (m, 2H), 3.79 (dd, J=10.6, 4.5 Hz, 1H), 3.45-3.27 (m, 2H ), 2.62 (d, J=36.6 Hz, 1H), 2.48 (d, J=13.2 Hz, 3H). ESI-MS (m/z): [M] ⁺ calculated for CzoH29SM07, 621.18; found 621.4.

Example 108. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-((2-chlorobenzyl)amino)-9H-purine-9-ul)3 - ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)-3-ethoxy-3-oxopropanoic acid

The title compound was prepared in a manner similar to that described in Example 59, except that only 5 equivalents of lithium hydroxide were used in step 8.

^1H NMR (400 MHz, CD3OD) δ 8.16 (s, 1H), 7.42 (m, 2H), 7.26 (m, 2H), 7.21 (m, 2H), 6.99 ( m, 3H), 5.97 (d, J=7.6 Hz, 1H), 4.96 (d, J=7.6 Hz, 1H), 4.82 (m, 2H), 4.28 ( m, 1H), 4.03 (m, 2H), 3.75 (m, 2H), 3.38 (m, 2H), 3.22 (m, 2H), 2.97 (s, 1H) 1 .29 (t, J=7.6 Hz, 3H). HPLC: 9.43 min, 96.2%. LC-MS: m/z=642, 597 (M-CO ₂ h); m/z=292 (M-ribose fragment).

Example 109. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-((pyridin-4-ylmethyl)amino)-9Hpurin-9-yl)-3 -ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)-3-ethoxy-3-oxopropanoic acid

The title compound was prepared in a manner similar to that described in Example 59, except that only 5 equivalents of lithium hydroxide were used in step 8. The compound was isolated as a ~2:1 mixture of diastereomers.

^1H NMR (400 MHz, DMSO-d6) main isomer: δ 8.73 (d, J=5.9 Hz, 2H), 8.15 (s, 0.61H), 7.98 (d, J= 5.9 Hz, 2H), 7.23 (m, 2H), 7.06 (m, 3H), 6.00 (m, 1H), 5.00 (m, 2H), 4.10 (m, 4H), 3.39 (m, 2H), 3.12 (s, 0.80H), 1.17 (m, 3H). ¹ H NMR (DMSO-d ₆ ) minor isomer: δ 8.73 (d, J=5.9 Hz, 2H), 8.31 (s, 0.32H), 7.98 (d, J=5, 9 Hz, 2H), 7.23 (m, 2H), 7.06 (m, 3H), 6.00 (m, 1H), 5.00 (m, 2H), 4.10 (m, 4H) , 3.39 (m, 2H), 3.01 (s, 0.41H), 1.17 (m, 3H). HPLC: 6.25 min, 94.8%. LC-MS: m/z=638 (M+), 594 (M-CO2H), m/z=259 (M-ribose fragment).

- 103 046381

Example 110. Synthesis of 2-(((2R,3S,4R,5R)-5-(2-chloro-6-(isopropylamino)-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2- yl)methoxy)-2-((5-methylisoxazol-3-yl)methyl)malonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that 3-(bromomethyl)-5-methylisoxazole was used instead of benzyl bromide in Step 4 and propan-2-amine was used instead of benzylamine in Step 7.

¹ H-NMR (400 MHz, CD3OD) δ 8.46 (s, 1H) 5.99 (d, J=7.6 Hz, 1H) 5.95 (s, 1H) 5.07 (d, J= 7.6 Hz, 1H) 4.41-4.38 (m, 1H) 4.31-4.29 (m, 1H) 4.07-4.05 (m, 1H) 3.99-3.88 (m, 1H) 3.50-3.38 (m, 2H) 2.95 (s, 1H) 2.05 (s, 3H) 1.27 (d, J=6.5 Hz, 6H). ESI-MS (m/z): [M] ⁺ calculated for C ₂₃ H ₂₅ ClN ₆ O ₉ , 564.93; found 566.1.

Example 111. Synthesis of 2-(((2R,3S,4R,5R)-5-(2-chloro-6-((4-fluorobenzyl)amino)-9H-purin-9-yl)-3ethynyl-3,4 -dihydroxytetrahydrofuran-2-yl)methoxy)-2-((5-methylisoxazol-3-yl)methyl)malonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that 3-(bromomethyl)-5-methylisoxazole was used instead of benzyl bromide in Step 4 and (4-fluorophenyl)methanamine was used instead of benzylamine in Step 7.

¹ H-NMR (400 MHz, CD3OD) δ 8.62-8.58 (br. s, 1H) 7.41-7.38 (m, 2H) 7.07-7.01 (m, 2H) 6 .01 (d, J=7.4 Hz, 1H) 5.96 (s, 1H) 5.09 (d, J=7.3 Hz, 1H) 4.82-4.71 (m, 2H) 4 .31-4.29 (m, 1H) 4.06 (dd, J=10.0, 3.8 Hz, 1H) 3.97 (dd, J=10.1, 3.1 Hz, 1H) 3 .44-3.32 (m, 2H) 3.18 (s, 3H) 2.96 (s, 1H).

Example 112. Synthesis of 2-(benzoY]thiazol-2-ylmethyl)-2-(((2R^,4R,5R)-5-(2-chloro-6-((4fluorobenzyl)amino)-9H-purine-9 -yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

The title compound was prepared in a manner similar to that described in Example 59, except that 2-(bromomethyl)benzothiazole was used instead of benzyl bromide in Step 4 and (4fluorophenyl)methanamine was used instead of benzylamine in Step 7.

¹ H-NMR (400 MHz, CD3OD) δ 8.57-8.54 (br.s, 1H) 7.74-7.70 (m, 2H) 7.45-7.41 (m, 2H) 7 .23-7.20 (m, 1H) 7.12-7.16 (m, 1H) 7.09-7.04 (m, 2H) 5.94 (d, J=7.3 Hz, 1H) 5.15 (d, >7.1Hz, 1H) 4.77-4.72 (m, 2H) 4.354.32 (m, 1H) 4.20 (dd, J=10.1, 2.7 Hz, 1H) 4.11 (dd, J=10.1, 3.1 Hz, 1H) 3.99-3.86 (m, 2H) 2.77 (s, 1H).

Example 113. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl) methoxy)-3-oxo-3-(pyrrolidin-1-yl)propanoic acid

The title compound was prepared analogously to Example 59, except that ethyl 2-diazo-3-oxo-3-(pyrrolidin-1-yl)propanoate was used instead of 1-ethyl-3-(prop-1-ene-1- il)-2

- 104 046381 diazomalonate in step 3, and ammonia was used instead of benzylamine in step 7. The product was isolated as a 55/45 mixture of diastereomers.

Main diastereomer Ή-NMR (400 MHz, CD ₃ OD) d 8.80 (s, 1H) 5.99 (d, J=7.4 MHz, 1H) 5.05 (d, J=7.4 MHz, 1H) 4.88 (s, 1H) 4.28-4.26 (m, 1H) 4.13 (dd, J=10.7, 2.5 Hz, 1H) 4.00 (dd, J=10 .7, 3.9 Hz, 1H) 3.663.39 (m, 4H) 3.17 (s, 1H) 1.89-1.79 (m, 4H). Minor diastereomer Ή-NMR (400 MHz, CD ₃ OD) δ 8.89 (s, 1H) 6.03 (d, J=7.4 Hz, 1H) 5.05 (d, J=7.4 Hz, 1H) 4.86 (s, 1H) 4.28-4.26 (m, 1H) 3.90-3.86 (m, 2H) 3.66-3.39 (m, 4H) 3.16 ( s, 1H) 1.89-1.79 (m, 4H). ESI-MS (m/z): [M]- calculated for Ci ₉ H2iClN ₆ O ₇ , 480.86; found 479.1.

Example 114. Synthesis of 2-(((2R,3S,4R,5R)-5-(2-chloro-6-(cyclopentylamino)-9H-purin-9-yl)-3-ethynyl3,4-dihydroxytetrahydrofuran-2- yl)methoxy)-3-oxo-3-(pyrrolidin-1-yl)propanoic acid

Pruggr 114

The title compound was prepared analogously to Example 59, except that ethyl 2-diazo-3-oxo-3-(pyrrolidin-1-yl)propanoate was used instead of 1-ethyl-3-(prop-1-ene-1- yl)-2diazomalonate in step 3, and cyclopentanamine was used instead of benzylamine in step 7. The product was isolated as a 55/45 mixture of diastereomers.

Main diastereomer Ή NMR (400 MHz, CD3OD) d 8.72 (s, 1H) 5.96 (d, J=7.4 Hz, 1H) 5.04 (d, J=7.4 Hz, 1H) 4 .87 (s, 1H) 4.52-4.48 (m, 1H) 4.28-4.25 (m, 1H) 4.14 (d, J=10.5 Hz, 1H) 4.01 ( dd, J=10.6, 5.2 Hz, 1H) 3.60-3.36 (m, 4H) 3.17 (s, 1H) 2.10-2.04 (m, 2H) 1.84 -1.77 (m, 6H) 1.69-1.58 (m, 4H) Minor diastereomer Ή NMR (400 MHz, CD3OD) δ 8.64 (s, 1H) 6.00 (d, J=7, 4 Hz, 1H) 5.01 (d, J=7.4 Hz, 1H) 4.85 (s, 1H) 4.52-4.48 (m, 1H) 4.28-4.25 (m, 1H) 3.92-3.86 (m, 2H) 3.60-3.36 (m, 4H) 3.16 (s, 1H) 2.10-2.04 (m, 2H) 1.841.77 ( m, 6H) 1.69-158 (m, 4H). ESI-MS (m/z): [m]- calculated for C ₂₄ H ₂₉ ClN ₆ O ₇ 548.98; found 547.3.

Example 115. Synthesis of 3-amino-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(cyclopentylamino)-9H-purin-9-yl)-3ethynyl-3,4- dihydroxytetrahydrofuran-2-yl)methoxy)-3-oxopropanoic acid

Example 115

Stage 1.

A suspension of 2,6-dichloroadenine (2.91 g, 15.4 mmol, 1.01 eq.) and N,O-6uc(trimethylsilyl)acetamide (4.87 ml, 19.6 mmol, 1.0) was taken under a nitrogen atmosphere. 29 equiv.) in anhydrous acetonitrile (90 ml). Then a solution of (2R,3R,4R,5R)-2,4-bis-(acetyloxy)-5-{[(tert-butyldiphenylsilyl)oxy]methyl}-4ethynyloxolan-3-yl acetate (8.2 g, 15. 22 mmol) in anhydrous acetonitrile (10 mL) followed by dropwise addition of trimethylsilyl trifluoromethanesulfonate (3.67 mL, 20.3 mmol, 1.33 eq.). The reaction mixture was heated to 50°C for 18 h, then cooled to room temperature. (The reaction starts out pale yellow and turns clear amber after 4 hours.) Saturated aqueous sodium bicarbonate (10 ml) was added and the mixture was stirred for ten minutes. The resulting mixture was extracted with ethyl acetate (3x100 ml) and the combined organic layer was dried (Na ₂ SO ₄ ), filtered and evaporated. The residue was dissolved in dichloromethane/ethyl acetate (~3 ml, 1:1), loaded onto a silica gel column (~300 cm ³ ) and eluted with 0-30% ethyl acetate in hexane to give (2R,3R,4R,5R)-2- (((tert-butyldiphenylsilyl)oxy)methyl)-5-(2,6-dichloro-9H-purin-9-yl)-3-ethynyltetrahydrofuran-3,4-diyldiacetate (8.2 g, 81%) as white solid.

Stage 2.

Solution of (2R,3R,4R,5R)-2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-(2,6-dichloro-9H-purin-9-yl)-3ethynyltetrahydrofuran-3,4-diyldiacetate (1.6 g, 2.4 mmol) in anhydrous THF (25 ml) was cooled to 0°C and treated with acetic acid (0.192 ml, 3.36 mmol, 1.4 eq.) 1 N solution of tetrabutylammonium fluoride in THF ( 3.36 ml, 3.36 mmol, 1.4 eq.). After addition was complete, the reaction mixture was warmed to room temperature and stirred for 3 hours. The reaction mixture was evaporated, purified by flash column chromatography (0 to 50% ethyl acetate in hexanes to give (2R,3R,4R,5R)-5- (2,6-dichloro-9H-purin-9-yl)-3-ethynyl-2-(hydroxymethyl)tetrahydrofuran-3,4diyldiacetate (0.88 g, 86%) as a white foam.

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Stage 3.

In a nitrogen atmosphere, a solution of ((2R,3R,4R,5R)-5-(2,6-dichloro-9H-purin-9-yl)-3-ethynyl-2(hydroxymethyl)tetrahydrofuran-3,4-diyldiacetate (172 mg, 0.40 mmol) and ethyl 3-(aminooxy)-2-diazo3-oxopropanoate (160 mg, 1.02 mmol, 2.5 eq.) in anhydrous toluene (3 ml) were treated with rhodium(II) acetate dimer (5 mg, 0.011 mmol, 2.8 mol%) and heated to 80°C for 5 hours. The reaction mixture was evaporated and purified by flash column chromatography (25-75% ethyl acetate in dichloromethane) to give (2R, 3R,4R,5R)-2-(((1-amino-3-ethoxy-1,3-dioxopropan-2-yl)oxy)methyl)-5-(2,6-dichloro-9Hpurin-9-yl) -3-ethynyltetrahydrofuran-3,4-diyldiacetate (101 mg, 45% in a mixture of diastereomers ~ 85:15) as colorless glass.

Stage 4.

In a nitrogen atmosphere, a solution of (2R,3R,4R,5R)-2-(((1-amino-3-ethoxy-1,3-dioxopropan-2yl)oxy)methyl)-5-(2,6-dichloro-9H -purin-9-yl)-3-ethynyltetrahydrofuran-3,4-diyldiacetate (157 mg, 0.28 mmol) in dioxane (2.5 ml) was treated with diisopropylethylamine (0.100 ml, 0.61 mmol, 2.2 eq. ) and cyclopentylamine (0.065 ml, 0.066 mmol, 2.33 eq.). After stirring at room temperature for 18 hours, the mixture was diluted with ethyl acetate (20 ml), washed with water (10 ml), dried (Na ₂ SO ₄ ), filtered and evaporated to give crude (2R,3R,4R,5R)-2- (((1-amino-3-ethoxy-1,3dioxopropan-2-yl)oxy)methyl)-5-(2-chloro-6-(cyclopentylamino)-9H-purin-9-yl)-3ethynyltetrahydrofuran-3, 4-diyldiacetate.

Stage 5.

The resulting residue from the previous step was stirred in ammonium hydroxide and ethanol (1:1/v:v, 10 ml) at room temperature overnight. The mixture was evaporated, dissolved in THF (2.5 ml) and treated with lithium hydroxide (23 mg, 0.96 mmol, 3.4 eq.) dissolved in water (2.5 ml). The mixture was stirred at room temperature, gently heated from time to time for ~3 h, then neutralized with 1 N HCl to pH ~ 6 and evaporated in vacuum. The crude product was dissolved in water and purified using

Reverse phase HPLC and freeze-dried to give the title compound (52 mg, 37% in 3 steps) as a bulk white solid.

1H NMR (400 MHz, D ₂ O) of the main isomer: δ 8.30 (s, 1H), 5.81 (m, 1H), 4.88 (d, J=6.6 Hz, 1H), 4, 80 (d, J=6.8 Hz, 1H), 4.24 (m, 2H), 4.20 (br. s, 1H), 3.83 (m, 2H), 3.01 (s, 1H ), 1.85 (m, 2H), 1.49 (m, 6H). 1H NMR (400 MHz D2O) minor isomer: δ 8.39 (s, 1H), 5.81 (m, 1H), 4.88 (d, J=6.6 Hz, 1H), 4.80 (d , J=6.8 Hz, 1H), 4.24 (m, 2H), 4.20 (br. s, 1H), 3.83 (m, 2H), 2.99 (s, 1H), 1 .85 (m, 2H), 1.49 (m, 6H). HPLC: Rt=7.08 min, 93.0%. ESI-MS for C ₂₀ H ₂₃ ClN ₆ O ₇ calculated 494.13, found 493.2 (M-); ESI-MS for C ₁₀ H _n ClN ₅ calculated 236.07, found 236.0 (M-ribose fragment).

Example 116. Synthesis of -2-(((2R,3S,4R,5R)-5-(2-chloro-6-(cyclopentylamino)-9H-purin-9-yl)-3-ethynyl3,4-dihydroxytetrahydrofuran-2 -yl)methoxy)malonamide

Iriver 11δ

The title compound was obtained as the second product from Step 5 of the synthesis of Example 115. The compound was isolated as a bulk white solid (8.5 mg, 6%).

1H NMR (400 MHz, DMSO-06): 8.57 (s, 0.5H), 8.31 (s, 1H), 7.73 (s, 1H), 7.38 (m, 1.5H) , 7.21 (m, 1.5H), 7.05 (m, 0.5H), 6.87 (m, 0.5H), 6.20 (m, 1H), 6.02 (d, J =7.0 Hz, 1H), 5.79 (d, J=7.8 Hz, 1H), 5.02 (s, 1H), 4.73 (s, 1H), 4.39 (m, 1H ), 4.18 (m, 1H), 3.79 (m, 1H), 3.68 (s, 1H), 1.85 (m, 2H), 1.49 (m, 6H). HPLC: Rt=6.64 min, 97.1%. ESI-MS for C ₂₀ H ₂₄ ClN ₇ O ₆ calculated 493.15, found 492.3 (M-); ESI-MS for C10H11QN5 calculated 236.1, found 236.1 (M-ribose fragment).

Example 117. Synthesis of 2-(((2R,3S,4R,5R)-5-(2-chloro-6-(methylamino)-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2- yl)methoxy)malonic acid

. G

I g ·. -I

ΠΐΕ.Κ I

The title compound was prepared in a manner similar to that described in Example 59, except that step 7 was omitted and step 4 was omitted.

1H NMR (400 MHz, CD3OD) δ 8.88 (s, 1H), 6.05 (d, J=7.6 Hz, 1H), 5.03 (d, J=7.6 Hz, 1H), 4.62 (s, 1H), 4.26 (m, 1H), 4.04 (m, 1H), 3.90 (m, 1H), 3.11 (s, 1H), 3.07 (s , 3H). HPLC: Rt=5.92 min, 97.9%. ESI-MS for

- 106 046381

C ₁₆ H ₁₆ ClN ₅ O ₈ calculated 441.07, found 442.5 (M+); ESI-MS for C ₆ H ₅ ClN ₅ calculated 182.02, found 184.2 (M-ribose fragment).

Example 118. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-(benzylamino)-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2- yl)methoxy)malonic acid.

The title compound was prepared in a manner similar to that described in Example 59, except that step 4 was omitted.

^1H NMR (400 MHz, CD3OD) δ 8.87 (s, 1H), 7.39 (m, 2H), 7.33 (m, 2H), 7.26 (m, 1H), 6.06 ( d, J=7.6 Hz, 1H), 5.02 (d, J=7.6 Hz, 1H), 4.76 (m, 2H), 4.62 (s, 1H), 4.26 ( s, 1H), 4.03 (m, 1H), 3.90 (m, 1H), 3.10 (s, 1H). HPLC: Rt=7.83 min, 98.2%. ESI-MS for C ₂₂ H ₂₀ ClN ₅ O ₈ calculated 517.10, found 516.7 (M-); ESI-MS for C ₁₂ H ₉ ClN ₅ calculated 258.05, found 258 (M-ribose fragment).

Example 119. Synthesis of 2-(((2R,3S,4R,5R)-5-(2-chloro-6-morpholino-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl) methoxy)-2-(phenylsulfonyl)acetic acid

The title compound was prepared analogously to Example 59, except that ethyl 2-diazo-2-(phenylsulfonyl)acetate was used instead of 1-ethyl 3-(prop-1-en-1-yl)-2-diazomalonate in step 3. Morpholine was used instead of benzylamine in step 7, and step 4 was omitted.

^1H NMR (400 MHz, DMSO-d6) main isomer: δ 8.40 (s, 1H), 7.83 (m, 2H), 7.66 (m, 2H), 7.53 (m, 1H) , 5.83 (m, 1H), 5.50 (d, J=7.8 Hz, 1H), 4.80 (d, J=7.8 Hz, 1H), 4.16 (m, 4H) , 3.98 (m, 1H), 3.76 (m, 4H), 3.63 (s, 1H). ^1H NMR (400 MHz, DMSO-d6) minor isomer: δ 8.41 (s, 1H), 7.83 (m, 2H), 7.66 (m, 2H), 7.53 (m, 1H) , 5.83 (m, 1H), 5.50 (d, J=7.8 Hz, 1H), 4.66 (d, J=7.8 Hz, 1H), 4.16 (m, 4H) , 3.98 (m, 1H), 3.76 (m, 4H), 3.61 (s, 1H). HPLC: Rt=7.83 (minor), 8.18 min (major), 99.6% (40:60). ESI-MS for C ₂₆ H ₂₆ ClN ₅ O ₉ calculated. 587.14, 588 found (M+); ESI-MS for C ₂₄ H ₂₄ ClN ₅ O ₉ S calculated. 593.10, found 592 (M-); ESI-MS for C ₂₃ H ₂₃ ClN ₅ O ₇ S calculated. 548.10, found 548 (M-CO2H); ESI-MS for C ₉ H ₉ ClN ₅ O calculated. 238.05, 240 found (M-ribose fragment).

Example 120. Synthesis of 2-(((2R,3S,4R,5R)-5-(2-chloro-6-(cyclopentylamino)-9H-purin-9-yl)-3-ethynyl3,4-dihydroxytetrahydrofuran-2- yl)methoxy)-2-(methylsulfonyl)acetic acid

The title compound was prepared analogously to Example 59, except that ethyl 2-diazo-2-(methylsulfonyl)acetate was used instead of 1-ethyl 3-(prop-1-en-1-yl)-2-diazomalonate in step 3. Cyclopentanamine was used instead of benzylamine in step 7, and step 4 was omitted.

^1H NMR (400 MHz, DMSO-d6) main isomer: δ 8.47 (s, 1H), 5.85 (m, 1H), 5.36 (m, 1H), 5.01 (m, 1H) , 4.86 (d, J=8.0 Hz, 1H), 4.42 (m, 2H), 4.33 (m, 1H), 4.25 (m, 2H), 4.14 (m, 1H), 4.00 (m, 1H), 3.37 (s, 1H), 3.05 (s, 3H), 1.97 (m, 2H), 1.72 (m, 2H), 1. 57 (m, 4H). ^1H NMR (400 MHz, DMSO-d6) minor isomer: δ 8.39 (s, 1H), 5.83 (m, 1H), 5.36 (m, 1H), 4.68 (d, J= 8.0 Hz, 1H), 4.42 (m, 2H), 4.33 (m, 1H), 4.25 (m, 2H), 4.14 (m, 1H), 4.00 (m, 1H), 3.37 (s, 1H), 3.05 (s, 3H), 1.97 (m, 2H), 1.72 (m, 2H), 1.57 (m, 4H). HPLC: Rt=8.02 min, 98.4%. ESI-MS for C ₂₀ H ₂₄ ClN ₅ O ₈ S calculated 529.10, found 530 (M+); ESI-MS for C ₁₀ H ₁₁ ClN ₅ calculated 236.07, found 238 (M-ribose fragment).

- 107 046381

Examples 121 and 122. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-carbamoyl-9H-purin-9-yl)-3-ethynyl3,4-dihydroxytetrahydrofuran-2- yl)methoxy)-2-benzyl-3-ethoxy-3-oxopropanoic acid and 2(((2R,3S,4R,5R)-5-(6-amino-2-carbamoyl-9H-purin-9-yl) -3-ethynyl-3,4-dihydroxytetrahydrofuran-2yl)methoxy)-2-benzylmalonic acid

aq. LiOH, THF

Stage 1.

To a solution of diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-(N,N'-bis-(tertbutoxycarbonyl)amino)-2-chloro 1 ,4-diazabicyclo[2.2.2]octane (128 μl, 1.17 mmol, 1 eq.) and NaCN (114.20 mg, 2.33 mmol, 2 eq.). The solution was stirred at 60°C for 3 hours, then diluted with water (15 ml) and extracted using ethyl acetate (3x15 ml). The combined organic layer was washed with water (50 ml), brine (50 ml), dried using Na ₂ SO ₄ , filtered and evaporated. The crude residue was purified by combi-flash chromatography (silica gel, 30-70% EtOAc in petroleum ether) to give diethyl-2-benzyl-2(((2R,3R,4R,5R)-3,4-diacetoxy-5-( 6-N,N'-(bis-(tert-butoxycarbonyl)amino)-2-cyano-9H-purin-9-yl)3-methyltetrahydrofuran-2-yl)methoxy)malonate (312 mg, 40% yield) in in the form of yellow gum.

Stage 2.

To a solution of diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-(N,N'-bis-(tert-butoxycarbonyl)amino)-2 -cyano-9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (50 mg, 75.23 µmol, 1 eq.) in DCM (1.7 ml) was added TFA (0.3 ml) at 0°C. The solution was stirred at 20°C for 1 hour before treatment with saturated aq. NaHCO ₃ to adjust pH to 9. The mixture was extracted using ethyl acetate (3x3 ml). The combined organic layer was evaporated to give crude diethyl 2-(((2R,3S,4R,5R)-5-(6-amino-2-cyano-9H-purin-9-yl)-3-ethynyl-3,4 -dihydroxytetrahydrofuran-2-yl)methoxy)-2-benzylmalonate (45 mg) as yellow gum.

Stage 3.

To a solution of crude diethyl 2-(((2R,3S,4R,5R)-5-(6-amino-2-cyano-9H-purin-9-yl)-3-ethynyl3,4-dihydroxytetrahydrofuran-2-yl )methoxy)-2-benzylmalonate (65 mg, 115.14 µmol, 1 eq.) in MeCN (2 ml), add 3,4,6,7,8,9-hexahydro-2H-pyrimido[1,2- α]-pyrimidine (TBD) (1 M aq, 461 µl, 4 eq). The reaction mixture was stirred at 20°C for 18 h before evaporation. The crude residue was purified by preparative HPLC and the fractions were lyophilized to give 2(((2R,3S,4R,5R)-5-(6-amino-2-carbamoyl-9H-purin-9-yl)-3-ethynyl -3,4-dihydroxytetrahydrofuran-2yl)methoxy)-2-benzyl-3-ethoxy-3-oxopropanoic acid (Example 121) (4.1 mg, 5% yield) as a white solid and 2-(((2R ,3S,4R,5R)-5-(6-amino-2-carbamoyl-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl)methoxy)-2-benzylmalonic acid (Example 122 ) (2.2 mg, 3% yield) as a white solid.

Example 121 ¹ H NMR (400 MHz, CD3OD) δ ppm. 8.19~8.39 (m, 1H) 7.26 (br. d, J=5.88 Hz, 2H) 7.037.12 (m, 3H) 6.20 (dd, J=9.69, 7 ,19 Hz, 1H) 4.91-4.97 (m, 1H) 4.29 (br. s, 1H) 3.98-4.24 (m, 4H) 3.41-3.53 (m, 1H) 3.31-3.40 (m, 1H) 2.97-3.08 (m, 1H) 1.18 (k, J=7.25 Hz, 3H); LC/MS [M+H] = 555.1.

Example 122 1H NMR (400 MHz, CD3OD) δ ppm. 8.504 (s, 1H) 7.16-7.28 (m, 2H) 7.07 (br. s, 3H) 6.20 (d, J=5.88 Hz, 1H) 4.92-4.99 (m, 1H) 4.37 (br.s, 1H) 3.97 (br.d, J=3.63 Hz, 2H) 3.32-3.48 (m, 2H) 3.01 (s, 1H); LC/MS [M+H] = 527.0.

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Example 123. Synthesis of (1-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl )methoxy)-2-methoxy-2-oxoethyl)phosphonic acid

Stage 1.

A mixture of (2R,3R,4R,5R)-5-(6-(N,N'-bis-(tert-butoxycarbonyl)amino)-2-chloro-9H-purin-9-yl)-3ethynyl-2-( Hydroxymethyl)tetrahydrofuran-3,4-diyldiacetate (565 mg, 0.926 mmol, 1.0 eq.) and methyl 2diazo-2-(dimethoxyphosphoryl) acetate (247 mg, 1.20 mmol, 1.3 eq.) were azeotroped twice distillation with toluene, and the resulting oil was redissolved in toluene (5.7 ml). The reaction solution was stirred at ambient temperature under an argon atmosphere and equipped with a jacketed reflux condenser. Rhodium(II) acetate (0.185 mmol, 82 mg, 0.2 eq) was added and the reaction mixture was heated at 75°C for 9 h, then cooled to room temperature. The reaction mixture was evaporated and the resulting oil was purified by silica gel flash column chromatography to give (2R,3R,4R,5R)-5-(6-(N,N'-bis-(tert-butoxycarbonyl)acetamido)-2 -chloro-9Hpurin-9-yl)-2-((1-(dimethoxyphosphoryl)-2-methoxy-2-oxoethoxy)methyl)-3-ethynyltetrahydrofuran-3,4diyldiacetate.

Stages 2, 3.

Deprotection of the product in the previous step was performed according to the procedure described for step 4 in example 121. Aq. NaOH solution was used instead of KOEt, which leads to the formation of carboxylic acid. The title compound was isolated as a white solid by preparative reverse phase HPLC.

1H NMR (CD ₃ OD, 300 MHz) δ 8.08 (s, 1H), 6.06 (br. s, 1H), 5.06-5.08 (d, J=5 Hz, 1H), 4 .28 (s, 1H), 3.90-4.10 (m, 2H), 3.79 (s, 3H), 3.98 (br. s, 2H), 3.13 (s, 1H); LC/MS [M+H] = 478.2.

Example 124. Synthesis of (1-(((2R,3S,4R,5R)-5-(2-chloro-6-morpholino-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl )methoxy)-2-ethoxy-2-oxoethyl)phosphonic acid

(1-(((2R,3S,4R,5R)-5-(2-chloro-6-morpholino-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy )-2-ethoxy-2-oxoethyl)phosphonic acid was prepared in a manner similar to that described in example 59, except that in step 3, ethyl 2-diazo-2-(diethoxyphosphoryl) acetate was used instead of 2-diazomalonate, and in step 7, instead of benzylamine Morpholine was used, and stage 4 was excluded. The compound was isolated as a 1:1 mixture of diastereomers

1H NMR (400 MHz, DMSO-d6) main isomer: δ 8.59 (s, 1H), 5.90 (m, 1H) 5.00 (d, J=7.0 Hz, 1H), 4.25 (m, 2H), 4.05 (m, 4H), 3.99 (m, 1H), 3.83 (m, 1H), 3.75 (m, 4H), 3.01 (s, 1H) 1.05 (t, J=7.1 Hz, 3H). 1H NMR (400 MHz, DMSO-d6) minor isomer: δ 8.48 (s, 1H), 5.88 (m, 1H), 4.94 (d, J=6.9 Hz, 1H), 4, 25 (m, 2H), 4.05 (m, 4H), 3.99 (m, 1H), 3.83 (m, 1H), 3.75 (m, 4H), 3.01 (s, 1H ), 0.99 (t, J=7.1 Hz, 3H). HPLC: minor isomer=6.63 min; main isomer=6.65 min, 98.6%. LC-MS: m/z=562 (M+); m/z=240 (M-ribose fragment).

- 109 046381

Example 125. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-3-ethynyl-3,4-dihydroxy-5-(5-(trifluoromethyl)-3Himidazo[4,5-b]pyridine -3-yl)tetrahydrofuran-2-yl)methoxy)malonic acid

Example 112

Proceeding as described in Example 15 above, but replacing 6-amino-2-chloroudenine with 5-(trifluoromethyl)3H-imidazo[4,5-b]pyridine, afforded the title compound as a white solid.

¹ H NMR (CD3OD, 300 MHz) δ 8.82 (br.s, 1H), 8.23 (d, J=8 Hz, 1H), 7.78 (d, J=8 Hz, 1H), 7 .29-7.26 (m, 2H), 7.03-7.00 (m, 3H), 6.32 (d, J=7 Hz, 1H), 5.10 (d, J=7 Hz, 1H), 4.37-4.36 (m, 1H), 4.08 (d, J=3 Hz, 2H), 3.42 (dd, J=15, 28 Hz, 2H), 2.98 ( s, 1H); LC/MS [M+H] = 535.2.

Example 126. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl) methoxy)-2-(thiazol-4-ylmethyl)malonic acid

Example 126

Proceeding as described in Example 15 above, but replacing the alylbromide with 4-(chloromethyl)thiazole, afforded the title compound as a white solid.

^1H NMR (CD3OD, 300 MHz) δ 8.76 (br.s, 1H), 8.57 (br.s, 1H), 7.36 (br.s, 1H), 6.00 (d, J =7 Hz, 1H), 5.00-4.95 (m, 1H), 4.35 (br. s, 1H), 4.08-4.04 (m, 2H), 3.66-3, 64 (m, 2H), 2.98 (s, 1H); LC/MS [M+H] = 524.9.

Example 127. Synthesis of 2-(((2R,3S,4R,5R)-5-(2-acetyl-6-amino-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl) methoxy)-2-benzylmalonic acid

Example 127

Stage 1.

To a suspension of 6-amino-2-chloroadenine (3.0 g, 17.69 mmol, 1 eq.) in DCM (60 ml) was added 4-DMAP (2.16 g, 17.69 mmol, 1 eq.), TEA (21.48 g, 212.30 mmol, 29.55 ml, 12 eq.) and (Boc) _2O (30.89 g, 141.53 mmol, 8 eq.). The suspension was stirred at 20°C for 18 hours before diluting with saturated aq. NH ₄ Cl solution (100 ml) and extracted with ethyl acetate (2x100 ml). The combined organic layer was washed with brine (200 ml), dried using Na ₂ SO ₄ , filtered and evaporated. The crude residue was purified by combi-flash chromatography (silica gel, 0-20% EtOAc in petroleum ether) to give tert-butyl 6-(HN-bis-(tert-butoxycarbonyl)amino)-2chloro-9H-purine-9 -carboxylate (904 mg, 11% yield) as yellow gum.

Stage 2.

To a solution of tert-butyl-6-(N,N'-bis-(tert-butoxycarbonyl)amino)-2-chloro-9H-purine-9-carboxylate (900 mg, 1.92 mmol, 1 eq.) in DMF (12 ml) added Pd(PPh ₃ ) ₂ Cl ₂ (134.43 mg, 191.52 µmol, 0.1 eq.) and tributyl(1-ethoxyvinyl) stannane (832 µl, 2.46 mmol, 1.29 eq.) in an N2 atmosphere. The suspension was stirred at 95°C for 3 hours before diluting with saturated aq. KF solution (8 ml) and stirred at 20°C for 1 hour. The mixture was extracted with ethyl acetate (2x8 ml). The combined organic layer was washed with water (20 ml), brine (15 ml), dried using Na ₂ SO ₄ , filtered and evaporated. The crude residue was purified by combi-flash chromatography (silica gel, 3070% EtOAc in petroleum ether) to give tert-butyl Na(tert-butoxycarbonyl)amino-(2-(1ethoxyvinyl)-9H-purin-6-yl)carboxymate ( 256 mg, yield 33%) as a white solid.

- 110 046381

Stage 3.

To a solution of the product obtained in the last step (60 mg, 109.38 µmol, 1 eq.), and diethyl-2benzyl-2-(((2R,3R,4R)-3,4,5-triacetoxy-3-ethynyltetrahydrofuran -2-yl)methoxy)malonate (53.22 mg, 131.26 µmol, 1.2 eq.) in MeCN (1 ml) was added with BSA (65 µl, 262.52 µmol, 2.4 eq.). The solution was stirred at 65°C for 0.5 h, then cooled to 25°C, after which TMSOTf (24 μl, 131.26 μmol, 1.2 eq.) was added. The resulting solution was stirred at 65°C for 1 hour before diluting with saturated aq. NaHCO ₃ solution (5 ml) and extracted using EtOAc (2x5 ml). The combined organic layer was evaporated. The crude residue was purified by preparative TLC (EtOAc) to give diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-amino-2-(1ethoxyvinyl) -9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (42 mg, 55% yield) as a colorless gum.

Stage 4.

To a solution of diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-amino-2-(1-ethoxyvinyl)-9Hpurin-9-yl)- 3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (40 mg, 57.66 µmol, 1 eq.) in THF (1.5 ml) was added 1 M aq. HCl. (0.5 ml, 8.67 eq.). The mixture was stirred at 20°C for 21 hours before diluting with saturated aq. NaHCO ₃ (5 ml) and extracted using ethyl acetate (3x5 ml). The combined organic layer was washed with Na ₂ SO ₄ , filtered and evaporated, yielding crude diethyl 2-benzyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(2-acetyl-6- amino-9H-purin-9-yl)-3ethynyltetrahydrofuran-2-yl)methoxy)malonate (40 mg) as a light yellow gum.

Stage 5.

To a solution of crude diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(2-acetyl-6-amino9H-purin-9-yl)-3-ethynyltetrahydrofuran -2-yl)methoxy)malonate (30 mg, 45.07 µmol, 1 eq.) in THF (4 ml) was added 1 M aq. LiOH (901 µl, 20 eq.). The mixture was stirred at 20°C for 6 hours, then acidified using 1 N aq. HCl solution to pH 6 and evaporated. The crude residue was purified by preparative HPLC and the fraction was dried by lyophilization to give the title compound (1.4 mg, 6% yield) as a white solid.

1H NMR (CD3OD, 300 MHz) δ 8.43 (br.s, 1H), 7.14-6.98 (m, 5H), 6.06 (d, J=6.4 Hz, 1H), 4 .89 (d, J=6.8 Hz, 1H), 4.26 (m, 1H), 3.94-3.91 (m, 2H), 3.30-3.21 (m, 2H), 2.91 (s, 1H), 2.61 (s, 3H); LC/MS [M+H] = 526.0.

Example 128. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl) methoxy)-2-(thiophen-2-ylmethyl)malonic acid

Example 128

Proceeding as described in Example 15 above, but replacing allyl bromide with 2-(bromomethyl)thiophene, afforded the title compound as a white solid.

1H NMR (CD3OD, 300 MHz) δ 8.37 (br.s, 1H), 7.10 (d, J=5 Hz, 1H), 6.93-6.72 (m, 2H), 6.00 (d, J=7.2 Hz, 1H), 4.98 (d, J=7.6 Hz, 1H), 4.23 (br.s, 1H), 4.06 (br.s, 2H) , 3.62-3.58 (m, 2H), 2.94 (s, 1H); LC/MS [M+H] = 523.9.

Example 129. Synthesis of 2-(((2R,3S,4R,5R)-5-(2-chloro-6-(cyclopentylamino)-9H-purin-9-yl)-3-ethynyl3,4-dihydroxytetrahydrofuran-2- yl)methoxy)-2-(phenylsulfonyl)acetic acid

Priaer 129

The title compound was prepared analogously to Example 59, except that ethyl 2-diazo-2-(phenylsulfonyl)acetate was used instead of 1-ethyl 3-(prop-1-en-1-yl)-2-diazomalonate in step 3. Cyclopentanamine was used instead of benzylamine in step 7, and step 4 was omitted.

LC/MS [M+H]=592.0.

- 111 046381

Example 130. Synthesis of 2-(((2R,3S,4R,5R)-5-(2-chloro-6-(methylamino)-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2- yl)methoxy)-2-(4-(1-ethyl-2-oxo-1,2-dihydropyridin-3-yl)benzyl)malonic acid

Proceeding as described in example 20 above, but replacing diethyl-2-(((2I,3I,4I,5I)-3,4-diacetoxy-5(6-UK-(bis-(tert-butoxycarbonyl) amino)-2-chloro-9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate on diethyl 2-(((2R,3R,4R,5R)-3,4-diacetoxy- 5-(6-((tert-butoxycarbonyl)(methyl)amino)2-chloro-9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate gave the title compound as a white solid substances.

¹ H NMR (400 MHz, CD ₃ OD) δ ppm. 8.04 (s, 1H), 7.58 (dd, J=6.7, 1.9 Hz, 1H), 7.27-7.38 (m, 5H), 6.35 (t, J= 6.9 Hz, 1H), 5.95 (d, J=7.8 Hz, 1H), 4.77 (d, J=7.8 Hz, 1H), 4.28 (t, J=2, 6 Hz, 1H), 3.96-4.13 (m, 4H), 3.38-3.57 (m, 2H), 3.05 (s, 1H) 2.99 (m, 3H), 1 .32 (t, J=7.3 Hz, 3H); LC/MS[M+H] = 653.1.

Example 131. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-chloro-3-methyl-1H-pyrazolo[3,4-b]pyridin-1-yl)-3ethynyl-3, 4-dihydroxytetrahydrofuran-2-yl)methoxy)-2-(4-(2-oxotetrahydropyrimidin-1(2H)yl)benzyl)malonic acid

Stage 1.

1- chloro-3isocyanatopropane (26.83 g, 224.44 mmol, 1 eq.). The reaction mixture became slightly exothermic and yellow in color as a precipitate formed within 15 minutes. The mixture was stirred for 1.5 hours, then hexanes (50 ml) were added. The mixture was stirred for an additional 15 minutes, after which the solid was collected by filtration, washing with a mixture of DCM and hexanes (5:1=v:v). After drying, 1-(3-chloropropyl)-3-(4-(hydroxymethyl)phenyl)urea (38.45 g) was obtained as a light yellow solid.

Stage 2.

To a solution of 1-(3-chloropropyl)-3-(4-(hydroxymethyl)phenyl)urea (30.00 g, 123.6 mmol, 1.0 eq.) in THF (300 ml) at 25°C dropwise add a solution of 1 M t-BuOK in THF (247.2 ml, 247.2 mmol, 2.0 eq.) with vigorous stirring with a mechanical stirrer. The resulting heterogeneous mixture was stirred at 25°C for 6 hours, then cooled to 0°C and acidified to pH 5-6 using 2 N aq. HCl. The VOCs were then removed under reduced pressure. Unrefined solid

- 112 046381 the substance was placed in MeOH (75 ml) and evaporated. The resulting solid mixture was washed with a solution of 7% MeOH in DCM (220 ml) under low heat and the solid was filtered. The solid was washed again with 7% MeOH in DCM (150 ml) and filtered. The combined filtrates of the washing liquid were evaporated to obtain the desired crude 1-(4(hydroxymethyl)phenyl)tetrahydropyrimidin-2(III)-one as a yellowish solid (27.68 g).

Stage 3.

To a suspension of crude 1-(4-(hydroxymethyl)phenyl)tetrahydropyrimidin-2(III)-one (15.00 g, 72.74 mmol, 1 eq.) in DCM (250 mL) was added a solution of thionyl chloride (10.61 mL , 145.48 mmol, 2 equiv.) at 25°C in an N2 atmosphere. The mixture was stirred at 25°C for 8 hours, then diluted with EtOAc (250 ml) and stirred for 30 minutes. The solid was collected by filtration, washed with EtOAc and dried to give crude 1-(4-(chloromethyl)phenyl)tetrahydropyrimidin-2(III)-one (15.00 g).

Stage 4.

To a solution of diethyl-2-(((3αR,5R,6R,6αR)-6-acetoxy-2,2-dimethyl-6-(prop-1-in-1-yl)tetrahydrofuro[2,3^][1 ,3]dioxol-5-yl)methoxy)malonate (7.04 g, 16.99 mmol, 1 eq.) in DMF (70 ml) was added Cs ₂ CO ₃ (11.07 g, 33.98 mmol, 2 eq.) and crude 1-(4-(chloromethyl)phenyl)tetrahydropyrimidin2(1H)-one (5.09 g, 25.49 mmol, 1.5 eq.) at 20°C. The mixture was stirred at 20°C for 5 hours, then diluted with H2O (300 ml) and extracted using EtOAc (3x100 ml). The combined organic layer was washed with brine (200 ml), dried over Na ₂ SO ₄ , filtered and evaporated. The crude product was purified by silica gel flash column chromatography (10-40% acetone in DCM) to form diethyl 2-(((3aR,5R,6R,6aR)-6-acetoxy-6-ethynyl-2,2- dimethyltetrahydrofuro[2,3d][1,3]dioxol-5-yl)methoxy)-2-(4-(2-oxotetrahydropyrimidin-1(2H)-yl)benzyl)malonate (9.62 g, 94% yield) in the form of a solid.

Stage 5.

To a solution of diethyl-2-(((3αR,5R,6R,6αR)-6-αcetoxy-6-ethynyl-2,2-dimethyltetrahydrofuro[2,3d][1,3]dioxol-5-yl)methoxy)- 2-(4-(2-oxotetrahydropyrimidin-1(2H)-yl)benzyl)malonate (17.31 g, 28.72 mmol, 1 eq.) in DCM (90 ml) was added with H2O (18 ml) and TFA ( 90 ml, 1.22 mol, 42 eq.) at 0°C. The reaction mixture was stirred at 20-25°C for 16 hours, then evaporated under reduced pressure. The residue was azeotroped with DCM (2x50 ml) under reduced pressure to give the crude product diethyl 2-(((2R,3S,4R)-3-αcetoxy-3-ethynyl-4,5-dihydroxytetraαhydrofuran-2yl)methoxy)- 2-(4-(2-oxotetrahydropyrimidin-1(2H)-yl)benzyl)malonate, which was used in the next step without further purification.

Stage 6.

To a solution of crude diethyl-2-(((2R,3S,4R)-3-acetoxy-3-ethynyl-4,5-dihydroxytetrahydrofuran-2-yl)methoxy)-2-(4-(2-oxotetrahydropyrimidine-1 CC -yl-enzyl-alonate (17.23 g, crude) in DCM (170 ml) was added 4-DMAP (374 mg, 3.06 mmol, 0.1 eq.), Ac ₂ O (17.21 ml, 183 .77 mmol, 6 equiv.) and pyridine (19.78 ml, 245.02 mmol, 8 equiv.) at 0 ° C. The reaction mixture was stirred at 20-25 ° C for 16 hours, then evaporated under reduced pressure The residue was redissolved in EtOAc (200 ml), washed with 1 N aq. HCl (150 ml) and 10% aq. Cu ₂ SO ₄ (150 ml), saturated aq. NaHCO ₃ (150 ml) and brine (150 ml). ml), dried over Na ₂ SO ₄ , filtered and evaporated, obtaining crude diethyl 2-(4-(2-oxotetrahydropyrimidin-1(2H)-yl)benzyl)-2-(((2R,3R,4R)3 ,4,5-triacetoxy-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (19.24 g) as a foam, which was used in the next step without further purification.

Stage 7.

To a suspension of crude diethyl ^-C-C-oxotetrahydropyrimidin LC-yl^ensyl)^(((2R,3R,4R)-3,4,5-triacetoxy-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (450 mg, 0.70 mmol, 1 eq.) and 6-chloro-3-methyl-1H-pyrazolo[3,4-b]pyridine (128 mg, 0.77 mmol, 1.1 eq.) in MeCN (4 ml) DBU (315 μL, 2.09 mmol, 3.0 eq.) was added at 0°C. The solution was stirred at 0°C for 5 min followed by the dropwise addition of a solution of TMSOTf (566 μL, 3.13 mmol, 4.5 eq.) in MeCN (2 mL). The solution was stirred at 0°C for 0.5 h and then stirred at 70°C for 16 h, after which it was allowed to cool to 25°C and adjusted to pH 9 with saturated aq. _NaHCO3 . The mixture was extracted using ethyl acetate (2x40 ml). The combined organic layer was dried using Na ₂ SO ₄ , filtered and evaporated. The crude residue was purified by combi-flash silica gel chromatography (0-10% MeOH in DCM) to give diethyl 2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6- chlor3-methyl-1H-pyrazolo[3,4-b]pyridin-1-yl)-3-ethynyltetrahydrofurαn-2-yl)methoxy)-2-(4-(2-oxotetrahydropyrimidin-1(2H)-yl)benzyl )malonate (255 mg, 49% yield) as yellow gum.

Stage 8.

To a solution of diethyl-2-(((2R,3R,4R,5R)-3,4-dicetoxy-5-(6-chloro-3-methyl-1H-pyrazolo[3,4-b]pyridin-1-yl )3-ethynyltetrahydrofuran-2-yl)methoxy)-2-(4-(2-oxotetrahydropyrimidin-1(2H)-yl)benzyl)malonate (296 mg, 392 µmol, 1 eq.) in THF (6 ml) was added aq. LiOH solution (2 M, 1.96 ml, 10 eq.). The solution was stirred at 50°C for 2 hours, after which volatile organic compounds were removed under reduced pressure. 1 N HCl was added to the aqueous layer to adjust the pH to 5-6. The mixture was evaporated to obtain crude

- 113 046381 product, which was purified using preparative HPLC (column: YMC-Actus Triart C18 150-30 mm-5 µm; mobile phase: [water (0.225%FA)-ACN]; B%: 25%-45%, 10 min). The product was isolated by lyophilization to obtain 2-(((2R,3S,4R,5R)-5-(6-chloro-3-methyl-N-pyrazolo[3,4-b]pyridin-1-yl)-3 - ethynyl3,4-dihydroxytetrahydrofuran-2-yl)methoxy)-2-(4-(2-oxotetrahydropyrimidin-1(2H)-yl)benzyl)malonic acid (32 mg) as a white solid.

1H NMR (400 MHz, DMSO-dg) δ ppm. 8.27 (d, J=8.28 Hz, 1H), 7.31 (d, J=8.28 Hz, 1H), 6.97 (d, J=8.53 Hz, 2H), 6, 81 (d, J=8.53 Hz, 2H), 6.49 (s, 1H), 6.20 (s, 1H), 6.13 (d, J=7.53 Hz, 1H), 5, 97 (s, 1H), 4.98 (d, J=7.53 Hz, 1H), 4.13 (dd, J=8.41, 2.64 Hz, 1H), 3.93-3.99 (m, 1H), 3.84-3.91 (m, 1H), 3.64 (s, 1H), 3.42-3.52 (t, J=5.60 Hz, 2H), 3, 17-3.23 (m, 2H), 3.05-3.16 (m, 2H), 2.45 (s, 3H), 1.90 (m, 2H); LC/MS [M+H] = 614.3.

Example 132. Synthesis of 2-(((2R,3S,4R,5R)-5-(2-chloro-6-oxo-1H-purin-9(6H)-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2 -yl)methoxy)-2-(4-(2-oxotetrahydropyrimidin-1(2H)yl)benzyl)malonic acid

Stage 1.

To a solution of 2,6-dichloro-9I-purine (379.97 mg, 2.01 mmol) in MeCN (5 mL) was added BSA (956 μL, 3.87 mmol) at 25°C. The reaction mixture was stirred at 65°C for 0.5 h and then cooled again to 25°C. To this mixture was added diethyl 2-(4-(2-oxotetrahydropyrimidin-1(2H)-yl)benzyl)-2(((2R,3R,4R)-3,4,5-triacetoxy-3-ethynyltetrahydrofuran-2 -yl)methoxy)malonate (1 g) in MeCN (5 ml) and TMSOTf (419 μl, 2.32 mmol) at 25°C and further stirred at 65°C for 5 hours. The reaction mixture was allowed to cool to 25° C, then quenched with saturated aq. NaHCO ₃ solution (10 ml) and extracted using EtOAc (3x5 ml). The combined organic layer was washed with brine (10 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated. The crude residue was purified by silica gel flash column chromatography (0-10% MeOH in DCM) to give diethyl 2-(((2R,3R,4R,5R)-3,4-di-acetoxy-5-(2 ,6-dichloro-9H-purin-9-yl)-3-ethynyltetrahydrofuran-2yl)methoxy)-2-(4-(2-oxotetrahydropyrimidin-1(2H)-yl)benzyl)malonate (365 mg) foam .

Stage 2.

To a solution of diethyl-2-(((2R,3R,4R,5R)-3,4-di-acetoxy-5-(2,6-dichloro-9H-purin-9-yl)-3-ethynyltetrahydrofuran-2- yl)methoxy)-2-(4-(2-oxotetrahydropyrimidin-1(2H)-yl)benzyl)malonate (180 mg) in THF (2 ml) was added LiOH-H ₂ O (97.39 mg, 2.32 mmol, 10 eq.) in H2O (1 ml) at 25°C. The reaction mixture was stirred at 40°C for 2 h before removing volatile organic compounds under reduced pressure. The aqueous phase was acidified to pH 5-6 with 1 N aq. HCl and evaporated under reduced pressure. The crude residue was purified by preparative HPLC (column: YMC-Actus Triart C18 150-30 mm-5 µm; mobile phase: [water (0.225%FA)-ACN]; B%: 13%-33%, 10 min) and dried by lyophilization to obtain 2-(((2R,3S,4R,5R)-5-(2-chloro-6-oxo-1H-purin-9(6H)-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2 -yl)methoxy)-2-(4-(2-oxotetrahydropyrimidin-1(2H)-yl)benzyl)malonic acid (15 mg) as a white solid.

1H NMR (400 MHz, CD3OD) δ ppm. 8.62 (s, 1H), 7.24 (d, J=8.31 Hz, 2H), 7.03 (d, J=8.31 Hz, 2H), 6.32 (d, J=6 .48 Hz, 1H), 4.61 (d, J=6.48 Hz, 1H), 4.28-4.34 (m, 1H), 3.92-4.04 (m, 2H), 3 .55-3.66 (m, 2H), 3.33-3.40 (m, 4H), 3.02 (s, 1H), 1.99-2.06 (m, 2H); LC/MS [M+H] = 617.2.

Example 133. Synthesis of 2-(((2R,3S,4R,5R)-3-ethynyl-3,4-dihydroxy-5-(5-methyl-2,4-dioxo-3,4dihydropyrimidin-1 CN-ylHetrahydrofuran^ -yl^ethoxy^-C-C-oxotetrahydropyrimidin-1 (2H)yl)benzyl)malonic acid

Example 133

Stage 1.

To a mixture of 5-methylpyrimidine-2,4(1H,3H)-dione (100 mg, 792.94 µmol, 1 eq.) in MeCN (2 ml) was added BSA (490 µl, 1.98 mmol, 2.5 eq. .). The mixture was stirred at 85°C for 0.5 hours. The mixture was cooled to 0°C, and then a solution of diethyl 2-(4-(2-oxotetrahydropyrimidin-1(2H)-yl)benzyl)-2((( 2R,3R,4R)-3,4,5-triacetoxy-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (385 mg) in MeCN (2 ml) and TMSOTf (430 µl, 2.38 mmol, 3.0 eq.). The mixture was stirred at 65°C under N2 for 5 hours, then allowed to cool to 25°C and quenched with saturated aq. NaHCO ₃ (10 ml). The mixture was extracted using EtOAc (3x5 ml). The organic layers were combined and dried over

- 114 046381

Na2SO ₄ , filtered and evaporated in vacuum. The crude compound was purified by silica gel flash column chromatography (0-5% MeOH in DCM) to give gn3Tn.i-2-(((2R.3R.4R.5R)-3.4diacetoxy-3-ethynyl-5-( 5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)methoxy)-2-(4-(2-oxotetrahydropyrimidin-1(2H)-yl)benzyl )malonate (208 mg, 37% yield) as a solid.

Stage 2.

To a solution of diethyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-3-ethynyl-5-(5-methyl-2,4-dioxo-3,4dihydropyrimidine-1(2H)- yl)tetrahydrofuran-2-yl)methoxy)-2-(4-(2-oxotetrahydropyrimidin-1(2H)yl)benzyl)malonate (200 mg, 280.62 µmol, 1 eq.) in THF (2 ml) was added LiOH-H ₂ O (58.88 mg, 1.40 mmol, 5 eq.) in H2O (1 ml) at 20-25°C. The reaction mixture was stirred at 40°C for 1 h before removing volatile organic compounds under reduced pressure. The aqueous phase was acidified to pH 5-6 with 1 N aq. HCl and evaporated under reduced pressure. The crude residue was purified by preparative HPLC (column: YMC-Actus Triart C18 150-30 mm-5 µm; mobile phase: [water (0.225%FA)-ACN]; B%: 13%-33%, 10 min) and then dried by lyophilization to obtain 2(((2R,3S,4R,5R)-3-ethynyl-3,4-dihydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidine-1(2H )-yl)tetrahydrofuran-2-yl)methoxy)-2-(4-(2-oxotetrahydropyrimidin-1(2H)-yl)benzyl)malonic acid (49 mg) as a white solid.

1H NMR (400 MHz, CD3OD) δ ppm. 7.83 (d, J=0.75 Hz, 1H), 7.33 (d, J=8.28 Hz, 2H), 7.11 (d, J=8.28 Hz, 2H), 6, 06 (d, J=7.78 Hz, 1H), 4.44 (d, J=7.78 Hz, 1H), 4.16 (t, J=2.26 Hz, 1H), 3.92- 4.05 (m, 2H), 3.47-3.65 (m, 3H), 3.33-3.41 (m, 3H), 2.98 (s, 1H), 2.04 (m, 2H), 1.62 (s, 3H); LC/MS [M+H] = 573.1.

Example 134. Synthesis of 2-(((2R,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-ethynyl3,4-dihydroxytetrahydrofuran-2 -yl)methoxy)-2-(4-(2-oxotetrahydropyrimidin-1(2H)-yl)benzyl)malonic acid

Proceeding as described in Example 133 above, but replacing thymine with uracil, afforded the title compound as a white solid.

1H NMR (400 MHz, CD ₃ OD) δ ppm. 7.91 (d, J=8.0 Hz, 1H), 7.31 (d, J=8.4 Hz, 2H), 7.15 (d, J=8.4 Hz, 2H), 6, 01 (d, J=7.5 Hz, 1H), 5.20 (d, J=8.0 Hz, 1H), 4.36 (d, J=7.5 Hz, 1H), 4.17 ( t, J=2.4 Hz, 1H), 3.99 (dd, J=18.8, 2.5 Hz, 2H), 3.55-3.66 (m, 2H), 3.44-3 .52 (m, 1H), 3.33-3.38 (m, 3H), 3.01 (s, 1H), 1.99-2.10 (m, 2H); LC/MS [M+H] = 559.1.

Example 135. Synthesis of 2-(((2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl)methoxy )-2-(4-(2-oxotetrahydropyrimidin-1(2H)yl)benzyl)malonic acid

Example 135

Proceeding as described in Example 133 above, but replacing thymine with cytosine, afforded the title compound as a white solid.

1H NMR (400 MHz, DMSO<) δ ppm. 7.92-8.05 (m, 1H), 7.76-7.92 (br.s, 1H), 7.45-7.62 (s, 1H), 7.03-7.19 (m , 4H), 6.55 (s, 1H), 5.91-5.99 (m, 1H), 5.87 (d, J=6.80 Hz, 1H), 5.80 (d, J= 6.00 Hz, 1H), 5.57 (d, J=7.20 Hz, 1H), 4.13 (t, J=6.80 Hz, 1H), 4.02-4.08 (m, 1H), 3.68-3.82 (m, 2H), 3.50-3.57 (m, 2H), 3.22 (s, 1H), 3.19-3.22 (m, 3H) , 1.82-1.99 (m, 2H); LC/MS [M+H] = 558.3.

Example 136. Synthesis of 2-(((2R,3S,4R,5R)-5-(2-chloro-6-((cyclopropylmethyl)amino)-9H-purin-9-yl)-3ethynyl-3,4-dihydroxytetrahydrofuran -2-yl)methoxy)-2-(4-(2-oxotetrahydropyrimidin-1(2H)-yl)benzyl)malonic acid

Prnmir 13®

- 115 046381

Proceeding as described in Example 133 above, but replacing thymine with 2-chloro-N-(cyclopropylmethyl)9H-purin-6-amine, afforded the title compound as a white solid.

1H NMR (400 MHz, CD3OD) δ ppm. 8.22 (s, 1H), 7.28 (br.d, J=8.03 Hz, 2H), 7.03 (d, J=8.28 Hz, 2H), 5.97 (d, J =7.28 Hz, 1H), 4.72-4.77 (m, 1H), 4.28 (s, 1H), 3.95-4.05 (m, 2H), 3.34-3, 52 (m, 8H), 3.05 (s, 1H), 1.92-2.02 (m, 2H), 1.11-1.20 (m, 1H), 0.51-0.59 ( m, 2H), 0.34 (k, J=4.85 Hz, 2H); LC/MS [M+H] = 670.1.

Example 137. Synthesis of 2-(((2R,3S,4R,5R)-5-(2-chloro-6-(methylamino)-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2- yl)methoxy)-2-(4-(3-(2-hydroxyethyl)-2-oxotetrahydropyrimidin-1(2H)yl)benzyl)malonic acid

Priper 137

Stage 1.

NaH (9, 89 g, 247.22 mmol, 60% in mineral oil, 12 eq.). The reaction mixture was stirred at 25°C for 1.5 hours, then TBDPSCl (6.80 g, 24.72 mmol, 1.2 eq.) was added and stirred for another 1.5 hours. Allyl bromide (9 .97 g, 82.41 mmol, 4 eq.) and stirred for an additional 16 hours. H2O (50 ml) was added to the reaction mixture and the resulting mixture was extracted using EtOAc (3x100 ml). The combined organic layer was washed with brine (100 ml), dried over Na ₂ SO ₄ , filtered and evaporated. The crude residue was purified by silica gel flash column chromatography and eluted with EtOAc in petroleum ether (0-30%) to give 1-allyl-3-(4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)tetrahydropyrimidine-2 (1H)-one (6 g, 60% yield) as an oil.

Stage 2.

To a solution of the compound 1-allyl-3-(4-(((tert-butyldiphenylsilyl)oxy)methyl)fennlDetrahydropyrimidin-CN-one (6 g, 12.38 mmol, 1 eq.) in a mixture of MeOH (60 ml) and DCM (30 ml) at -78°C, ozone (15 psi) was introduced until the blue color of the solution remained for 20 minutes. Excess ozone was removed by bubbling nitrogen gas for 10 minutes. To this reaction mixture was added NaBH ₄ (937 mg, 24.76 mmol, 2 eq.), the mixture was allowed to warm to 0° C. and stirred for 15 hours at 25° C. The mixture was poured into 1 N aq. HCl (50 ml) and extracted using EtOAc (2x100 ml) The combined organic layer was washed with water (100 ml), brine (100 ml), dried (Na2SO4), filtered and evaporated to give crude 1-(4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl) -3-(2-hydroxyethyl)tetrahydropyrimidin-2(1H)-one (6.6 g) as an oil, which was used in the next step.

Stage 3.

To a mixture of crude 1-(4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)-3-(2hydroxyethyl)tetrahydropyrimidin-2(1H)-one (12.38 mmol, 1 eq.) in DCM (40 ml ) and pyridine (3.00 ml, 37.14 mmol, 3 eq.) at 25°C was added Ac ₂ O (2.32 ml, 24.76 mmol, 2 eq.) and 4-DMAP (151 mg, 1 .24 mmol, 0.1 eq.). The mixture was stirred for 2 hours and then quenched with H2O (60 ml). The mixture was extracted using EtOAc (3x50 ml). The combined organic layer was washed with water (2x30 ml), brine (30 ml), dried over Na2SO4, filtered and evaporated to give crude 2-(3-(4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)-2 -oxotetrahydropyrimidin-1(2H)-yl)ethyl acetate, which was directly used in the next step.

Stage 4.

To a solution of crude 2-(3-(4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)-2oxotetrahydropyrimidin-1(2H)-yl)ethyl acetate (6.57 g, 12.38 mmol, 1 eq.) in THF (40 ml) was added a 1 M solution of TBAF in THF (18.57 ml, 1.5 eq.) at 0°C. The mixture was stirred at 25°C for 1 hour before adding H2O (100 ml). The reaction mixture was extracted using EtOAc (4x100 ml). Volume

- 116 046381 The dined organic layer was washed with brine (50 ml), dried over Na ₂ SO ₄ , filtered and evaporated. The aqueous phase was further extracted using CH2Cl2 (4x100 ml). The combined organic layer was washed with brine (50 ml), dried over Na ₂ SO ₄ , filtered and evaporated to obtain more product. Both batches of crude product were combined and further purified by silica gel column chromatography (40-100% EtOAc in petroleum ether) to give 2-(3-(4-(hydroxymethyl)phenyl)-2-oxotetrahydropyrimidin-1(2H)yl )ethyl acetate (3.14 g) as a white solid.

Stage 5.

To a solution of crude 2-(3-(4-(hydroxymethyl)phenyl)-2-oxotetrahydropyrimidin-1(2H)yl)ethyl acetate (1.28 g, 4.38 mmol, 1 eq.) and DMF (3.37 µl , 43.79 µmol, 0.01 eq.) SOCl ₂ (5 ml, 68.92 mmol, 15.74 eq.) was added to DCM (25 ml) at 0°C. The mixture was stirred at 50°C for 2 hours, then evaporated to give crude 2-(3-(4-(chloromethyl)phenyl)-2-oxotetrahydropyrimidin-1(2H)yl)ethyl acetate (1.51 g), which was used at the next stage without additional purification.

Stage 6.

To a solution of crude 2-(3-(4-(chloromethyl)phenyl)-2-oxotetrahydropyrimidin-1(2H)yl)ethyl acetate (1.51 g, 4.38 mmol, 1 eq.) and diethyl 2-(( (3aR,5R,6R,6aR)-6-acetoxy-2,2-dimethyl-6-(prop1-in-1-yl)tetrahydrofuro[2,3D][1,3]dioxol-5-yl)methoxy) malonate (1.91 g, 4.60 mmol, 1.05 eq.) in DMF (8 ml) was added Cs ₂ CO ₃ (4.28 g, 13.14 mmol, 3 eq.). The mixture was stirred at 25°C for 16 hours, then diluted with H2O (40 ml) and extracted using EtOAc (3x30 ml). The combined organic layer was washed with sat. aq. NH ₄ Cl (2x15 ml), water (2x15 ml), saline (15 ml), dried over Na ₂ SO ₄ , filtered and evaporated. The residue was purified by silica gel column chromatography (25-70% EtOAc in petroleum ether) to give diethyl 2-(((3aR,5R,6R,6aR)-6-acetoxy-6ethynyl-2,2-dimethyltetrahydrofuro[2, 3D][1,3]dioxol-5-yl)methoxy)-2-(4-(3-(2-acetoxyethyl)-2-oxotetrahydropyrimidin-1(2H)-yl)benzyl)malonate (2.20 g, yield 68%) in the form of yellow gum.

Stage 7.

To a solution of diethyl-2-(((3aR,5R,6R,6aR)-6-acetoxy-6-ethynyl-2,2-dimethyltetrahydrofuro[2,3d][1,3]dioxol-5-yl)methoxy)- 2-(4-(3-(2-acetoxyethyl)-2-oxotetrahydropyrimidin-1-Cl-yl-enzyl-alonate (2.20 g, 3.19 mmol, 1 eq.) in DCM (7.5 ml) was added TFA (7.5 ml, 101.30 mmol, 32 eq.) and H2O (1.5 ml).The mixture was stirred at 25°C for 16 hours, then diluted with water (20 ml).The mixture was then adjusted to pH 7 -8 solid NaHCO _3. The mixture was then extracted with CH ₂ Cl ₂ (4x20 ml).The organic phase was then washed with brine (10 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated to obtain crude diethyl-2-(( (2R,3S,4R)-3-acetoxy-3-ethynyl-4,5-dihydroxy-tetrahydrofuran-2-yl)methoxy)-2-(4-(3 -(2-acetoxyethyl)-2-oxotetrahydropyrimidine-1 CI-yl-enzyl-alonate (2.1 g) in the form of yellow gum.

To a solution of the above crude product (2.1 g, 3.19 mmol, 1 eq.) in DCM (15 ml) was added Ac ₂ O (1.79 ml, 19.14 mmol, 6 eq.), pyridine (2 .06 ml, 25.52 mmol, 8 equiv.) and 4-DMAP (38.97 mg, 319.00 µmol, 0.1 equiv.). The mixture was stirred at 25°C for 2 hours, then diluted with EtOAc (100 ml), washed successively with 1 N aq. HCl (2x30 ml). The organic extract was washed with water (20 ml), saturated aq. NaHCO ₃ solution (2x20 ml), water (20 ml) and saline solution (10 ml). The organic layer was dried over MgSO ₄ and evaporated to give crude diethyl 2-(4-(3-(2-acetoxyethyl)2-oxotetrahydropyrimidin-1(2H)-yl)benzyl)-2-(((2R,3R,4R )-3,4,5-triacetoxy-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (2.3 g) as a yellow foam.

Stage 8.

To a solution of 2-chloro-N-methyl-9H-purin-6-amine (151.59 mg, 825.68 µmol, 1.1 eq.) in MeCN (3 ml) was added BSA (408.19 µl, 1. 65 mmol, 2.2 eq.). The mixture was stirred at 65°C for 0.5 h, then cooled to 0°C and diethyl 2-(4-(3-(2-acetoxyethyl)-2-oxotetrahydropyrimidin-1(2H)yl)benzyl)- 2-(((2R,3R,4R)-3,4,5-triacetoxy-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (550 mg, crude) in MeCN (3 ml) and TMSOTf (406.91 µl , 2.25 mmol, 3 equiv.). The mixture was stirred at 65°C for 3 hours, then allowed to cool to 25°C and quenched with saturated aq. NaHCO ₃ (20 ml). The reaction mixture was extracted using EtOAc (4x20 ml). The combined organic layer was washed with water (10 ml), brine (10 ml), dried over Na ₂ SO ₄ , filtered and evaporated. The crude product was first purified by silica gel flash column chromatography (3070% EtOAc in petroleum ether) and then further purified by preparative TLC (7% MeOH in DCM) to give diethyl-2-(4-(3-(2 -acetoxyethyl)-2-oxotetrahydropyrimidin-1(2H)yl)benzyl)-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(2-chloro-6-(methylamino)- 9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (180 mg, 22% yield) as foam.

Stage 9.

To a solution of crude diethyl 2-(4-(3-(2-acetoxyethyl)-2-oxotetrahydropyrimidin-1(2H)yl)benzyl)-2-(((2R,3R,4R,5R)-3,4- diacetoxy-5-(2-chloro-6-(methylamino)-9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (180 mg, 210.21 µmol, 1 eq.) in THF (1 ml) added saturated aq. LiOH solution (1.5 ml). The mixture was stirred at 50°C for 2 hours, after which volatiles were removed

- 117 046381 organic substances under reduced pressure. The pH of this mixture was adjusted to 2-3 using 6N aq. HCl and then evaporated. The crude residue was purified by preparative HPLC (column: YMC-Actus ODS-AQ 150-30 5u; mobile phase: [water (0.225%FA)-ACN]; B%: 15%-35%, 15 min) to give 2 -(((2R,3S,4R,5R)-5-(2-chloro-6-(methylamino)-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy )-2-(4-(3-(2-hydroxyethyl)-2-oxotetrahydropyrimidin-1(2H)-yl)benzyl)malonic acid (71.8 mg, 50% yield) as a white solid.

¹ H NMR (400 MHz, CD3OD) δ ppm. 8.14 (s, 1H), 7.27 (d, J=8.0 Hz, 2H), 6.98 (d, J=8.0 Hz, 2H), 5.96 (d, J=7 .5 Hz, 1H), 4.76 (d, J=7.4 Hz, 1H), 4.26 (br. s, 1H), 4.04 (s, 2H), 3.66 (t, J =5.6 Hz, 2H), 3.36-3.55 (m, 8H), 3.05 (m, 4H), 1.96-2.04 (m, 2H); LC/MS [M+H] = 674.1.

Example 138. Synthesis of 2-(((2R,3S,4R,5R)-5-(2-chloro-6-((cyclopropylmethyl)amino)-9H-purin-9-yl)-3ethynyl-3,4-dihydroxytetrahydrofuran -2-yl)methoxy)-2-(4-(3-(2-hydroxyethyl)-2-oxotetrahydropyrimidin-1(2H)-yl)benzyl)malonic acid

Prizhr 135

Proceeding as described in Example 137 above, but replacing 2-chloro-N-methyl-9H-purin-6-amine with 2chloro-N-(cyclopropylmethyl)-9H-purin-6-amine, the title compound was obtained as white solid.

¹ H NMR (400 MHz, CD3OD) δ ppm. 8.09 (s, 1H), 7.28 (d, J=8.3 Hz, 2H), 7.01 (d, J=8.3 Hz, 2H), 5.96 (d, J=7 .5 Hz, 1H), 4.74 (d, J=7.3 Hz, 1H), 4.26 (t, J=2.8 Hz, 1H), 4.04 (d, J=2.3 Hz, 2H), 3.62-3.69 (m, 2H), 3.45-3.54 (m, 4H), 3.34-3.42 (m, 6H), 3.06 (s, 1H) 1.97-2.05 (m, 2H), 1.09-1.21 (m, 1H), 0.52-0.61 (m, 2H), 0.34 (k, J=4 ,8 Hz, 2H); LC/MS [M+H] = 714.1

Example 139. Synthesis of 2-(((2R,3S,4R,5R)-5-(2-chloro-6-(isopropylamino)-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2- yl)methoxy)-2-(4-(3-(2-hydroxyethyl)-2-oxotetrahydropyrimidin-1(2H)yl)benzyl)malonic acid

Proceeding as described in Example 137 above, but replacing 2-chloro-N-methyl-9H-purin-6-amine with 2chloro-N-isopropyl-9H-purin-6-amine, afforded the title compound as a white solid. substances.

1H NMR (400 MHz, CD ₃ OD) δ ppm. 8.08 (s, 1H), 7.29 (d, J=8.3 Hz, 2H), 7.02 (d, J=8.3 Hz, 2H), 5.95 (d, J=7 .5 Hz, 1H), 4.73 (d, J=7.5 Hz, 1H), 4.39 (br. s, 1H), 4.26 (t, J=2.5 Hz, 1H), 4.04 (d, J=2.0 Hz, 2H), 3.66 (t, J=5.8 Hz, 2H), 3.37-3.52 (m, 9H), 1.97-2 .04 (m, 2H), 1.30 (d, J=6.3 Hz, 6H); LC/MS [M+H] = 702.1.

Example 140. Synthesis of 2-(((2R,3S,4R,5R)-5-(2-chloro-6-(isopropylamino)-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2- yl)methoxy)-2-(4-(2-oxotetrahydropyrimidin-1(2H)-yl)benzyl)malonic acid

Proceeding as described in Example 133 above, but replacing thymine with 2-chloro-Nisopropyl-9H-purine-6amine, afforded the title compound as a white solid.

1H NMR (400 MHz, CD3OD) δ ppm. 8.16 (s, 1H) 7.32 (d, J=8.44 Hz, 2H) 7.08 (br.d, J=8.31 Hz, 2H) 5.99 (d, J=7, 46 Hz, 1H) 4.81 (d, J=7.46 Hz, 1H) 4.41 (br. s, 1H) 4.26-4.31 (m, 1H) 3.97-4.12 ( m, 2H) 3.40-3.57 (m, 4H), 3.33-3.37 (m, 2H), 3.03 (s, 1H), 1.99 (m, 2H), 1. 31 (m, 6H); LC/MS [M+H] = 658.3.

- 118 046381

Example 141. Synthesis of 2-(((2R,3S,4R,5R)-5-(2-chloro-6-(methylamino)-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2- yl)methoxy)-2-(4-(2-oxotetrahydropyrimidin-1(2H)-yl)benzyl)malonic acid

Proceeding as described in Example 133 above, but replacing thymine with 2-chloro-N-methyl-9H-purine-6-amine, afforded the title compound as a white solid.

¹ H NMR (400 MHz, CD3OD) δ ppm. 8.20 (s, 1H), 7.27 (d, J=8.13 Hz, 2H), 7.00 (d, J=8.00 Hz, 2H), 5.97 (d, J=7 .38 Hz, 1H), 4.80 (d, J=7.38 Hz, 1H), 4.27 (s, 1H), 4.04 (m, 2H), 3.37-3.50 (m , 4H), 3.31 (d, J=1.13 Hz, 3H), 3.06 (s, 2H) 3.04 (s, 1H), 1.90-2.00 (m, 2H); LC/MS [M+H] = 630.2.

Example 142. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl) methoxy)-2-(thiazol-2-ylmethyl)malonic acid

Proceeding as described in Example 15 above, but replacing allyl bromide with 2-(bromomethyl)thiazole, afforded the title compound as a white solid.

1H NMR (400 MHz, CD3OD) δ ppm. 8.56 (s, 1H), 7.56 (d, J=3.4 Hz, 1H), 7.33 (d, J=3.4 Hz, 1H), 6.00 (d, J=5 .6 Hz, 1H), 4.68-4.73 (m, 1H), 4.38 (dd, J=6.3, 3.4 Hz, 1H), 3.98-4.07 (m, 2H), 3.85 (s, 2H), 3.00 (s, 1H); LC/MS [M+H] = 524.9.

Example 143. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl) methoxy)-2-(thiazol-5-ylmethyl)malonic acid

Proceeding as described in Example 15 above, but replacing the alylbromide with 5-(chloromethyl)thiazole, afforded the title compound as a white solid.

1H NMR (400 MHz, CD3OD) δ ppm. 8.75 (s, 1H), 8.47 (s, 1H), 7.72 (s, 1H), 6.03 (d, J=7.3 Hz, 1H), 4.97 (d, J =7.3 Hz, 1H), 4.36 (dd, J=4.3, 3.0 Hz, 1H), 4.12-4.18 (m, 1H), 4.04-4.10 ( m, 1H), 3.63-3.79 (m, 2H), 3.00 (s, 1H); LC/MS [M+H] = 524.9.

Example 144. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl) methoxy)-2-(4-(pyrrolidin-1-yl)benzyl)malonic acid

144

Stage 1.

To a solution of diethyl-2-(((3aR,5R,6R,6aR)-6-acetoxy-6-ethynyl-2,2-dimethyltetraαhydrofuro-[2,3d][1,3]dioxol-5-yl)methoxy) malonate (10.0 g, 24.13 mmol, 1 eq.) in DMF (100 ml) was added Cs ₂ CO ₃ (23.59 g, 72.39 mmol, 3 eq.) and 1-(bromomethyl)-4 -iodobenzene (10.75 g, 36.20 mmol, 1.5 eq.). The suspension was stirred at 20°C for 1 hour, then diluted with water (200 ml). The resulting mixture is extragyro

- 119 046381 using EtOAc (4x50 ml). The combined organic layer was washed with water (2x200 ml), brine (200 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated. The crude residue was purified by silica gel column chromatography (5-20% EtOAc in petroleum ether) to give diethyl 2-(((3αR,5R,6R,6αR)-6-acetoxy-6-ethynyl-2,2-dimethyltetrahydrofuro [2,3d][1,3]dioxol-5-yl)methoxy)-2-(4-iodobenzyl)malonate (11.83 g, 74% yield) as a white solid.

Stage 2.

To a solution of diethyl-2-(((3αR,5R,6R,6αR)-6-αcetoxy-6-ethynyl-2,2-dimethyltetrahydrofuro[2,3d][1,3]dioxol-5-yl)methoxy)- 2-(4-iodobenzyl)malonate (2.00 g, 3.17 mmol, 1 eq.) in DMA (22 ml) was added K ₂ CO ₃ (1.32 g, 9.52 mmol, 3 eq.), CuI (120.84 mg, 634.50 µmol, 0.2 equiv.) and proline (438.30 mg, 3.81 mmol, 1.2 equiv.). The green suspension was stirred at 80°C under N2 for 16 hours, after which it was allowed to cool and poured into water (40 ml) and 2 N aq. LiOH solution (1 ml). The mixture was extracted using ethyl acetate (2x30 ml). The resulting aq. the layer was acidified to pH 5 with 1 N aq. HCl solution and then extracted using ethyl acetate (2x40 ml). The combined organic layer was washed with water (80 ml), brine (80 ml), dried over Na ₂ SO ₄ , filtered and evaporated to give crude (S)-1-(4-(2-(((3αR,5R,6R ,6αR)-6-αcetoxy-6-ethynyl-2,2-dimethyltetrahydrofuro[2,3D][1,3]dioxol-5-yl)methoxy)-3-ethoxy-2-(ethoxycarbonyl)-3-oxopropyl) phenyl)pyrrolidine-2-carboxylic acid (390 mg) as yellow gum.

Stage 3.

To a solution of (S)-1-(4-(2-(((3αR,5R,6R,6αR)-6-αcetoxy-6-ethynyl-2,2-dimethyltetrahydrofuro[2,3d][1,3]dioxol -5-yl)methoxy)-3-ethoxy-2-(ethoxycarbonyl)-3-oxopropyl)phenyl)pyrrolidine-2-carboxylic acid (420 mg, 680.01 µmol, 1 eq.) in DMF (5 ml) was added K ₂ CO ₃ (282 mg, 2.04 mmol, 3 eq.) and EtI (81.58 μl, 1.02 mmol, 1.5 eq.). The mixture was stirred at 20°C for 0.5 h, then diluted with water (10 ml) and extracted using ethyl acetate (2x10 ml). The combined organic layer was washed with water (20 ml), brine (20 ml), dried over Na ₂ SO ₄ , filtered and evaporated to give crude diethyl 2-(((3αR,5R,6R,6αR)-6-acetoxy- 6-ethynyl-2,2-dimethyltetrahydrofuro[2,3d][1,3]dioxol-5-yl)methoxy)-2-(4-((S)-2-(ethoxycarbonyl)pyrrolidin-1-yl)benzyl )malonate (330 mg) as a yellow foam.

Stage 4.

To a solution of diethyl-2-(((3αR,5R,6R,6αR)-6-acetoxy-6-ethynyl-2,2-dimethyltetrahydrofuro[2,3d][1,3]dioxol-5-yl)methoxy)- 2-(4-((S)-2-(ethoxycarbonyl)pyrrolidin-1-yl)benzyl)malonate (330 mg, crude) in DCM (4 ml) added H2O (0.8 ml) and TFA (4 ml) at 0°C. The solution was stirred at 20°C for 4.5 hours before quenching with saturated aq. NaHCO ₃ to adjust pH to 9. The mixture was extracted using ethyl acetate (2x8 ml). The combined organic layer was washed with brine (20 ml), dried over Na ₂ SO ₄ , filtered and evaporated to give crude diethyl 2-(((2S,3S,4R)-3-acetoxy-3-ethynyl-4,5-dihydroxytetrahydrofuran- 2-yl)methoxy)-2-(4-(^)-2-(ethoxycarbonyl)pyrrolidin-1-yl)benzyl)malonate (285 mg) as a yellow foam.

Stage 5.

To a mixture of crude diethyl-2-(((2S,3S,4R)-3-acetoxy-3-ethynyl-4,5-dihydroxytetraαgyndrofuran2-yl)methoxy)-2-(4-(^)-2-(ethoxycarbonyl) pyrrolidin-1-yl)benzyl)malonate (285 mg) in pyridine (4 ml) was added with 4-DMAP (172 mg, 1.41 mmol, 3 eq.) and Ac ₂ O (352.60 μl, 3.76 mmol , 8 eq.). The solution was stirred at 20°C for 16 hours, then diluted with water (10 ml) and extracted using ethyl acetate (3x10 ml). The combined organic layer was washed with brine (20 ml), dried using Na2SO4, filtered and evaporated. The crude residue was purified by silica gel flash column chromatography (20-50% ethyl acetate in petroleum ether) to give diethyl-2(4-((S)-2-(ethoxycarbonyl)pyroludin-1-yl)benzyl)-2- (((2R,3R,4R)-3,4,5-triacetoxy-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (220 mg, 51% yield for four steps) as yellow gum.

Stage 6.

To a suspension of diethyl-2-(4-((S)-2-(ethoxycarbonyl)pnrolndin-1-yl)benzyl)-2-(((2R,3R,4R)-3,4,5triacetoxy-3-ethynyltetrahydrofuran- 2-yl)methoxy)malonate (50 mg, 72.50 µmol, 1 equiv.) and 6-chloropurine (15 mg, 86.99 µmol, 1.2 equiv.) in MeCN (1 ml) was added BSA (44.80 µl , 181.24 µmol, 2.5 eq.). The suspension was stirred at 65°C for 0.5 h, then cooled to 0°C, after which TMSOTf (32.75 μl, 181.24 μmol, 2.5 eq.) was added dropwise. The mixture was stirred at 65°C for 1 hour, then poured into saturated aq. NaHCO3 (3 ml). The reaction mixture was extracted using ethyl acetate (3x3 ml). The combined organic layer was evaporated. The crude residue was purified by preparative TLC (petroleum ether: EtOAc=2:1) to give diethyl 2(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-amino-2-chloro -9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)-2-(4-(^)-2-(ethoxycarbonyl)pnrrolndin-1-yl)benzyl)malonate (33 mg, yield 57 %) in the form of yellow gum.

Stage 7.

To a solution of diethyl-2-(((2R,3R,4R,5R)-3,4-dicetoxy-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl

- 120 046381 tetrahydrofuran-2-yl)methoxy)-2-(4-((b)-2-(ethoxycarbonyl)pyrrolidin-1-yl)benzyl)malonate (28 mg, 35.03 µmol, 1 eq.) in THF (2 ml) was added 1 M aq. LiOH solution (701 µl, 20 eq.). The mixture was stirred at 20°C for 4.5 hours, after which volatile organic compounds were removed under reduced pressure. Vodn. the layer was acidified to pH 6 using 1 N aq. HCl solution and then evaporated. The crude residue was purified by preparative HPLC (column: YMC-Triart Prep C18 150-40 mm-7 µm; mobile phase: [water (0.225%FA)-ACN]; B%: 15% - 35%, 10 min) and dried by lyophilization to obtain 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl) methoxy)-2-(4-(pyrrolidin-1-yl)benzyl)malonic acid (2.6 mg) as a white solid.

Ή NMR (400 MHz, CD3OD) δ ppm. 8.21 (s, 1H), 7.05 (d, J=8.53 Hz, 2H), 6.26 (d, J=8.53 Hz, 2H), 5.98 (d, J=7 .53 Hz, 1H), 4.92-5.02 (m, 1H), 4.29-4.33 (m, 1H), 4.09 (dd, J=10.16, 2.38 Hz, 1H), 3.97 (dd, J=10.04, 3.01 Hz, 1H), 3.38 (d, J=14.56 Hz, 1H), 3.23 (d, J=14.56 Hz, 1H), 3.02-3.09 (m, 4H), 3.00 (s, 1H), 1.89-1.97 (m, 4H); LC/MS [M+H] = 587.1.

Example 145. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-(ethylthio)-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2- yl)methoxy)-2-benzylmalonic acid nh ₂

Przher 145

Stage 1.

To a mixture of diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-(bis-(Ίret-butoxycarbonyl)amino)-2-chloro-9H- purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (300 mg, 349.53 µmol, 1 eq.) in DMF (3 ml) was added NaSEt (88.20 mg, 1.05 mmol, 3 eq.). The mixture was stirred at 20°C for 20 hours before partitioning it between water (15 ml) and EtOAc (15 ml). The aqueous phase was extracted using EtOAc (2x10 ml). The combined organic layer was washed with brine (10 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure to obtain crude diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3, 4-diacetoxy-5-(6-amino-2-(ethylthio)9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (310 mg) as an oil, which was used in the next step without additional cleaning.

Stage 2.

To a mixture of crude diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-amino-2-(ethylthio)9H-purin-9-yl)- 3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (310 mg) in DCM (3 ml) was added with TFA (1.5 ml, 20.26 mmol). The mixture was stirred at 20°C for 2 hours, then neutralized to pH 7 ~ 8 with saturated aq. _NaHCO3 . The reaction mixture was extracted using EtOAc (3x20 ml). The combined extract was washed with brine (15 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure to obtain crude diethyl-2-benzyl-2-(((2R,3R,4R,5R)3,4- diacetoxy-5-(6-amino-2-(ethylthio)-9H-purin-9-yl)-3-ethynyltetrahydrofuran-2yl)methoxy)malonate in the form of foam.

Stage 3.

To a mixture of crude diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-amino-2-(ethylthio)9H-purin-9-yl)- 3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (280 mg, crude) in THF (3 ml) was added saturated aq. LiOH (3 ml). The mixture was stirred at 55°C for 1 hour, after which it was left to cool to room temperature. The reaction mixture was extracted using EtOAc (3x8 ml). The aqueous phase was brought to pH 2-3 using 2M aq. HCl, then extracted with EtOAc (4x10 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated. The crude product was purified by preparative HPLC (column: YMC-Triart Prep C18 150-40 mm-7 µm; mobile phase: [water (0.225%FA)-ACN]; B%: 23% - 43%, 11 min) and dried by lyophilization to obtain 2-(((2R,3S,4R,5R)-5-(6-amino-2-(ethylthio)-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2- yl)methoxy)-2-benzylmalonic acid (4.4 mg) as a white powder.

Ή NMR (400 MHz, DMSOO δ ppm 12.59-14.32 (m, 2H), 8.30 (s, 1H), 7.33 (s, 2H), 7.00-7, 21 (m, 5H), 6.15 (s, 1H), 6.00 (d, J=6.78 Hz, 1H), 5.82 (d, J=7.53 Hz, 1H), 5, 01 (s, 1H), 4.14 (dd, J=6.40, 2.64 Hz, 1H), 3.99 (d, J=13.05 Hz, 1H), 3.83 (s, 1H ), 3.58 (s, 1H), 3.17-3.18 (m, 2H), 2.99-3.13 (m, 2H), 1.31 (t, J=7.28 Hz, 3H); LC/MS [M+H] = 544.0.

- 121 046381

Example 146. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-nurin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl) methoxy)-2-(4-(1-(2-methoxyethyl)-2-oxo-1,2-dihydropyridin-3-yl)benzyl)malonic acid

Proceeding as described in Example 11 above, but replacing chloro(methoxy)methane with 1-chloro-2methoxyethane, afforded the title compound as a white solid.

1H NMR (400 MHz, DMSO-de) δ ppm. 8.41 (s, 1H), 7.80 (s, 2H), 7.60 (dd, J=6.78, 2.01 Hz, 1H), 7.46 (dd, J=7.03, 2.01 Hz, 1H), 7.35 (d, J=8.28 Hz, 2H), 7.21 (d, J=8.28 Hz, 2H), 7.19-7.24 (m, 1H), 6.26-6.31 (m, 1H), 5.82 (d, J=7.78 Hz, 1H,) 4.86 (d, J=7.78 Hz, 1H), 4 .17 (d, J=1.76 Hz, 1H), 4.10 (t, J=5.14 Hz, 2H), 4.02 (dd, J=10.29, 4.77 Hz, 1H) , 3.78-3.85 (m, 1H), 3.54-3.64 (m, 3H), 3.29 (d, J=2.01 Hz, 2H), 3.24 (s, 3H ); LC/MS [M+H] = 669.0.

Example 147. Synthesis of 2-(((2R,3S,4R,5R)-5-(2-chloro-6-(methylamino)-9H-nurin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran- 2-yl)methoxy)-2-(4-(2-oxo-1,2-dihydropyridin-3-yl)benzyl)malonic acid

Stage 1.

To a solution of diethyl-2-(((3aR,5R,6R,6aR)-6-acetoxy-6-ethynyl-2,2-dimethyltetrahydrofuro[2,3d][1,3]dioxol-5-yl)methoxy)- 2-(4-iodobenzyl) malonate (2.0 g, 3.17 mmol, 1 eq.) in DCM (15 ml) was added with H2O (3 ml) and TFA (15 ml) at 0°C. The solution was stirred at 25°C for 16 hours before quenching with saturated aq. NaHCO ₃ (150 ml). The mixture was extracted using ethyl acetate (2x100 ml). The combined organic layer was washed with brine (200 ml), dried over Na ₂ SO ₄ , filtered and evaporated to give crude diethyl 2-(((2R,3S,4R)-3-ethynyl-3,4,5trihydroxytetrahydrofuran-2- yl)methoxy)-2-(4-iodobenzyl)malonate (1.74 g) as a yellow foam.

Stage 2.

To a solution of diethyl 2-(((2R,3S,4R)-3-ethynyl-3,4,5-trihydroxytetrahydrofuran-2-yl)methoxy)-2-(4iodobenzyl)malonate (1.74 g, 3.17 mmol, 1 eq.) in pyridine (20 ml) was added 4-DMAP (1.16 g, 9.52 mmol, 3 eq.) and Ac ₂ O (2.38 ml, 25.39 mmol, 8 eq.) . The solution was stirred at 25°C for 3 hours, then diluted with water (60 ml) and extracted using ethyl acetate (2x60 ml). The combined organic layer was washed with water (100 ml), brine (100 ml), dried over Na ₂ SO ₄ , filtered and evaporated. The crude residue was purified by silica gel flash column chromatography (10-50% ethyl acetate in petroleum ether) to give diethyl 2-(4-iodobenzyl)-2(((2R,3R,4R)-3,4,5- triacetoxy-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (1.85 g, 86% yield in two steps) as a yellow foam.

Stage 3.

To a solution of diethyl 2-(4-iodobenzyl)-2-(((2R,3R,4R)-3,4,5-triacetoxy-3-ethynyltetrahydrofuran-2yl)methoxy)malonate (1.00 g, 1.48 mmol, 1 equiv.) to MeCN (12 ml) was added 2-chloro-N-methyl-9H-purine-6amine (327 mg, 1.78 mmol, 1.2 equiv.) and BSA (916 μl, 3.71 mmol, 2.5 eq.). The suspension was stirred at 65°C for 0.5 h, then cooled to 0°C, after which TMSOTf (804 μl, 4.45 mmol, 3 eq.) was added. The resulting mixture was stirred at 65°C for 1.5 hours, then diluted with saturated aq.

- 122 046381

NaHCO ₃ solution (10 ml) and extracted using ethyl acetate (2x10 ml). The combined organic layer was washed with brine (25 ml), dried over Na ₂ SO ₄ , filtered and evaporated. The crude residue was purified by silica gel flash chromatography (10-50% ethyl acetate in petroleum ether) to give diethyl 2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(2-chloro -6-(methylαamino)9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)-2-(4-iodobenzyl)malonate (595 mg, 50% yield) as a yellow solid.

Stage 4.

To a solution of diethyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(2-chloro-6-(methylamino)-9H-purin-9-yl)-3ethynyltetrahydrofuran-2- yl)methoxy)-2-(4-iodobenzyl)malonate (592 mg, 741.88 µmol, 1 eq.) in DCM (8 ml) and added 4-DMAP (18 mg, 148.38 µmol, 0.2 eq. .), TEA (413 µl, 2.97 mmol, 4 eq.) and (Boc) ₂ O (324 mg, 1.48 mmol, 2 eq.). The solution was stirred at 20°C for 2 hours, then diluted with saturated aq. NH ₄ Cl solution (20 ml) and extracted with ethyl acetate (2x20 ml). The combined organic layer was washed with brine (40 ml), dried over Na ₂ SO ₄ , filtered and evaporated. The crude product was purified by silica gel flash column chromatography (15-50% ethyl acetate in petroleum ether) to give diethyl 2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6- ((tertbutoxycarbonyl)(methyl)amino)-2-chloro-9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)-2-(4iodobenzyl)malonate (560 mg, 84% yield) as foam.

Stage 5.

To a mixture of diethyl 2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-((tert-butoxycarbonyl)(methyl)amino)-2chloro-9H-purin-9-yl )-3-ethynyltetrahydrofurαn-2-yl)methoxy)-2-(4-iodobenzyl)malonate (660 mg, 734.89 µmol, 1 eq.) and (2-hydroxypyridin-3-yl)boronic acid (204.18 mg, 1.47 mmol, 2 eq.) in dioxane (6 ml) and H2O (2 ml) added Pd(dppf)Cl ₂ (53.77 mg, 73.49 µmol, 0.1 eq.) and K ₂ CO ₃ (304.70 mg, 2.20 mmol, 3 eq.). The yellow mixture was degassed with N2 gas for 10 minutes, then the mixture was stirred at 70°C for 2.5 hours. The mixture was diluted with water (5 ml) and extracted using ethyl acetate (3x5 ml). The combined organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and evaporated. The crude product was purified by silica gel flash column chromatography (50-100% ethyl acetate in petroleum ether) to give diethyl 2-(((2R,3R,4R,5R)-3,4-dicetoxy-5(6-( (tert-butoxycarbonyl)(methyl)amino)-2-chloro-9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)-2-(4-(2-oxo-1,2-dihydropyridine -3-yl)benzyl)malonate (146 mg) as foam.

Stage 6.

To a solution of diethyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-((mret-butoxykarbonyl)(methyl)amino)-2-chloro9H-nurin-9-yl )-3-ethynylmetragugrofuran-2-yl)methoxy)-2-(4-(2-oxo-1,2-guhydronurugun-3-yl)benzyl)malonate (145 mg, 167.58 µmol, 1 eq.) in DCM (6 ml) was added TFA (1.5 ml, 20.26 mmol, 121 eq.). The solution was stirred at 25°C for 1 hour, then neutralized with saturated aq. NaHCO ₃ solution. The mixture was extracted using ethyl acetate (3x12 ml). The combined organic layer was washed with brine (30 ml), dried over Na ₂ SO ₄ , filtered and evaporated. The crude product was purified by preparative TLC (ethyl acetate) to form diethyl 2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(2chloro-6-(methylamino)-9H-purine-9 -yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)-2-(4-(2-oxo-1,2-dihydropyridin-3-yl)benzyl)malonate (110 mg) as a solid.

Stage 7.

To a solution of diethyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(2-chloro-6-(methylamino)-9H-purin-9-yl)-3ethynyltetrahydrofuran-2- yl)methoxy)-2-(4-(2-oxo-1,2-dihydropyridin-3-yl)benzyl)malonate (105 mg, 137.23 µmol, 1 eq.) in THF (4.5 ml) was added aq. LiOH solution (1 M, 1.5 ml, 11 eq.). The mixture was stirred at 25°C for 4 hours, after which volatile organic compounds were removed under reduced pressure. Vodn. the layer was acidified to pH 6 using 1 N aq. HCl solution and then evaporated. The crude residue was purified by preparative HPLC (column: YMC-Triart Prep C18 150-40 mm-7 µm; mobile phase: [water (0.225%FA)-ACN]; B%: 20% - 40%, 10 min) and dried by lyophilization to obtain 2-(((2R,3S,4R,5R)-5-(2-chloro-6-(methylamino)-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2- yl)methoxy)-2-(4-(2-oxo-1,2-dihydropyridin-3-yl)benzyl)malonic acid (6.6 mg) as a white solid.

1H NMR (400 MHz, DMSO-d ₆ ) δ ppm. 8.44 (s, 1H), 8.23 (d, J=3.75 Hz, 1H), 7.48 (d, J=6.63 Hz, 1H), 7.39 (d, J=7 .00 Hz, 2H), 7.34 (d, J=5.63 Hz, 1H), 7.13-7.22 (m, 2H), 6.15-6.29 (m, 2H), 6 .00 (d, J=6.75 Hz, 1H), 5.82 (d, J=7.38 Hz, 1H), 4.71-4.89 (m, 1H), 4.16 (dd, J=4.94, 2.56 Hz, 1H), 3.86-4.05 (m, 1H), 3.53-3.83 (m, 1H), 3.51 (s, 1H), 3 .48-3.30 (m, 5H overlapped by the water peak), 2.90 (d, J=4.38 Hz, 3H); LC/MS [M+H] = 624.9.

- 123 046381

Example 148. Synthesis of 2-(((2R,3S,4R,5R)-5-(2-chloro-6-(methylamino)-9H-nurin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran- 2-yl)methoxy)-2-(4-(3-methyl-2-oxotetrahydropyrimidin-1(2H)-yl)benzyl)malonic acid

Priif 14Ϊ

Stage 1.

To a solution of 1 -(4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)tetrahydropyrimidin-2(III)-one (7.33 g, 16.48 mmol, 1 eq.) in DMF at 0°C was added NaH (725 mg, 60% in mineral oil, 18.13 mmol, 1.1 eq.). The mixture was stirred for 15 minutes and then CH ₃ I (4.10 mL, 65.92 mmol, 4 eq.) was added. The reaction mixture was stirred at 0 to 25°C for 16 hours, then diluted with H ₂ O (100 ml) and extracted using EtOAc (3x50 ml). The combined organic layer was washed with brine (100 ml), dried over Na ₂ SO ₄ , filtered and evaporated. The crude product was purified by silica gel flash column chromatography (0-50% EtOAc in petroleum ether) to give 1-(4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)-3methyltetrahydropyrimidin-2(III)-one (3.68 g, 48% yield) as a colorless gum.

Stage 2.

To a solution of 1-(4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)-3-methyltetrahydropyrimidin2(III)-one (3.68 g, 8.02 mmol, 1 eq.) in THF (35 ml) add TBAF in THF (1.5 M, 10.70 ml, 2 eq.) at 0°C. The reaction mixture was stirred at 25°C for 1.5 hours, then diluted with H ₂ O (20 ml) and extracted using EtOAc (3x50 ml). The combined organic layer was washed with brine (50 ml), dried over Na ₂ SO ₄ , filtered and evaporated. The crude product was purified by silica gel flash column chromatography (0-5% MeOH in DCM) to give 1-(4(hydroxymethyl)phenyl)-3-methyltetrahydropyrimidin-2(1H)-one (1.06 g, 60% yield %) as a yellow solid.

Stage 3.

To a solution of 1-(4-(hydroxymethyl)phenyl)-3-methyltetrahydropyrimidin-2(1H)-one (1.06 g, 4.81 mmol, 1 eq.) in DCM (10 ml) and DMF (0.1 ml) SOCl ₂ (698 μl, 9.62 mmol, 2 eq.) was added at 2025°C. The reaction mixture was stirred for 0.5 h and additional SOCl2 (419 µl, 5.77 mmol, 1.2 eq.) was added. The reaction mixture was stirred at 40°C for 1 hour and then evaporated. The residue was azeotroped with DCM (3x10 ml) under reduced pressure to give crude 1-(4-(chloromethyl)phenyl)-3-methyltetrahydropyrimidin-2(III)-one, which was used in the next step without further purification.

Stage 4.

To a solution of diethyl-2-(((3aR,5R,6R,6aR)-6-acetoxy-6-ethynyl-2,2-dimethyltetrahydrofuro-[2,3d][1,3]dioxol-5-yl)methoxy) malonate (1.78 g, 4.30 mmol, 1 eq.) in DMF (20 ml) was added Cs ₂ CO ₃ (4.21 g, 12.91 mmol, 3 eq.) at 25°C. The reaction mixture was stirred for 0.5 h and then crude 1-(4-(chloromethyl)phenyl)-3-methyltetrahydropyrimidin-2(III)-one (1.13 g) was added. The reaction mixture was stirred at 25°C for 16 hours, then diluted with H ₂ O (50 ml) and extracted using EtOAc (3x20 ml). The combined organic layer was washed with brine (30 ml), dried over Na ₂ SO ₄ , filtered and evaporated. The residue was purified by silica gel column chromatography (0-10% MeOH in DCM) to give diethyl 2-(((3aR,5R,6R,6aR)-6-acetoxy-6ethynyl-2,2-dimethyltetrahydrofuro[2,3D ][1,3]dioxol-5-yl)methoxy)-2-(4-(3-methyl-2-oxotetrahydropyrimidine LC-yl-benzyl-alonate) (2.63 g, 78% yield) as a brown foam.

Stage 5.

To a solution of diethyl-2-(((3aR,5R,6R,6aR)-6-acetoxy-6-ethynyl-2,2-dimethyltetrahydrofuro[2,3d][1,3]dioxol-5-yl)methoxy)- 2-(4-(3-methyl-2-oxotetrahydropyrimidin-1(2H)-yl)benzyl)malonate (2.62

- 124 046381 g, 4.25 mmol, 1 eq.) in DCM (25 ml) at 0°C was added TFA (25 ml, 337.65 mmol, 79 eq.) and H ₂ O (2.5 ml, 138 .77 mmol, 33 eq.). The reaction mixture was stirred at 20-25°C for 16 hours, after which it was evaporated under reduced pressure. The residue was azeotroped with DCM (3x20 ml) under reduced pressure to give crude diethyl 2-(((2R,3S,4R)-3-acetoxy-3-ethynyl-4,5dihydroxytetrahydrofuran-2-yl)methoxy)-2 -(4-(3-methyl-2-oxotetrahydropyrimidin-1(2H)-yl)benzyl)malonate (2.57 g), which was used in the next step without further purification.

Stage 6.

To a solution of crude diethyl-2-(((2R,3S,4R)-3-acetoxy-3-ethynyl-4,5-dihydroxytetrahydrofuran-2-yl)methoxy)-2-(4-(3-methyl-2- Ac ₂ O (2.39 mL, 25.50 mmol, 6 eq.) was added to oxotetrahydropyrimidin-1(2H)-yl)benzyl)malonate (2.57 g) in DCM (25 mL) at 20-25°C. 4-DMAP (51.92 mg, 425.00 µmol, 0.1 equiv.) and pyridine (2.74 ml, 34.00 mmol, 8 equiv.). The reaction mixture was stirred at 25°C for 16 hours, after which it was evaporated under reduced pressure. The residue was redissolved in EtOAc (50 ml), washed with 1 N aq. HCl (40 ml), 10% aq. Cu ₂ SO ₄ (40 ml), saturated aq. NaHCO ₃ (40 ml) and brine (40 ml), dried over Na ₂ SO ₄ , filtered and evaporated to form crude diethyl-2-(4-(3-methyl-2-oxotetrahydropyrimidin-1(2H)-yl) benzyl)-2-(((2R,3R,4R)-3,4,5triacetoxy-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (2.62 g, 53% yield in two steps), which was used in the following stage without additional purification.

Stage 7.

BSA (468 µL, 1 .89 mmol, 2.5 eq.) at 20-25°C. The reaction mixture was stirred at 65°C for 0.5 h and then cooled to 25°C. To this mixture was added crude diethyl 2-(4-(3-methyl-2-oxotetrahydropyrimidin-1(2H)-yl)benzyl)-2-(((2R,3R,4R)-3,4,5-triacetoxy-3 -ethynyltetrahydrofuran-2-yl)methoxy)malonate (500 mg, 756.81 µmol, 1 eq.) in MeCN (2.5 ml) and TMSOTf (205 µl, 1.14 mmol, 1.5 eq.) and stirred at 65°C for 5 hours before quenching with saturated aq. NaHCO ₃ (10 ml). The mixture was then extracted using EtOAc (3x10 ml). The combined organic layer was washed with brine (20 ml), dried over anhydrous Na2SO4, filtered and evaporated. The crude product was purified by silica gel column chromatography (010% MeOH in DCM) to give diethyl 2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(2-chloro-6-( methylamino)9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)-2-(4-(3-methyl-2-oxotetrahydropyrimidin1(2H)-yl)benzyl)malonate (530 mg, yield 60 %) in the form of foam.

Stage 8.

To a solution of diethyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(2-chloro-6-(methylamino)-9H-purin-9-yl)-3ethynyltetrahydrofuran-2- yl)methoxy)-2-(4-(3-methyl-2-oxotetrahydropyrimidin-1(2H)-yl)benzyl)malonate (540 mg, 688.59 µmol, 1 eq.) in THF (6 ml) added LiOH .H ₂ O (288.96 mg, 6.89 mmol, 10 eq.) in H2O (3 ml) at 25°C. The reaction mixture was stirred at 40°C for 2 h before removing volatile organic compounds under reduced pressure. The aqueous phase was acidified to pH 2-3 using 1 N aq. HCl and then evaporated. The crude product was purified by preparative HPLC (column: YMC-Actus Triart C18 150-30 mm-5 µm; mobile phase: [water (0.225%FA)-ACN]; B%: 23%-43%, 10 min) and dried by lyophilization to obtain 2-(((2R,3S,4R,5R)-5-(2-chloro-6(methylamino)-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2 -yl)methoxy)-2-(4-(3-methyl-2oxotetrahydropyrimidin-1(2H)-yl)benzyl)malonic acid (77.6 mg, 17% yield) as a white solid.

1H NMR (400 MHz, DMSO-d6) δ ppm. 13.00-13.93 (s, 2H), 8.37 (s, 1H), 8.23 (d, J=5.01 Hz, 1H), 7.11 (d, J=8.31 Hz , 2H), 6.90 (d, J=8.19 Hz, 2H), 6.15 (s, 1H), 5.98 (s, 1H), 5.81 (d, J=7.58 Hz , 1H), 4.82 (s, 1H), 4.15 (dd, J=4.34, 3.00 Hz, 1H), 3.96 (m, 1H), 3.78 (m, 1H) , 3.53 (s, 1H), 3.43-3.52 (m, 2H), 3.23 (m, 2H), 3.203.10 (m, 4H overlapped with aqueous solvent peak), 2.91 (m , 2H), 2.81 (s, 3H), 1.95 (m, 2H); LC/MS [M+H] = 644.1.

Example 149. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl) methoxy)-2-(4-(3-methyl-2-oxotetrahydropyrimidin-1(2H)-yl)benzyl)malonic acid

Example 149

Proceeding as described in Example 148 above, but replacing 2-chloro-N-methyl-9H-purin-6-amine with 2chloro-9H-purin-6-amine, afforded the title compound as a white solid.

1H NMR (400 MHz, CD3OD) δ ppm. 8.21 (s, 1H), 7.26 (m, J=8.28 Hz, 2H), 6.96 (m, J=8.28 Hz, 2H), 5.97 (d, J=7 .53 Hz, 1H), 4.70-4.83 (m, 1H), 4.27 (t, J=2.76 Hz, 1H), 3.99-4.07 (m, 2H), 3 .32-3.52 (m, 6H), 3.05 (s, 1H), 2.87 (s, 3H), 2.00 (quin, J=5.83 Hz, 2H); LC/MS [M+H] = 630.2.

- 125 046381

Example 150. Synthesis of 2-(((2R,3S,4R,5R)-5-(2-chloro-6-((cyclopropylmethyl)amino)-9H-purin-9-yl)-3ethynyl-3,4-digvdroxytetrahydrofuran -2-yl)methoxy)-2-(4-(3-methyl-2-oxotetravdropyrimvdin-1(2H)yl)benzyl)malonic acid

Proceeding as described in Example 148 above, but replacing 2-chloro-N-methyl-9H-purin-6-amine with 2chloro-N-(cyclopropylmethyl)-9H-purin-6-amine, the title compound was obtained as white solid.

¹ H NMR (400 MHz, CD3OD) δ ppm. 8.12 (s, 1H), 7.27 (d, J=8.28 Hz, 2H), 7.00 (d, J=7.68 Hz, 2H), 5.96 (d, J=7 .53 Hz, 1H), 4.70 (d, J=7.53 Hz, 1H), 4.26 (t, J=2.89 Hz, 1H), 4.03 (br. s, 2H), 3.33-3.54 (m, 8H), 3.05 (s, 1H), 2.88 (s, 3H), 1.97-2.04 (m, 2H), 1.11-1, 20 (m, 1H), 0.53-0.59 (m, 2H), 0.34 (m, 2H); LC/MS [M+H] = 684.3.

Example 151. Synthesis of 2-(((2R,3S,4R,5R)-5-(2-chloro-6-(isopropylamino)-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran^-yl Schetoxy ^-i-i-methyl^-oxotetrahydropyrimidine I^N-yl^ensyl)malonic acid

Proceeding as described in Example 148 above, but replacing 2-chloro-N-methyl-9H-purin-6-amine with 2chloro-N-isopropyl-9H-purin-6-amine, afforded the title compound as a white solid. substances.

¹ H NMR (400 MHz, CD3OD) δ ppm. 8.08 (s, 1H), 7.28 (d, J=8.28 Hz, 2H), 7.00 (d, J=8.53 Hz, 2H), 5.95 (d, J=7 .53 Hz, 1H), 4.66-4.80 (m, 1H), 4.32-4.48 (m, 1H), 4.25 (t, J=2.89 Hz, 1H), 4 .00-4.08 (m, 2H), 3.34-3.53 (m, 6H), 3.05 (s, 1H), 2.88 (s, 3H), 1.94-2.06 (m, 2H), 1.24-1.35 (m, 6H); LC/MS [M+H] = 672.1.

Example 152. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl) methoxy)-2-(4-(2-oxo-3-propyltetrahydropyrimidin-1(2H)-yl)benzyl)malonic acid

Proceeding as described in Example 148 above, but replacing MeI and 2-chloro-N-methyl-9H-purin-6-amine with 1-bromopropane and 2-chloro-9H-purin-6-amine, gave the title compound as a white solid.

¹ H NMR (400 MHz, CD3OD) δ ppm. 8.15 (s, 1H), 7.28 (d, J=8.4 Hz, 2H), 6.99 (d, J=8.4 Hz, 2H), 5.97 (d, J=7 .5 Hz, 1H), 4.75 (d, J=7.6 Hz, 1H), 4.27 (s, 1H), 3.99-4.11 (m, 2H), 3.46-3 .54 (m, 2H), 3.35-3.44 (m, 4H), 3.16-3.27 (m, 2H), 3.05 (s, 1H), 1.97-2.03 (m, 2H), 1.50-1.58 (m, 2H), 0.88 (t, J=7.4 Hz, 3H); LC/MS [M+H] = 658.1.

- 126 046381

example 153 yl)methoxy)-2-(4-(1-(2-hydroxyethyl)-2-oxo-1,2-dihydropyridin-3yl)benzyl)malonic acid

- g--*· _ J..

· - \ _-G .1 _ cheg

K,CO,,t«F

I

Stage 1.

To a solution of diethyl 2-(((3aR,5R,6R,6aR)-6-acetoxy-6-ethynyl-2,2-dimethyltetrahydrofuro[2,3d][1,3]dioxol-5-yl)methoxy)malonate (4.13 g, 9.97 mmol, 1 eq.) in DCM (40 ml) at 20°C was added TFA (40 ml, 540.24 mmol, 54 eq.) and H ₂ O (4 ml, 222, 03 mmol, 22 eq.). The mixture was stirred at 20°C for 15 hours, then quenched with saturated aq. NaHCO ₃ solution (200 ml) and extracted using EtOAc (5x50 ml). The combined organic layer was washed with brine (200 ml), dried over Na ₂ SO ₄ , filtered and evaporated to give crude diethyl 2-(((2R,3S,4R)-3-acetoxy3-ethynyl-4,5-dihydroxytetrahydrofuran-2 -yl)methoxy)malonate (4.14 g) as a light yellow syrup, which was directly used in the next step.

To a solution of the above crude product (4.14 g, 12.46 mmol, 1 eq.) in pyridine (40 ml) was added Ac ₂ O (9.33 ml, 99.67 mmol, 8 eq.) and 4-DMAP (3.81 g, 31.15 mmol, 2.5 eq.). The mixture was stirred at 20°C for 16 hours, then quenched with water (150 ml) and the resulting solution was extracted using EtOAc (4 x 50 ml). The combined organic layer was washed with 0.5 N aq. HCl (120 ml), water (2x100 ml) and brine (100 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated. The crude residue was purified by silica gel flash column chromatography (30-50% EtOAc in petroleum ether) to give diethyl 2-(((2R,3R,4R)-3,4,5-triacetoxy-3ethynyltetrahydrofuran-2-yl )methoxy)malonate (2.60 g) as syrup.

Stage 2.

To a solution of diethyl 2-(((2R,3R,4R)-3,4,5-triacetoxy-3-ethynyltetrahydrofuran-2yl)methoxy)malonate (1.2 g, 2.62 mmol, 1 eq.) and 2 -chloro-N-isopropyl-9H-purin-6-amine (665 mg, 3.14 mmol, 1.2 eq.) in MeCN (15 ml) was added BSA (1.62 ml, 6.54 mmol, 2. 5 eq.). The suspension was stirred at 65°C for 0.5 h, then cooled to 0°C. TMSOTf (1.45 g, 6.54 mmol, 1.18 mL, 2.5 eq.) was added to this solution. The mixture was then stirred at 65°C for 2.5 hours, after which it was quenched with saturated aq. NaHCO3 solution (50 ml) and the resulting solution was extracted using EtOAc (4x30 ml). The combined organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and evaporated. The residue was purified by silica gel flash column chromatography (30-50% EtOA in petroleum ether) to give diethyl 2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(2-chloro -6(isopropylamino)-9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (814 mg, 51% yield).

Stage 3.

To a solution of diethyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(2-chloro-6-(isopropylamino)-9H-purin-9yl)-3-ethynyltetrahydrofuran-2- yl)methoxy)malonate (120 mg, 197 µmol, 1 eq.) in DMF (1 ml) and added K ₂ CO ₃ (81.56 mg, 590.15 µmol, 3 eq.) and 3-(4-( bromomethyl)phenyl)-1-(2-((tert-butyldiphenyl^xy^tyl^iridine^Shuon (161 mg, 295 µmol, 1.5 eq.). The mixture was stirred at 20°C for 1.5 h, then diluted water (10 ml) and extracted using EtOAc (4x5 ml).The combined organic layer was washed with water (2x30 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated. The residue was purified using silica gel flash column chromatography (3060% EtOA in petroleum ether) to form diethyl-2-(4-(1-(2-((tert-butyldiphenylsilyl)oxy)ethyl)-2-oxo-1,2-dihydropyridin-3-yl)benzyl)-2- (((2R,3R,4R,5R)-3,4-diacetoxy-5-(2chloro-6-(isopropylamino)-9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate ( 154 mg) as a colorless oil.

Stage 4.

To a solution of diethyl-2-(4-( 1 -(2-((tert-butyldiphenylsilyl)oxy)ethyl)-2-oxo-1,2-dihydropyridin-3 yl)benzyl)-2-(((2R,3R ,4R,5R)-3,4-diacetoxy-5-(2-chloro-6-(isopropylamino)-9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (150 mg, 139 µmol, 1 eq.) in THF (1 ml) was added TBAF (1 M, 209 µl, 1.5 eq.) and AcOH (5.98 µl, 104.59 µmol, 0.75 eq.) at 0°C . The mixture was stirred at 20°C in

- 127 046381 for 16 hours, then diluted with water (5 ml) and the resulting solution was extracted using EtOAc (3x5 ml). The combined organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and evaporated. The residue was purified by preparative TLC (petroleum ether: EtOAc=1:4) to give diethyl 2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(2-chloro-6-(isopropylamino) -9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)-2-(4-(1-(2-hydroxyethyl)-2-oxo-1,2-dihydropyridin-3-yl)benzyl )malonate (51 mg, 32% yield) as a white solid.

Stage 5.

To a solution of diethyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(2-chloro-6-(isopropylamino)-9H-purin-9yl)-3-ethynyltetrahydrofuran-2- yl)methoxy)-2-(4-( 1 -(2-hydroxyethyl)-2-oxo-1,2-dihydropyridin-3 yl)benzyl)malonate (50 mg, 60 µmol, 1 eq.) in THF (0 .5 ml) added saturated aq. LiOH (2.51 mg, 60 µmol, 0.5 ml, 1 eq.). The mixture was stirred at 20°C for 2.5 hours, after which volatile organic compounds were removed under reduced pressure. The resulting aq. the layer was acidified to pH 2 using 2 N aq. HCl solution and then evaporated. The residue was further purified by preparative HPLC (column: YMC-Triart Prep C18 150-40 mm-7 µm; mobile phase: [water (0.225%FA)-ACN]; B%: 28%-48%, 10 min) with producing 2-(((2R,3S,4R,5R)-5-(2-^op-6(isopropylαamino)-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxymetrahydrofuran-2-yl )methoxy)-2-(4-(1-(2hydroxyethyl)-2-oxo-1,2-dihydropyridin-3-yl)benzyl)malonic acid (10.8 mg, 26% yield) as a white solid.

1H NMR (400 MHz, CD ₃ OD) δ ppm. 8.08 (s, 1H), 7.55 (dd, J=6.7, 1.9 Hz, 1H), 7.21-7.42 (m, 5H), 6.34 (t, J= 6.8 Hz, 1H), 5.95 (d, J=7.3 Hz, 1H), 4.74 (d, J=7.3 Hz, 1H), 4.24-4.39 (m, 2H), 4.00-4.14 (m, 4H), 3.763.87 (m, 2H), 3.40-3.54 (m, 2H), 3.06 (s, 1H), 1.24 (dd, J=6.4, 2.9 Hz, 6H); LC/MS [M+H] = 697.0.

Examples 154 and 155. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2- yl)methoxy)-2-(4-(4-methyl-2-oxopiperazin-1-yl)benzyl)malonic acid and 2-(((2R,3S,4R,5R)-5-(6-αmino-2 -chloro-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxymetrahydrofuran2-yl)methoxy)-2-(4-((2-((carboxymethyl)(methyl)amino)ethyl)amino)benzyl )malonic acid

‘ - L;.? to 1:- L;,:.·. <

Stage 1.

To a mixture of tert-butyl-3-oxopiperazine-1-carboxylate (8.7 g, 43.45 mmol, 1.2 eq.) and (4-iodophenyl)methanol (8.5 g, 36.32 mmol, 1 eq. .) in DMF (10 ml) and dioxane (90 ml) was added CuI (1.03 g, 5.43 mmol, 0.15 eq.), 2-(hydroxymethyl)-2-methylpropane-1,3-diol ( 653 mg, 5.43 mmol, 0.15 eq.) and Cs ₂ CO3 (35.39 g, 108.62 mmol, 3 eq.). The mixture was stirred at 110°C under N2 for 14 hours and then cooled. The inorganic solid was filtered off and the filtrate was evaporated in vacuo. The residue was diluted with water (50 ml) and extracted using ethyl acetate (3x50 ml). The combined organic layer was washed with brine (50 ml), dried with Na ₂ SO ₄ , filtered and evaporated. The crude residue was purified by silica gel flash chromatography (0-10% EtOAc in petroleum ether) to give tert-butyl 4-(4-(hydroxymethyl)phenyl)-3oxopiperazine-1-carboxylate (6.1 g, 55% yield %) as a white solid.

Stage 2.

To a solution of tert-butyl 4-(4-(hydroxymethyl)phenyl)-3-oxopiperazine-1-carboxylate (2 g, 6.53 mmol, 1 eq.) in DCM (10 mL) was added TFA (5.00 mL , 67.53 mmol, 10.34 eq.) at 0°C. The mixture was stirred at 25°C for 3 hours, then it was evaporated. The residue was diluted with water (20 ml) and extracted with a mixture of DCM and MeOH (50:1=v:v, 2x20 ml). The combined organic layer was evaporated to give crude 1-(4-(hydroxymethyl)phenyl)piperazin-2-one (2.45 g) as a colorless oil.

Stage Z.

HCHO (720 μL, 26.12 mmol, 2.2 eq.), AcOH (5 ml, 87.42 mmol, 7.4 eq.).

- 128 046381

The mixture was stirred at 25°C under N ₂ for 15 hours before NaBH ₃ CN (2.05 g, 32.65 mmol, 2.75 eq.) was added and the resulting mixture was stirred for 2 hours. The reaction mixture was evaporated in vacuo . The residue was purified by silica gel flash column chromatography (10-40% MeOH in EtOAc) to give 1-(4-(hydroxymethyl)phenyl)-4-methylpiperazin-2-one (1.23 g) as a colorless oil.

Stage 4.

To a solution of 1-(4-(hydroxymethyl)phenyl)-4-methylpiperazin-2-one (1.23 g, 5.58 mmol, 1 eq.) in DCM (2 mL) was added DMF (0.2 mL) and SOCl ₂ (810 µl, 11.17 mmol, 2 eq.). The mixture was stirred at 25°C for 30 min to obtain a white suspension, then it was evaporated under reduced pressure to obtain crude 1-(4-(chloromethyl)phenyl)-4-methylpiperazin-2-one (1.27 g, 95% yield %) as a white solid, which was directly used in the next step.

Stage 5.

To a solution of diethyl-2-(((3aR,5R,6R,6aR)-6-acetoxy-6-ethynyl-2,2-dimethyltetrahydrofuro-[2,3d][1,3]dioxol-5-yl)methoxy) malonate (1.4 g, 3.38 mmol, 1 eq.) in DMF (15 ml) was added Cs ₂ CO ₃ (3.30 g, 10.14 mmol, 3 eq.) and crude 1-(4-( chloromethyl)phenyl)-4-methylpiperazin-2-one (1.21 g, 5.07 mmol, 1.5 eq.). The mixture was stirred at 25°C for 1 hour, then filtered and the filtrate was evaporated. The crude residue was purified by silica gel column chromatography (0-50% MeOH in EtOAc) to give diethyl 2-(((3aR,5R,6R,6aR)-6-acetoxy-6-ethynyl-2,2-dimethyltetrahydrofuro [2,3d][1,3]dioxol-5-yl)methoxy)-2-(4-(4-methyl-2-oxoniperazin-1-yl)benzyl)malonate (1.02 g).

Stage 6.

To a solution of diethyl-2-(((3aR,5R,6R,6aR)-6-acetoxy-6-ethynyl-2,2-dimethyltetrahydrofuro[2,3d][1,3]dioxol-5-yl)methoxy)- 2-(4-(4-methyl-2-oxopiperazin-1-yl)benzyl)malonate (1.02 g, 1.65 mmol, 1 eq.) in DCM (5 ml) and water (1 ml, 55, 51 mmol, 34 eq.) TFA (4.99 mL, 67.36 mmol, 41 eq.) was added. The mixture was stirred at 25°C for 20 hours, then it was evaporated to obtain crude diethyl-2(((2R,3S,4R)-3-ethynyl-3,4,5-trihydroxytetrahydrofuran-2-yl)methoxy)-2 -(4-(4-methyl-2-oxopiperazin1-yl)benzyl)malonate (1.13 g) as an oil.

To a solution of diethyl-2-[[(2R,3S,4R)-3-ethynyl-3,4,5-trihydroxytetrahydrofuran-2-yl]methoxy]-2-[[4(4-methyl-2-oxo-piperazine -1-yl)phenyl]methyl]propanedioate (1.13 g, 2.11 mmol, 1 eq.) in DCM (10 ml) was added 4-DMAP (25.78 mg, 211.00 µmol, 0.1 eq. .), pyridine (1.07 ml, 13.2 mmol, 6.3 eq.) and Ac ₂ O (927 μl, 9.9 mmol, 4.7 eq.). The mixture was stirred at 25°C for 16 hours and then evaporated. The residue was diluted with EtOAc (20 ml) and 1 N aq. HCl (10 ml). The organic layer was separated and the aqueous layer was extracted using EtOAc (4x20 ml). The combined organic layer was washed with water (50 ml), brine (50 ml) and dried over anhydrous Na ₂ SO ₄ , filtered and evaporated to give crude diethyl 2-(4-(4-methyl-2-oxopiperazin-1-yl )benzyl)-2-(((2R,3R,4R)-3,4,5-triacetoxy-3ethynyltetrahydrofuran-2-yl)methoxy)malonate (674 mg) as syrup.

Stage 7.

To a solution of 2-chloro-9H-nurin-6-amine (76 mg, 450.46 µmol, 1.2 eq.) in dichloroethane (3 ml) was added BSA (204 µl, 825.84 µmol, 2.2 eq. ). The mixture was stirred at 65°C for 0.5 h, then cooled to 0°C and diethyl 2-(4-(4-methyl-2-oxopiperazin-1-yl)benzyl)-2-(((2R ,3R,4R)-3,4,5triacetoxy-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (248 mg, 375.38 µmol, 1 eq.) in dichloroethane (1 ml) and added TMSOTf (102 µl, 563 .07 µmol, 1.5 eq.) The mixture was stirred at 65°C for 2 hours under N ₂ before quenching with saturated aq. NaHCO ₃ solution (15 ml) and extracted using EtOAc (4x20 ml). The combined organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and evaporated. The residue was purified by silica gel flash column chromatography (0-30% MeOH in EtOAc) to give diethyl 2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-amino2 -chloro-9H-nurin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)-2-(4-(4-methyl-2-oxopiperazin-1yl)benzyl)malonate (62 mg) as a white solid substances.

Stage 8.

To a solution of diethyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-amino-2-chloro-9H-nurin-9-yl)-3ethynyltetrahydrofuran-2-yl) methoxy)-2-(4-(4-methyl-2-oxopiperazin-1-yl)benzyl)malonate (171 mg, 222.02 µmol, 1 eq.) in THF (2 ml) was added aq. LiOH (5.32 mg, 222.02 µmol, 2 ml, 1 eq.). The mixture was stirred at 25°C for 2.5 hours, then acidified to pH 2-3 using 2 N aq. HCl. The mixture was evaporated under reduced pressure. The crude residue was further purified by preparative HPLC to give 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3, 4dihydroxytetrahydrofuran-2-yl)methoxy)-2-(4-(4-methyl-2-oxopiperazin-1-yl)benzyl)malonic acid (3.3 mg) as an off-white solid and 2-(((2R ,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl)methoxy)-2-(4-(( 2-((carboxymethyl)(methyl)amino)ethyl)amino)benzyl)malonic acid (3.7 mg) as a white solid.

2-(((2R,3S,4R,5R)-5-(6-Amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2yl)methoxy)-2 -(4-(4-methyl-2-oxopiperazin-1-yl)benzyl)malonic acid: ¹ H NMR (400 MHz, CD3OD) δ ppm. 8.35 (s, 1H), 7.35 (d, J=8.3 Hz, 2H), 6.97 (d, J=8.5 Hz, 2H), 5.95 (d, J=5 .5 Hz, 1H), 4.70-4.79 (m, 1H),

- 129 046381

4.33 (dd, J=9.0, 3.3 Hz, 1H), 3.96-4.14 (m, 2H), 3.54-3.66 (m, 2H), 3.49 ( s, 2H), 3.35 (s, 1H), 3.21 (s, 2H), 2.83 (t, J=5.4 Hz, 2H), 2.32-2.48 (m, 3H ), 1.89 (s, 3H); LC/MS [M+H] = 630.2.

2-(((2R,3S,4R,5R)-5-(6-Amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2yl)methoxy)-2 -(4-((2-((carboxymethyl)(methyl)amino)ethyl)amino)benzyl)malonic acid: 1H NMR (400 MHz, CD ₃ OD) δ ppm. 8.56 (s, 1H), 7.03 (d, J=8.5 Hz, 2H), 6.49 (d, J=8.3 Hz, 2H), 6.01 (d, J=6 .0 Hz, 1H), 4.70 (d, J=6.0 Hz, 1H), 4.34 (br.d, J=4.3 Hz, 1H), 3.94 (dd, J=9 ,7, 6.9 Hz, 1H), 3.83 (dd, J=9.9, 2.4 Hz, 1H), 3.59 (s, 2H), 3.34-3.42 (m, 2H), 3.22-3.27 (m, 2H), 3.20 (s, 2H), 3.04 (s, 1H), 2.83 (s, 3H); LC/MS [M+H] = 649.3.

Example 156. Synthesis of 2-(((2R,3S,4R,5R)-5-(5-chloro-7-((2,4-dimethoxybenzyl)amino)-3H-imidazo[4,5b]pyridine-3- yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)-2-(4-(2-oxotetrahydropyrimidin-1 CN-yl-benzyl) acid

Stage 1.

To a mixture of 5,7-dichloro-3H-imidazo[4,5-b]pyridine (317 mg, 1.69 mmol, 1 eq.) d MeCN (6 ml) was added BSA (1.04 ml, 4.22 mmol , 2.5 eq.). The mixture was stirred at 65°C under N2 for 0.5 hours, then cooled to 0°C. Diethyld-C-C-oxotetrahydropyrimidinNCl-yl-ensyl)2-(((2R,3R,4R)-3,4,5-triacetoxy-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (1.20) was added to the mixture g, 1.85 mmol, 1.1 equiv.) and TMSOTf (913.99 μl, 5.06 mmol, 3 equiv.). The mixture was stirred at 65°C under N2 for 6 hours, then it was quenched with NaHCO ₃ (15 ml). The reaction mixture was extracted using EtOAc (3x15 ml). The combined organic layer was washed with brine (2x5 ml), dried (Na ₂ SO ₄ ), filtered under reduced pressure. The residue was purified by silica gel flash column chromatography (0-100% EtOAc in petroleum ether) to give diethyl 2(((2R,3R,4R,5R)-3,4-diacetoxy-5-(5,7-dichloro-3H -imidazo[4,5-b]pyridin-3-yl)-3-ethynyltetrahydrofurand-yl Schetoxy^-C-C-oxotetrahydropyrimidin NAC-yl^ensyl schalonate (752 mg) in the form of foam.

Stage 2.

A solution of diethyl 2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(5,7-dichloro-3H-imidazo[4,5-b]pyridin-3-yl)-3ethynyltetrahydrofurand- yl-N-C-C-oxotetrahydropyrimidine NAC-yl-enzyl Chalonate (752 mg, 970.82 µmol, 1 eq.) and 2,4-dimethoxybenzylamine (292 µl, 1.94 mmol, 2 eq.) and DIEA (507 µl, 2.91 mmol, 3 eq.) was placed in a microwave reaction tube in NMP (4 ml).The sealed tube was irradiated in a microwave reactor at 130°C for 2 hours, then diluted with H2O (10 ml) and extracted using EtOAc (3x15 ml). The combined organic layer was washed with brine (10 ml), dried over Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The crude residue was purified using silica gel flash column chromatography (0-100% EtOAc in petroleum ether) to give diethyl-2(((2R,3S,4R,5R)-5-(5-chloro-7-((2,4-dimethoxybenzyl)amino)-3H-imidazo[4,5-b]pyridine -3-yl)-3-ethynyl3,4-dihydroxytetrahydrofuran-2-yl)methoxy)-2-(4-(2-oxotetrahydropyrimidin-1(2H)-yl)benzyl)malonate (249 mg, 26% yield) in in the form of foam.

Stage 3.

To a mixture of diethyl-2-(((2R,3S,4R,5R)-5-(5-chloro-7-((2,4-dimethoxybenzyl)amino)-3H-imidazo[4,5b]pyridine-3- yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)-2-(4-(2-oxotetrahydropyrimidine NAC-yl-ensylchalonate (239 mg, 291.01 µmol, 1 equiv.) in THF (4 ml) and H2O (3 ml) LiOH (69.69 mg, 2.91 mmol, 10 eq.) was added.The mixture was stirred at 25°C for 20 hours, then acidified to pH 6-7 with 2 N aq. HCl and evaporated under reduced pressure.The crude residue was purified by preparative HPLC (column: Phenomenex Gemini-NX 150-30 mm5 µm; mobile phase: [water (0.1%TFA)-ACN]; B%: 25% - 65%, 10 min) and dried by lyophilization to obtain 2-(((2R,3S,4R,5R)-5-(5-chloro-7-((2,4-dimethoxybenzyl)amino)-3H-imidazo[4,5- b]pyridin-3-yl)3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)-2-(4-(2-oxotetrahydropyrimidin-1(2H)yl)benzyl)malonic acid (9.5 mg ) as a white solid.

¹ H NMR (400 MHz, CD3OD) δ ppm. 8.23 (s, 1H), 7.27 (d, J=8.44 Hz, 2H), 7.21 (d, J=8.31 Hz, 1H), 7.00 (d, J=8 .44 Hz, 2H), 6.58 (d, J=2.32 Hz, 1H), 6.46-6.51 (m, 2H), 6.04 (d, J=7.34 Hz, 1H ), 4.73 (d, J=7.34 Hz, 1H),

- 130 046381

4.43 (s, 2H), 4.27 (t, J=3.06 Hz, 1H), 4.01 (d, J=2.93 Hz, 2H), 3.87 (s, 3H), 3.78 (s, 3H), 3.37-3.47 (m, 2H), 3.31-3.37 (m, 2H), 3.22 (t, J=5.87 Hz, 2H) , 3.03 (s, 1H), 1.78-1.85 (m, 2H); LC/MS [M+H] = 765.1.

Example 157. Synthesis of 2-(((2R,3S,4R,5R)-5-(7-amino-5-chloro-3H-imidazo[4,5-b]pyridin-3-yl)-3ethynyl-3, 4-dihydroxytetrahydrofuran-2-yl)methoxy)-2-(4-(2-oxotetrahydropyrimidin-1(2H)-yl)benzyl)malonic acid 'Λ J dscht'. ;ί...../ O U] , at DDU”! ¹ — % 'Lch/'' ⁵ '

NN, NN u ’· Priyr 157

To a mixture of 2-(((2R,3S,4R,5R)-5-(5-chloro-7-((2,4-dimethoxybenzyl)amino)-3H-imidazo[4,5-b]pyridin-3yl) -3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)-2-(4-(2-oxotetrahydropyrimidin-1(2H)yl)benzyl)malonic acid (160 mg, 209.11 µmol, 1 eq. ) in DCM (3 ml) was added TFA (1 ml, 13.51 mmol, 64.59 eq.). The mixture was stirred at 25°C for 2 hours and then evaporated. The residue was purified by preparative HPLC (column: YMC-Actus Triart C18 150-30 mm-5 µm; mobile phase: [water (0.225%FA)-ACN]; B%: 20% - 40%, 10 min) and dried lyophilization to obtain 2-(((2R,3S,4R,5R)5 -(7-amino-5-chloro-3H-imidazo[4,5-b]pyridin-3-yl)-3-ethynyl-3, 4-Dihydroxytetrahydrofuran-2yl^ethoxy^-^-C-oxotetrahydropyrimidine DSCH-yl^ensyl^alonic acid (5.4 mg) as a white solid.

¹ H NMR (400 MHz, CD3OD) δ ppm. 8.34 (s, 1H), 7.28 (d, J=8.31 Hz, 2H), 7.02 (d, J=8.44 Hz, 2H), 6.49 (s, 1H), 6.07 (d, J=7.21 Hz, 1H), 4.78 (d, J=7.21 Hz, 1H), 4.30 (t, J=3.18 Hz, 1H), 3, 93-4.08 (m, 2H), 3.45-3.54 (m, 2H), 3.35-3.49 (m, 4H), 3.04 (s, 1H), 1.94- 2.00 (m, 2H); LC/MS [M+H] = 615.1.

Example 158. Synthesis of 2-((III-pyrazol-5-yl)methyl)-2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9yl) -3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid g Ί N . A _h ⁴ . > n t . 1 TF- G U I. III d-;'' ¹ '7. .A ;-A ¹ / ^{j3r hF} - 77' _Λ

Przher 15V Sy-m

Proceeding as described in Example 1 above, but replacing benzyl bromide with tert-butyl 5-(bromomethyl)N-pyrazole-1-carboxylate, afforded the title compound as a white solid.

1H NMR (400 MHz, CD3OD) δ ppm. 8.46 (s, 1H), 7.32 (s, 1H), 6.17 (s, 1H), 6.02 (d, J=7.13 Hz, 1H), 4.83 (s, 1H ), 4.34 (s, 1H), 3.98-4.11 (m, 2H), 3.43-3.54 (m, 2H), 2.95 (s, 1H); LC/MS [M+H] = 508.1.

Example 159. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(1-tosyl-1H-pyrazol-4-yl)-9H-purin-9yl )-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

Priif 159

Stage 1.

To a mixture of diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(2,6-dichloro-9H-purin-9-yl)-3ethynyltetrahydrofuran-2- yl)methoxy)malonate (1.3 g, 1.92 mmol, 1 eq.) and 4-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)-1-tosyl-III -pyrazole (669 mg, 1.92 mmol, 1 eq.) in dioxane (10 ml) and H2O (3 ml) under N2 atmosphere was added Pd(PPh ₃ ) ₄ (222 mg, 192 µmol, 0.1 eq.) and Cs ₂ CO ₃ (1.88 g, 5.76 mmol, 3 eq.). The mixture was stirred at 100°C for 3 hours, then filtered and the filtrate was evaporated under reduced pressure. The crude residue was purified by silica gel flash column chromatography (0-50% EtOA in petroleum ether) to give diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3,4diacetoxy-5-( 2-chloro-6-(1-tosyl-1H-pyrazol-4-yl)-9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (170 mg) as foam.

Stage 2.

To a solution of diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(2-chloro-6-(1-tosyl-1H-pyrazol-4yl)-9H -purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (50 mg, 58 µmol, 1 eq.) in THF (1 ml) was added 1 M aq. LiOH solution (1 µl, 18 eq.). The mixture was stirred at 25°C for 14 hours and then diluted with EtOAc (10 ml) and water (10 ml). The aqueous phase was brought to pH 2-3 using 2M aq. HCl solution, extracted with EtOAc (2x40 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The crude residue was purified using preparative HPLC (column: YMC-Triart Prep C18 150-40 mm-7 µm; mobile phase: [water (0.225%FA) - 131 046381

ACN]; B%: 40% - 60%, 10 min) to give 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(1-tosyl-1Hpyrazole-4- yl)-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid (2.0 mg) as a white solid.

1H NMR (400 MHz, CD3OD) δ ppm. 8.98 (s, 1H), 8.69 (s, 2H) 7.47 (d, J=8.25 Hz, 2H), 7.30 (d, J=6.88 Hz, 2H), 7 .07-7.19 (m, 4H), 6.92 (d, J=8.00 Hz, 2H), 6.24 (d, J=7.63 Hz, 1H), 5.85 (d, J=7.63 Hz, 1H), 4.35 (t, J=2.56 Hz, 1H), 4.03-4.09 (m, 1H), 3.90 (d, J=10.63 Hz, 1H), 3.40-3.50 (m, 1H), 2.92 (s, 1H), 2.05 (s, 3H); LC/MS [M+H] = 723.2.

Example 160. Synthesis of 2-(((2R,3S,4R,5R)-5-(2-chloro-6-(methylamino)-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2- yl)methoxy)-2-(4-(2-oxo-1-propyl-1,2-dihydropyridin-3yl)benzyl)malonic acid

Proceeding as described in Example 19 above, but substituting diethyl 2-(((2R,3R,4R,5R)-3,4-diacetoxy-5(6-N,N'-(bis-(tert-butoxycarbonyl) )amino)-2-chloro-9H-nurin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate on diethyl 2-(((2R,3R,4R,5R)-3,4-diacetoxy -5-(6-((tert-butoxycarbonyl)(methyl)amino)2-chloro-9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate, gave the title compound as white solid matter.

1H NMR (400 MHz, DMSOO δ ppm 8.44 (s, 1H), 8.25 (d, J=4.3 Hz, 1H), 7.68 (dd, J=6.7, 1.8 Hz, 1H), 7.29-7.51 (m, 3H), 7.18 (d, J=7.5 Hz, 2H), 6.28 (t, J=6.8 Hz, 1H), 6.21 (s, 1H), 6.01 (d, J=6.8 Hz, 1H), 5.83 (d, J=7.3 Hz, 1H), 4.82 (s, 1H), 4.17 (dd, J=5.1, 2.6 Hz, 1H), 3.96 (s, 1H), 3.89 (t, J=7.3 Hz, 2H), 3, 79 (s, 1H), 3.58 (s, 1H), 3.25 (s, 1H), 2.90 (d, J=4.5 Hz, 3H), 1.67 (smt, J=7 .3 Hz, 2H), 0.88 (t, J=7.4 Hz, 3H); LC/MS [M+H] = 667.1.

Example 161. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-(1-hydroxyethyl)-9H-nurin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran- 2-yl)methoxy)-2-benzylmalonic acid

Example I1

Stage 1.

To a solution of diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(2-acetyl-6-amino-9H-nurin-9yl)-3-ethyltetrahydrofuran- 2-yl)methoxy)malonate (48 mg, 72.11 µmol, 1 eq.) in MeOH (2 ml) at 0°C was added NaBH ₄ (4.09 mg, 108.17 µmol, 1.5 eq.) . The solution was stirred at 0°C for 1 hour. Additional NaBH ₄ (4.1 mg) was added to the reaction mixture and stirred at 0°C for 0.5 hour, then diluted with water (6 ml) and extracted with ethyl acetate (3x6 ml). The combined organic layer was dried using Na ₂ SO ₄ , filtered and evaporated. The crude residue was purified by preparative TLC (ethyl acetate) to give diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3,4diacetoxy-5-(6-amino-2-(1-hydroxyethyl) -9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (20 mg) as syrup.

Stage 2.

To a solution of diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-amino-2-(1-hydroxyethyl)-9Hpurin-9-yl)- 3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (16 mg, 23.96 µmol, 1 eq.) in THF (2 ml) was added 1 M aq. LiOH solution (0.5 ml, 21 eq.). The mixture was stirred at 20°C for 3 hours, then acidified to pH 5 using 1 N aq. HCl solution. The mixture was extracted using ethyl acetate (5x3 ml). The combined organic layer was evaporated. The crude residue was purified by preparative HPLC (column: YMC-Triart Prep C18 150-40 mm-7 µm; mobile phase: [water (0.225%FA)-ACN]; B%: 13% - 33%, 10 min) and dried by lyophilization to obtain 2-(((2R,3S,4R,5R)-5(6-amino-2-( 1 -hydroxyethyl)-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran -2-yl)methoxy)-2benzylmalonic acid (4.0 mg) as a white solid.

1H NMR (400 MHz, CD3OD) δ ppm. 8.49 (d, J=8.88 Hz, 1H), 7.18-7.25 (m, 2H), 7.07-7.14 (m, 3H), 6.09 (dd, J= 9.82, 6.94 Hz, 1H), 4.76-4.84 (m, 2H), 4.29-4.36 (m, 1H), 3.89-4.03 (m, 2H) , 3.32-3.45 (m, 1H), 3.24-3.28

- 132 046381 (m, 1H), 3.02 (d, J=10.13 Hz, 1H), 1.52 (d, J=6.50 Hz, 3H); LC/MS [M+H] = 528.1.

Example 162. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl) methoxy)-2-(4-( 1 -methyl-2-oxo-1,2-dihydropyridin-3 -yl)benzyl)malonic acid

Proceeding as described in example 19 above, but replacing 3-(4-(bromomethyl)phenyl)-1-propylpyridin2(1H)-one with 3-(4-(bromomethyl)phenyl)-1-methylpyridin-2(III )-one, the title compound was obtained as a white solid.

1H NMR (400 MHz, CD3OD) δ ppm. 8.12 (s, 1H), 7.55-7.53 (m, 1H), 7.38-7.28 (m, 5H), 6.33 (t, J=6.8 Hz, 1H) , 5.94 (d, J=7.6 Hz, 1H), 4.75 (d, J=7.6 Hz, 1H), 4.27-4.26 (m, 1H), 4.09- 4.01 (m, 2H), 3.55 (s, 3H), 3.533.44 (m, 2H), 3.05 (s, 1H); LC/MS [M+H] = 625.0.

Example 163. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(6-(3-carboxypropyl)-2-chloro-9H-purin-9-yl)-3ethynyl-3, 4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

Prinr IZ aq. - aq.

Stage 1.

To a solution of 2,6-dichloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purine (4.00 g, 14.65 mmol, 1 eq.) and Pd(Ph ₃ P) 4 (1 .69 g, 1.46 mmol, 0.1 equiv.) in THF (30 ml) in an N2 atmosphere at 0°C, a solution of 0.5 M (4-ethoxy-4-oxobutyl)zinc(II) bromide was added dropwise (73.23 ml, 36.61 mmol, 2.5 eq.). The mixture was stirred at 0 to 25°C for 16 hours, then cooled to 0°C and quenched using 0.5 N aq. HCl solution. The reaction mixture was extracted using EtOAc (3x100 ml). The combined organic extract was washed with H2O (100 ml), brine (50 ml), dried over Na ₂ SO ₄ , filtered and evaporated. The crude residue was purified by silica gel flash column chromatography (10-75% EtOAc in petroleum ether) to give ethyl 4-(2-chloro-9-(tetrahydro-2H-pyran-2yl)-9H-purine-6- yl)butanoate (2.83 g).

Stage 2.

To a solution of tyl 4-(2-chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-yl)butanoate (1.50 g, 4.25 mmol) in DCM (15 ml ) TFA (10 ml) was added. The mixture was stirred at 25°C for 7 hours and then evaporated under reduced pressure. The residue was again dissolved in H ₂ O (50 ml) and neutralized to pH 7 with saturated aq. _NaHCO3 . The resulting mixture was extracted using EtOAc (3x75 ml). The combined organic layer was washed with brine, dried over Na ₂ SO ₄ , filtered and evaporated to give ethyl 4-(2-chloro-9H-purin-6-yl)butanoate (1.05 g).

Stages 3-4.

Proceeding as described in Example 5 above, but replacing uracil with 4-(2-chloro-9H-purin-6-yl)butanoate, afforded the title compound as a white solid.

1H NMR (400 MHz, CD ₃ OD) δ ppm. 8.74 (s, 1H), 7.27-7.24 (m, 2H), 7.09-7.08 (m, 3H), 6.09 (d, J=7.6 Hz, 1H) , 5.05 (d, J=7.6 Hz, 1H), 4.31-4.14 (m, 1H), 4.10-4.06 (m, 2H), 3.18-3.16 (m, 3H), 2.41 (t, J=7.2 Hz, 2H),

- 133 046381

2.17-2.15 (m, 2H), 1.22 (t, J=7.6 Hz, 2H); LC/MS [M+H] = 589.1.

Examples 164 and 165. Synthesis of (R)-2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3, 4dihydroxytetrahydrofuran-2-yl)methoxy)-3-ethoxy-3-oxo-2-(4-(2-oxotetrahydropyrimidin-1 (2H)yl)benzyl)propanoic acid and (S)-2-(((2R,3S ,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl)methoxy)-3-ethoxy-3-oxo-2 -(4-(2-oxotetrahydropyrimidin-1(2H)yl)benzyl)propanoic acid

Stage 1

N,O-bis(trimethylsilyl) was added to a suspension of 2-chloro-9H-purin-6-amine (1.04 g, 6.15 mmol, 1.7 eq.) in MeCN (10 ml) at 25°C. acetamide (BSA) (3.1 ml, 0.0127 mol, 3.5 eq.) The resulting suspension was heated at 85°C for 30 min until it became clear. The reaction mixture was allowed to cool to 25°C, then a solution of diethyl 2-(4-(2-oxotetraαhydropyrimidin-1(2H)-yl)benzyl)2-(((2R,3R,4R)-3,4 ,5-triacetoxy-3-ethynyltetrahydrofuran-2-yl)methoxy) malonate (2.34 g, 0.0036 mol, 1.0 eq.) in MeCN (10 ml) and TMSOTf (1.12 ml, 0.00615 mol, 1.7 eq.). The reaction mixture was then heated at 70-80°C overnight as all the starting material was consumed. The reaction mixture was allowed to cool to 25°C, then diluted with MeCN (100 ml) and quenched with saturated aq. NaHCO ₃ solution (150 ml). The insoluble matter was removed by filtration. The organic layer of the filtrate was separated, washed with H2O (50 ml), brine (50 ml), dried over Na ₂ SO ₄ and evaporated. The crude residue was purified by silica gel flash column chromatography (0-5% MeOH in CH ₂ Cl ₂ ) to form diethyl 2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-( 6-amino-2-chloro-9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)-2-(4-(2-oxotetrahydropyrimudin-1(2H)-yl)benzyl)malonate (1 .1 g, 40% yield) as a white solid.

Stage 2.

To a solution of diethyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-amino-2-chloro-9H-purin-9-yl)-3ethynyltetrahydrofuran^-yl^ethoxy LiOH monohydrate (89 mg, 2 .12 mmol, 4 eq.) The resulting mixture was stirred at room temperature overnight before removing VOCs under reduced pressure.The mixture was cooled to 0°C and acidified to pH 6 using 1 N aq. HCl solution and evaporated under reduced pressure. The crude residue was purified by preparative reverse phase HPLC to give a pair of diastereomers as a white solid: (R)-2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro -9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl)methoxy)-3-ethoxy-3-oxo-2-(4-(2-oxotetrahydropyrimidin-1 (2H)yl)benzyl )propanoic acid and (S)-2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran- 2-yl)methoxy)-3-ethoxy-3-oxo-2-(4-(2-oxotetrahydropyrimidin-1 (2H)yl)benzyl)propanoic acid, which are arbitrarily assigned a stereo configuration. Additionally, Example 9 was also isolated as a white solid.

(R)-2-(((2R,3S,4R,5R)-5-(6-Amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrophen ran-2 -yl)methoxy)-3-ethoxy-3-oxo-2-(4-(2-oxotetrahydropyrimidin-1(2H)-yl)benzyl)propanoic acid: 1H NMR (300 MHz, CD3OD) δ 8.32 (s , 1H), 7.28 (d, J=8.1 Hz, 2H), 7.01 (d, J=8.1 Hz, 2H), 6.00 (d, J=7.5 Hz, 1H ), 4.84 (d, J=7.5, 1H), 4.28-4.06 (m, 3H), 3.99-3.95 (m, 2H), 3.52-3.35 (m, 6H), 3.08 (s, 1H), 2.021.97 (m, 2H), 1.21 (t, J=7.1, 3H); LC/MS [M+H] = 644.05.

(S)-2-(((2R,3S,4R,5R)-5-(6-Amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2- yl)methoxy)-3-ethoxy-3-oxo-2-(4-(2-oxotetrahydropyrimidin-1(2H)-yl)benzyl)propanoic acid: 1H NMR (300 MHz, CD3OD) δ 8.08 (s, 1H), 7.26-7.29 (d, J=6.8 Hz, 2H), 7.03-7.01 (d, J=7.23 Hz, 2H), 5.96-5.99 (d, J=7.14 Hz, 1H), 4.75-4.77 (d, J=7.5, 1H), 4.02-4.24 (m, 5H), 3.32-3 .66 (m, 6H), 3.15 (s, 1H), 1.952.19 (m, 2H), 1.22-1.27 (m, 3H); LC/MS [M+H] = 644.05.

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Examples 166 and 167. Synthesis of (R)-2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3, 4dihydroxytetrahydrofuran-2-yl)methoxy)-3 -(4-(2-oxotetrahydropyrimidin-1 (2H)-yl)phenyl)propanoic acid and (S)-2-(((2R,3S,4R,5R)-5 -(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl)methoxy)-3 -(4-(2-oxotetrahydropyrimidin-1 (2H-yl) phenyl)propanoic acid

The crude product of example 9 after treatment was dried in a vacuum oven at 60°C for 2 days, after which it was purified using preparative HPLC to obtain a pair of diastereomers: (R)-2(((2R,3S,4R,5R)-5 -(6-αmino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)-3-(4-(2-oxotetrahydropyrimidine-1(2H) -yl)phenyl)propanoic acid and (S)-2-(((2R,3S,4R,5R)-5(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3 ,4-dihydroxytetrahydrofuran-2-yl)methoxy)-3-(4-(2-oxotetrahydropyrimidin-1(2H)-yl)phenyl)propanoic acid, which are arbitrarily assigned a stereoconfiguration. Both compounds were isolated as white solids.

(R)-2-(((2R,3S,4R,5R)-5-(6-Amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2- yl)methoxy)-3-(4-(2-oxotetrahydropyrimidin-1(2H)-yl)phenyl)propanoic acid: 1H NMR (300 MHz, CD ₃ OD) δ 8.38 (s, 1H), 7.22 (d, J=8.3 Hz, 2H), 7.04 (d, J=8.3 Hz, 2H), 5.92 (d, J=7.3 Hz, 1H), 4.364.32 (m , 1H), 4.30 (d, J=7.3 Hz, 1H), 4.17 (t, J=2.3 Hz, 1H), 4.07-4.03 (m, 1H), 3 .80-3.75 (m, 1H), 3.54-3.49 (m, 2H), 3.33-3.31 (m, 2H), 3.25-3.19 (m, 1H) , 3.12 (s, 1H), 3.09-3.02 (m, 1H), 2.02-1.97 (m, 2H); LC/MS [M+H] = 572.0.

(S)-2-(((2R,3S,4R,5R)-5-(6-Amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2- yl)methoxy)-3-(4-(2-oxotetrahydropyrimidin-1(2H)-yl)phenyl)propanoic acid: 1H NMR (300 MHz, CD3OD) δ 8.47 (s, 1H), 7.26 (d , J=8.2 Hz, 2H), 7.15 (d, J=8.3 Hz, 2H), 5.97 (d, J=7.0 Hz, 1H), 4.97 (d, J =7.0 Hz, 1H), 4.31 (t, J=6.4 Hz, 1H), 4.20 (t, J=3.4 Hz, 1H), 3.91 (d, J=3 .4 Hz, 1H), 3.65 (t, J=6.0 Hz, 2H), 3.39-3.33 (m, 2H), 3.23-3.17 (m, 2H), 3 .08 (s, 1H), 3.06-3.01 (m, 1H), 2.10-1.95 (m, 2H); LC/MS [M+H] = 572.0.

Example 168. Synthesis of 2-benzyl-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(hydroxyamino)-9H-purin-9-yl)-3ethynyl-3,4- Dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid

Przhir 1-vZ

Stage 1.

To a solution of diethyl-2-benzyl-2-(((2R,3S,4R,5R)-5-(5-chloro-7-(hydroxyamino)-3H-imidazo[4,5b]pyridin-3-yl)- 3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonate (98 mg, 0.144 mmol) in dioxane (2 ml) was added with an aqueous solution of hydroxylamine (0.1 ml, 1.6 mmol, 16 M) and Et ₃ N (35 µl, 0.16 mmol). The reaction mixture was stirred for 2.5 hours and then diluted with EtOAc (15 ml) and H2O (5 ml). The organic layer was separated, washed with H2O (20 ml), brine (20 ml), dried over Na ₂ SO ₄ and evaporated to give crude diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3 ,4-diacetoxy-5(2-chloro-6-(hydroxyamino)-9H-purin-9-yl)-3-ethynyltetrahydrofuran-2-yl)methoxy)malonate (88 mg) as an inorganic solid, which was used on next stage without additional purification.

Stage 2.

To a solution of crude diethyl-2-benzyl-2-(((2R,3R,4R,5R)-3,4-diacetoxy-5-(2-chloro-6(hydroxyamino)-9H-purin-9-yl)- LiOH-H ₂ O ( 31 mg, 0.75 mmol). The resulting mixture was stirred at 25°C for 5.5 hours, after which volatile organic compounds were removed under reduced pressure. Vodn. The layer was cooled to 0°C and acidified to pH 6.5 using 1 N aq. HCl solution and then evaporated. The crude residue was purified by preparative reverse phase HPLC to give 2-benzyl-2-(((2R,3S,4R,5R)-5-(5-chloro-7-(hydroxyamino)-3Himidazo[4,5-b ]pyridin-3-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)malonic acid (17 mg) as a reddish solid.

1H NMR (CD3OD, 300 MHz) δ 8.25 (s, 1H), 7.25-7.28 (m, 2H), 7.05 (m, 3H), 6.43 (s, 1H), 6 .06-6.08 (d, J=7.17 Hz, 1H), 4.95-4.98 (d, J=7.05 Hz, 1H), 4.32 (s, 1H), 4. 05-4.11 (m, 2H), 3.89-3.93 (m, 1H), 3.31-3.39 (m, 2H), 2.99 (s, 1H), 1.30- 1.33 (m, 6H); LC/MS [M+H] = 533.1.

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Example 169. Synthesis of 2-(((2R,3S,4R,5R)-5-(2-chloro-6-(isopropylamino)-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2- yl)methoxy)-2-(thiazol-4-yl)-3-(thiophen-3-yl)propanoic acid

Stage 1.

To a solution of ethyl 2-(thiazol-4-yl) acetate (2 g, 11.7 mmol) in CH ₃ CN (15 ml) at 0°C was added DBU (2.62 ml, 17.6 mmol) and 4 acetamidibenzenesulfonyl azide (3.4 g, 14.1 mmol) in CH3CN (10 ml). The reaction mixture was stirred at room temperature for 1.5 hours, then evaporated to dryness under reduced pressure. The resulting crude product was purified by silica gel column chromatography (0-40% EtOAc in hexanes) to give ethyl 2-diazo-2-(thiazol-4-yl) acetate (2.0 g).

Stage 2.

To a mixture of (3aR,5R,6R,6aR)-6-ethynyl-5-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[2,3d][1,3]dioxol-6-yl acetate (7 g, 27.32 mmol , 1 eq.) in DCE (15 ml) was added dropwise Rh(OAc)2 (603.69 mg, 2.73 mmol, 0.1 eq.) and ethyl 2-diazo-2-(thiazol-4- yl)acetate (6.46 g, 32.78 mmol, 1.2 eq.) in DCE (15 ml) at 0°C. The mixture was stirred at 25°C under N2 for 14 hours, then the insoluble product was filtered and the filtrate was evaporated under reduced pressure. The crude residue was purified by silica gel flash column chromatography (0-50% EtOAc in petroleum ether) to give ethyl 2-(((3aR,5R,6R,6aR)-6-acetoxy-6-ethynyl-2,2 -dimethyltetrahydrofuro[2,3-d][1,3]dioxol5-yl)methoxy)-2-(thiazol-4-yl)acetate (10.81 g, 93% yield) as an oil.

Stage 3.

To a mixture of ethyl 2-(((3aR,5R,6R,6aR)-6-acetoxy-6-ethynyl-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5yl)methoxy)- 2-(thiazol-4-yl)acetate (2.69 g, 6.33 mmol, 1 eq.) in DMF (5 ml) was added Cs ₂ CO ₃ (6.18 g, 18.98 mmol, 3 eq. ). The mixture was stirred at 25°C under N2 for 0.5 h, after which 3(bromomethyl)thiophene (2.8 g, 15.81 mmol, 2.5 eq.) was added. The resulting mixture was stirred at 25°C for 14 h, then the insoluble product was filtered off and the filtrate was diluted with H2O (15 ml) and extracted using EtOAc (3x15 ml). The combined organic layer was washed with sat. aq. NH4Cl solution (3x15 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The crude residue was purified by silica gel flash column chromatography (0-50% EtOAc in petroleum ether) to give ethyl 2-(((3aR,5R,6R,6aR)-6-acetoxy-6-ethynyl-2,2 -dimethyltetrahydrofuro[2,3d][1,3]dioxol-5-yl)methoxy)-2-(thiazol-4-yl)-3-(thiophen-3-yl)npropanoate (2.27 g, 69% yield ) in the form of oil.

Stage 4.

To a mixture of ethyl 2-(((3aR,5R,6R,6aR)-6-acetoxy-6-ethynyl-2,2-dimethyltetrahydrofuro[2,3d][1,3]dioxol-5-yl)methoxy)- 2-(thiazol-4-yl)-3-(thiophen-3-yl)npropanoate (2.27 g, 4.35 mmol, 1 eq.) in DCM (5 ml) and H2O (0.5 ml) was added TFA (5 ml, 67.53 mmol, 15.5 eq.). The mixture was stirred at 15°C under N2 for 14 hours, then the pH was adjusted to 7-8 with saturated aq. NaHCO ₃ (50 ml) and evaporated under reduced pressure. The residue was diluted with H ₂ O (5 ml), extracted using EtOAc (4x15 ml). The combined organic layer was washed with brine (10 ml), dried over Na ₂ SO ₄ , filtered and evaporated under reduced pressure to give crude ethyl 2[[(2S,3S,4R)-3-acetoxy-3-ethynyl-4, 5-dihydroxytetrahydrofuran-2-yl]methoxy]-2-thiazol-4-yl-3-(3-thienyl)propanoate (1.82 g) as syrup.

To a solution of ethyl 2-[[(2R,3S,4R)-3-acetoxy-3-ethynyl-4,5-dihydroxymetrahydrofuran-2yl]methoxy]-2-thiazol-4-yl-3-(3-thienyl) propanoate (1.82 g, 3.78 mmol, 1 eq.) in pyridine (8 ml) under N2 at 0°C, add 4-DMAP (1.39 g, 11.34 mmol, 3 eq.) and Ac ₂ O (2.83 ml, 30.24 mmol, 8 eq.). The mixture was stirred at 15°C for 15 hours, then diluted with H2O (20 ml) and extracted using EtOAc (3x15 ml). The combined organic layer was washed with 10% CuSO ₄ solution (2x15 ml), dried over Na ₂ SO ₄ , filtered and evaporated under reduced pressure to give crude (3R,4R,5R)-5-(((1-ethoxy-1 -oxo-2-(thiazol-4-yl)-3-(thiophen-3-yl)npropan-2-yl)oxy)methyl)-4ethynyltetrahydrofuran-2,3,4-triyltriacetate (2.42 g) as syrup.

Stage 5.

To a mixture of 2-chloro-N-isonropyl-9H-nurine-6-amine (606.20 mg, 2.86 mmol, 1 eq.) in DCE (20 ml) was added BSA (1.77 ml, 7.16 mmol , 2.5 eq.). The mixture was stirred at 85°C under N2 atmosphere for

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0.5 h, after which it was allowed to cool to 0°C and then crude (3R,4R,5R)-5-(((1-3TOkch-1oxo-2-(thiazol-4-yl)-3-( thiophen-3-yl)propan-2-yl)oxy)methyl)-4-ethynyltetrahydrofuran-2,3,4-triyltriacetate (1.62 g, 2.86 mmol, 1 eq.) and TMSOTf (1.55 ml , 8.59 mmol, 3 equiv.). The resulting mixture was stirred at 65°C under N ₂ for 14 hours before quenching with saturated aq. NaHCO ₃ (20 ml). The reaction mixture was diluted with H2O (10 ml), and extracted using DCM (3x20 ml). The combined organic layer was washed with brine (3x15 ml), dried over Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The crude residue was purified by silica gel flash column chromatography (0-50% EtOA in petroleum ether) to give (2R,3R,4R,5R)-5-(2chloro-6-(isopropylamino)-9H-purin-9-yl) -2-(((1-ethoxy-1-oxo-2-(thiazol-4-yl)-3-(thiophen-3-yl)propan-2yl)oxy)methyl)-3-ethynyltetrahydrofuran-3,4- diyl diacetate (402 mg, crude) as syrup.

Stage 6.

To a mixture of (2R,3R,4R,5R)-5-(2-chloro-6-(isopropylamino)-9H-purin-9-yl)-2-(((1-ethoxy-1-oxo-2(thiazole -4-yl)-3-(thiophen-3-yl)propan-2-yl)oxy)methyl)-3-ethynyltetrahydrofuran-3,4-diyldiacetate (384 mg, crude) in THF (2 ml) and H2O ( 1 ml) LiOH (128 mg, 5.35 mmol) was added. The mixture was stirred at 50°C for 6 hours, then diluted with H2O (40 ml) and extracted using EtOAc (10 ml). The aqueous phase was acidified to pH 2-3 using 2 N aqueous HCl to pH ~ 2-3 and then evaporated under reduced pressure. The crude residue was purified by preparative HPLC (column: YMC-Actus Triart C18 150-30 mm-5 µm; mobile phase: [water (0.225%FA)-ACN]; B%: 40% - 60%, 10 min) and dried by lyophilization to obtain a diastereomeric mixture (approximately 1:1) 2(((2R,3S,4R,5R)-5-(2-chloro-6-(isopropylamino)-9H-purin-9-yl)-3-ethynyl -3,4-dihydroxytetrahydrofuran-2-yl)methoxy)-2-(thiazol-4-yl)-3-(thiophen-3-yl)propanoic acid (17.5 mg) as a white solid.

¹ H NMR (400 MHz, CD ₃ OD) δ ppm. 8.92-9.01 (m, 1H), 8.04-8.28 (m, 1H), 7.60-7.71 (m, 1H), 7.10-7.15 (m, 1H ), 6.98-7.06 (m, 1H), 6.77-6.94 (m, 1H), 5.90-6.02 (m, 1H), 4.91-5.06 (m , 2H), 4.40 (br. s, 1H), 4.19-4.32 (m, 1H), 3.89-3.99 (m, 1H), 3.65-3.87 (m , 3H), 2.89-3.02 (m, 1H), 1.25-1.35 (m, 6H); LC/MS [M+H] = 605.2.

Examples 170 and 171. Synthesis of (S)-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(isopropylamino)-9H-purin-9-yl)-3ethynyl-3, 4-dihydroxytetrahydrofuran-2-yl)methoxy)-3-phenyl-2-(thiazol-4-yl)propanoic acid and (R)-2-(((2R,3S,4R,5R)-5-(2- chloro-6-(isopropylamino)-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)-3-phenyl-2-(thiazol-4-yl)propanoic acid

Prism 1' 171

Proceeding as described in Example 169 above, but replacing 3-(bromomethyl)thiophene with benzyl bromide, a pair of diastereomeric products was obtained, the stereoconfiguration of which was arbitrary. Both products were purified by preparative HPLC and isolated as white solids.

(S)-2-(((2R,3S,4R,5R)-5-(2-Chloro-6-(isonropylamino)-9H-nurin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran- 2-yl)methoxy)-3-phenyl-2-(thiazol-4-yl)propanoic acid: ^1H NMR (CD3OD, 300 MHz) δ 8.99 (s, 1H), 7.91 (s, 1H) , 7.70 (s, 1H), 7.07-7.23 (m, 5H), 5.91-5.94 (d, J=6.9 Hz, 1H), 4.87-4.90 (d, J=7.0 Hz, 1H), 4.21-4.45 (m, 2H), 3.593.94 (m, 4H), 3.02 (s, 1H), 1.29-1, 31 (d, J=6.48 Hz, 6H); LC/MS [M+H] = 599.0.

(R)-2-(((2R,3S,4R,5R)-5-(2-Chloro-6-(isopropylamino)-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran- 2-yl)methoxy)-3-phenyl-2-(thiazol-4-yl)propanoic acid: ¹ H NMR (CD3OD, 300 MHz) δ 8.97-8.98 (d, J=1.83 Hz, 1H), 8.07 (s, 1H), 7.573-7.579 (d, J=1.86 Hz, 1H), 6.94-7.09 (m, 5H), 5.97-5.99 (d , J=7.17 Hz, 1H), 4.98-5.00 (d, J=7.23 Hz, 1H), 4.40-4.42 (m, 1H), 4.27-4, 29 (t, J=3.84 Hz, 1H), 3.93-3.97 (m, 2H), 3.59-3.81 (k, J=14.31, 37.47 Hz, 2H) , 2.95 (s, 1H), 1.29-1.33 (d, J=6.39 Hz, 6H); LC/MS [M+H] = 599.0.

Examples 172 and 173. Synthesis of (S)-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(cyclopropylmethyl)-9H-purin-9-yl)3-ethynyl-3 ,4-dihydroxytetrahydrofuran-2-yl)methoxy)-3-phenyl-2-(thiazol-4-yl)propanoic acid and (R)-2-(((2R,3S,4R,5R)-5-(2 -chloro-6-((cyclopropylmethyl)amino)-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)-3-phenyl-2-(thiazol-4-yl )propanoic acid

Prizer 172 Prizer 173

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Proceeding as described in Examples 170 and 171 above, but replacing 2-chloro-N-(cyclopropylmethyl)-9H-purine-6-amine with 2-chloro-N-(cyclopropylmethyl)-9H-purine-6-amine, a pair of diastereomeric products is obtained , the stereo configuration of which was specified arbitrarily. Both products were purified by preparative HPLC and isolated as white solids.

(S)-2-(((2R,3S,4R,5R)-5-(2-Chloro-6-((cyclopronylmethyl)amino)-9H-purin-9-yl)-3-ethynyl-3,4 -dihydroxytetrahydrofuran-2-yl)methoxy)-3-phenyl-2-(thiazol-4-yl)propanoic acid: ¹ H NMR (CD ₃ OD, 300 MHz) δ 8.99-8.99 (d, J= 1.95 Hz, 1H), 7.93 (s, 1H), 7.69-7.70 (d, J=1.86 Hz, 1H), 7.03-7.23 (m, 5H), 5.92-5.94 (d, J=6.96 Hz, 1H), 4.87-4.89 (d, J=7.11 Hz, 1H), 4.19-4.22 (m, 1H), 3.59-3.94 (m, 4H), 3.42-3.43 (m, 2H), 3.02 (s, 1H), 1.12-1.22 (m, 1H) , 0.54-0.61 (m, 2H), 0.32-0.37 (m, 2H); LC/MS [M+H] = 611.0.

(R)-2-(((2R,3S,4R,5R)-5-(2-Chloro-6-((cyclopropylmethyl)amino)-9H-purin-9-yl)-3-ethynyl-3,4 -dihydroxytetrahydrofuran-2-yl)methoxy)-3-phenyl-2-(thiazol-4-yl)propanoic acid: 1H NMR (CD3OD, 300 MHz) δ 8.96-8.97 (d, J=1.89 Hz, 1H), 8.07 (s, 1H), 7.55-7.56 (d, J=2.07 Hz, 1H), 6.94-7.11 (m, 5H), 5.975.99 (d, J=7.17 Hz, 1H), 4.98-5.00 (d, J=7.29 Hz, 1H), 4.27-4.29 (t, J=3.66 Hz, 1H), 3.94-3.95 (m, 2H), 3.593.80 (k, J=14.64, 32.46 Hz, 2H), 3.39-3.50 (m, 2H), 2 .96 (s, 1H), 1.13-1.23 (m, 1H), 0.55-0.62 (m, 2H), 0.32-0.38 (m, 2H); LC/MS [M+H] = 611.0.

Example 174. Synthesis of 2-(((2R,3S,4R,5R)-5-(2-chloro-6-(isopropylamino)-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2- yl)methoxy)-2-(thiazol-4-yl)-3-(4-(trifluoromethoxy)phenyl)propanoic acid

174

Proceeding as described in Example 169 above, but replacing 3-(bromomethyl)thiophene with 1-(bromomethyl)-4(trifluoromethoxy)benzene, the title compound was obtained as a mixture of diastereomers (approximately 1:1) and isolated as white solid matter.

1H NMR (400 MHz, CD3OD) δ ppm. 8.95-9.02 (m, 1H), 8.05-8.27 (m, 1H), 7.59-7.74 (m, 1H), 7.15-7.32 (m, 2H ), 6.78-6.98 (m, 2H), 5.89-6.00 (m, 1H), 4.92-5.10 (m, 1H), 4.32-4.46 (m , 1H), 4.22-4.32 (m, 1H), 3.763.98 (m, 2H), 3.57-3.71 (m, 2H), 2.98-3.04 (m, 1H ), 1.26-1.32 (m, 6H); LC/MS [M+H] = 682.8.

Example 175. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl) methoxy)-3-(4-(2-oxotetrahydronirimidin-1(2H)-yl)phenyl)-2(thiazol-4-yl)propanoic acid

Stage 1.

Mixture of 1-(bromomethyl)-4-nitrobenzene (7.62 g, 35.26 mmol, 3 eq.) and NaI (352.31 mg, 2.35 mmol, 0.2 eq.) in DMF (50 ml) stirred at 15°C for 30 min. This mixture was then added to a solution of ethyl 2-(((3aR,5R,6R,6aR)-6-acetoxy-6-ethynyl-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5 -yl)methoxy)-2-(thiazol-4-yl)acetate (5 g, 11.75 mmol, 1 eq.) and Cs ₂ CO ₃ (19.15 g, 58.76 mmol, 5 eq.) in DMF

- 138 046381 (50 ml) at 15°C and stirred for 30 minutes. The resulting mixture was stirred for 8 hours and then quenched with water (200 ml). The mixture was extracted using EtOAc (4x30 ml). The combined organic layer was washed with water (3x100 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated. The residue was purified by silica gel flash column chromatography (0-40% EtOAc in petroleum ether) to give ethyl 2-(((3aR,5R,6R,6aR)-6-acetoxy-6-ethynyl-2,2- dimethyltetrahydrofuro[2,3d][1,3]dioxol-5-yl)methoxy)-3-(4-nitrophenyl)-2-(thiazol-4-yl)propanoate (4.89 g) as syrup.

Stage 2.

To a solution of ethyl 2-(((3aR,5R,6R,6aR)-6-acetoxy-6-ethynyl-2,2-dimethyltetrahydrofuro[2,3d][1,3]dioxol-5-yl)methoxy)- 3-(4-nitrophenyl)-2-(thiazol-4-yl)propanoate (4.89 g, crude) in DCM (25 ml) and H2O (2.5 ml) added TFA (25 ml, 337.65 mmol ). The mixture was stirred at 30°C for 23 hours, then diluted with water (100 ml) and the resulting mixture was extracted using DCM (6x30 ml). The combined organic layer was washed with sat. aq. NaHCO ₃ (2x100 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated to give crude ethyl 2-(((2R,3S,4R)-3ethynyl-3,4,5-trihydroxytetrahydrofuran-2-yl)methoxy )-3-(4-nitrophenyl)-2-(thiazol-4-yl)propanoate (4.56 g) as a brown oil.

To a solution of crude ethyl 2-(((2S,3S,4R)-3-ethynyl-3,4,5-trihydroxytetrahydrofuran-2yl)methoxy)-3-(4-nitrophenyl)-2-(thiazol-4-yl )propanoate (4.56 g) in DCM (50 ml) was added 4-DMAP (232.86 mg, 1.91 mmol), pyridine (6.15 ml, 76.24 mmol) and Ac ₂ O (8.93 ml, 95.30 mmol). The mixture was stirred at 15°C for 19 hours, then quenched with water (100 ml) and the resulting mixture was extracted using DCM (4x30 ml). The combined organic layer was washed with water (3x100 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated. The crude residue was purified by silica gel flash column chromatography (10-55% EtOA in petroleum ether) to give (3R,4R,5R)-5(((1-ethoxy-3-(4-nitrophenyl)-1-oxo -2-(thiazol-4-yl)propan-2-yl)oxy)methyl)-4-ethynyltetrahydrofuran2,3,4-triyltriacetate (1.02 g) as a yellow oil.

Stage 3.

To a solution of 2,6-dichloro-9H-purine (382.64 mg, 2.02 mmol) in MeCN (5 ml) was added BSA (1.04 ml, 4.22 mmol). The suspension was stirred at 65°C for 0.5 h until it became clear. The resulting solution was cooled to 0°C, and then a solution of (3R,4R,5R)-5-(((1^tokcu-3-(4nitrophenyl)-1-oxo-2-(thiazol-4-yl)propan- 2-yl)oxy)methyl)-4-ethynyltetrahydrofuran-2,3,4triyltriacetate (1.02 g) in MeCN (5 ml) and TMSOTf (4.22 mmol, 762.15 µl). The mixture was then stirred at 65°C for 1 hour before quenching with saturated aq. NaHCO ₃ solution (40 ml) and the resulting mixture was extracted using EtOAc (4x20 ml). The combined organic layer was dried over Na ₂ SO ₄ , filtered and evaporated to give crude (2R,3R,4R,5R)-5-(2,6-dichloro-9Hpurin-9-yl)-2-(((1-ethoxy -3-(4-nitrophenyl)-1-oxo-2-(thiazol-4-yl)propan-2-yl)oxy)methyl)-3ethynyltetrahydrofuran-3,4-diyldiacetate (1.71 g) as a yellow solid substances.

Stage 4.

To a solution of crude (2R,3R,4R,5R)-5-(2,6-dichloro-9H-purin-9-yl)-2-(((1-ethoxy-3-(4nitrophenyl)-1-oxo- 2-(thiazol-4-yl)propan-2-yl)oxy)methyl)-3-ethynyltetrahydrofuran-3,4-diyldiacetate (1.0 g) in MeOH (20 ml) NH ₄ .OH ( 28.04 mmol, 4.00 ml, concentration 27%). The mixture was sealed and stirred at 100°C for 1.5 hours, after which it was allowed to cool and diluted with water (20 ml), and the resulting mixture was extracted using EtOAc (3x10 ml). The combined organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and evaporated to give crude ethyl 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purine-9- yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl)methoxy)-3-(4-nitrophenyl)-2-(thiazol-4-yl)propanoate (752 mg) as a yellow solid.

Stage 5.

To a solution of crude ethyl 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl )methoxy)-3-(4-nitrophenyl)-2-(thiazol-4-yl)propanoate (752 mg), 4DMAP (58.33 mg, 477.44 µmol) and Et ₃ N (7.16 mmol, 996 .81 µl) in DMF (8 ml) at 0°C was added Boc ₂ O (1.04 g, 4.77 mmol). The mixture was stirred at 20°C for 2 hours, then diluted with water (40 ml) and the resulting mixture was extracted using EtOAc (4x20 ml). The combined organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and evaporated to give crude ethyl-2(((2R,3R,4R,5R)-5-(6-(bis-(tert-butoxycarbonyl)amino)-2- chloro-9H-purin-9-yl)-4-((tert-butoxycarbonyl)oxy)-3-ethynyl-3-hydroxytetrahydrofuran-2-yl)methoxy)-3-(4-nitrophenyl)-2-(thiazol- 4-yl)propanoate (911 mg) as a brown solid.

Stage 6.

To a solution of crude ethyl 2-(((2R,3R,4R,5R)-5-(6-(bis-(tert-butoxycarbonyl)amino)-2-chloro9H-purin-9-yl)-4-(( tert-butoxycarbonyl)oxy)-3-ethynyl-3-hydroxytetrahydrofuran-2-yl)methoxy)-3(4-nitrophenyl)-2-(thiazol-4-yl)propanoate (711 mg) in EtOH (7 ml) was added saturated aq. NH ₄ Cl solution (764.21 µmol, 7 ml) and iron (426.77 mg, 7.64 mmol). The mixture was stirred at 50°C for 2 hours, then filtered through a celite pad and the filtrate was evaporated. Then the unrefined residue

- 139 046381 was dissolved in water (20 ml) and the resulting mixture was extracted using EtOAc (3x15 ml). The combined organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and evaporated to give crude ethyl 3-(4-aminophenyl)-2-(((2R,3R,4R,5R)-5-(6-(bis-( tert-butoxycarbonyl)amino)-2-chloro9H-purin-9-yl)-4-((tert-butoxycarbonyl)oxy)-3-ethynyl-3-hydroxytetrahydrofuran-2-yl)methoxy)-2(thiazol-4- yl)propanoate (552 mg) as a brown solid.

Stage 7.

To a solution of crude ethyl-3-(4-aminophenyl)-2-(((2R,3R,4R,5R)-5-(6-(bis-(tertbutoxycarbonyl)amino)-2-chloro-9H-purine-9 -yl)-4-((tert-butoxycarbonyl)oxy)-3-ethynyl-3-hydroxytetrahydrofuran-2-yl)methoxy)-2-(thiazol-4-yl)propanoate (552 mg) in DCM (5 ml) 1-chloro-3-isocyanatopropane (109.94 mg, 919.60 µmol) was added. The mixture was stirred at 15°C for 16 hours and then evaporated under reduced pressure. The crude residue was purified by silica gel flash column chromatography (20-100% EtOAc in petroleum ether) to give ethyl-2(((2R,3R,4R,5R)-5-(6-(bis-(tert-butoxycarbonyl)amino )-2-chloro-9H-purin-9-yl)-4-((tert-butoxycarbonyl)oxy)-3-ethynyl-3-hydroxytetrahydrofuran-2-yl)methoxy)-3 -(4-(3 -( 3-chloropropyl)ureido)phenyl)-2(thiazol-4-yl)propanoate (283 mg) as an off-white solid.

Stage 8.

To a solution of ethyl 2-(((2R,3R,4R,5R)-5-(6-(bis-(tert-butoxycarbonyl)amino)-2-chloro-9H-purin-9-yl)4-(( tert-butoxycarbonyl)oxy)-3-ethynyl-3-hydroxytetrahydrofuran-2-yl)methoxy)-3 -(4-(3 -(3-chloropropyl)ureido)phenyl)-2-(thiazol-4-yl)propanoate (283 mg, 277.47 µmol, 1 eq.) to THF (3 ml) was added NaH (55.49 mg, 1.39 mmol, 60% in mineral oil, 5 eq.). The mixture was stirred at 15°C for 5 hours and then quenched with H2O (1.5 ml). NaOH (166.48 mg, 4.16 mmol, 15 eq.) was added to this mixture, and the resulting mixture was stirred at 40°C for 48 hours, then VOCs were removed under reduced pressure. Vodn. the layer was acidified using 2 N aq. HCl (1 ml) and evaporated under reduced pressure to obtain crude 2-(((2R,3S,4R,5R)-5-(6-((tert-butoxycarbonyl)amino)2-chloro-9H-purine-9- yl)-3 -ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)-3 -(4-(2-oxotetrahydropyrimidin-1(2H)-yl)phenyl)-2-(thiazol-4-yl)propane acid (238 mg) as a yellow solid.

Stage 9.

To a mixture of crude 2-(((2R,3S,4R,5R)-5-(6-((tert-butoxycarbonyl)amino)-2-chloro-9H-purin-9yl)-3-ethynyl-3,4- Dihydroxytetrahydrofuran-2-yl)methoxy)-3-(4-(2-oxotetrahydropyrimidin-1(2H)yl)phenyl)-2-(thiazol-4-yl)propanoic acid (238 mg) in DCM (2 ml) was added TFA (9.45 mmol, 0.7 ml). The mixture was stirred at 15°C for 2 hours and then evaporated under reduced pressure. The crude residue was purified by preparative HPLC ([water (0.225% FA)-ACN]; B%: 20%40%, 10 min) to give a diastereomeric mixture (ca. 1:1) 2-(((2R,3S,4R ,5R)-5-(6-amino-2chloro-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)-3 -(4-(2-oxotetrahydropyrimidine-1 (2H)-yl)phenyl)-2-(thiazol-4-yl)propanoic acid (22.9 mg) as a white solid.

1H NMR (400 MHz, CD3OD) δ ppm. 9.01 (m, 1H), 7.98-8.32 (m, 1H), 7.59-7.83 (m, 1H), 6.91-7.34 (m, 4H), 5. 88-6.07 (m, 1H), 4.72-4.96 (m, 1H), 4.13-4.32 (m, 1H), 3.60-4.00 (m, 4H), 3.43-3.56 (m, 2H), 3.35-3.42 (m, 2H), 2.96-3.14 (m, 1H), 1.89-2.08 (m, 2H ); LC/MS [M+H] = 655.3.

Example 176. Synthesis of 2-(((2R,3S,4R,5R)-5-(2-chloro-6-(isopropylamino)-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2- yl)methoxy)-3 -(4-(2-oxotetrahydropyrimidin-1^N-yl^enyl)^(thiazol-4-yl)propanoic acid

Shchavir 17"

Stage 1.

To a solution of crude (2R,3R,4R,5R)-5-(2,6-dichloro-9H-purin-9-yl)-2-(((1-ethoxy-3-(4nitrophenyl)-1-oxo- 2-(thiazol-4-yl)propan-2-yl)oxy)methyl)-3-ethynyltetrahydrofuran-3,4-diyldiacetate (1.17 g) in MeCN (10 ml) was added propan-2-amine (1. 0 ml, 11.64 mmol) and DIEA (0.9 ml). The mixture was stirred at 15°C for 16 hours, then diluted with water (30 ml) and the resulting mixture was extracted

- 140 046381 using EtOAc (4x20 ml). The combined organic layer was dried over Na ₂ SO ₄ , filtered and evaporated to give crude (2R,3R,4R,5R)-5-(2-chloro-6-(isopropylamino)-9H-purin-9-yl)2-( ((1-ethoxy-3-(4-nitrophenyl)-1-oxo-2-(thiazol-4-yl)propan-2-yl)oxy)methyl)-3-ethynyltetrahydrofuran-3,4-diyldiacetate (1, 16 g) as a yellow solid.

Stage 2.

To a solution of crude (2R,3R,4R,5R)-5-(2-chloro-6-(isopropylamino)-9H-purin-9-yl)-2-(((1ethoxy-3-(4-nitrophenyl)- 1-oxo-2-(thiazol-4-yl)propan-2-yl)oxy)methyl)-3-ethynyltetrahydrofuran-3,4diyldiacetate (1.16 g) in EtOH (5 ml) added Fe powder (856.75 mg, 15.34 mmol) and saturated aq. NH ₄ Cl (1.53 mmol, 5 ml). The mixture was stirred at 50°C for 2 hours, then it was filtered through a celite pad and the filtrate was evaporated to give crude (2R,3R,4R,5R)-2-(((3-(4-aminophenyl)1-ethoxy- 1-oxo-2-(thiazol-4-yl)propan-2-yl)oxy)methyl)-5-(2-chloro-6-(isopropylamino)-9H-purin-9-yl)3-ethynyltetrahydrofuran-3 ,4-diyldiacetate (1.05 g) as a yellow solid.

Stage 3.

To a solution of crude (2R,3R,4R,5R)-2-(((3-(4-aminophenyl)-1-ethoxy-1-oxo-2-(thiazol-4yl)propan-2-yl)oxy)methyl )-5-(2-chloro-6-(isopropylamino)-9H-purin-9-yl)-3-ethynyltetrahydrofuran3,4-diyldiacetate (1.05 g) in DCM (10 ml) was added 1-chloro-3- isocyanatopropane (172.85 mg, 1.45 mmol). The mixture was stirred at 15°C for 16 hours, then quenched with water (20 ml) and the resulting mixture was extracted using DCM (3x10 ml). The combined organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and evaporated to give crude ethyl 2-(((2R,3S,4R,5R)-5-(2-chloro-6(isopropylamino)-9H-purine-9 -yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)-3 -(4-(3 -(3 chloropropyl)-ureido)phenyl)-2-(thiazol-4-yl)propanoate ( 1.33 g) as a yellow solid.

Stage 4.

To a solution of crude ethyl 2-(((2R,3S,4R,5R)-5-(2-chloro-6-(isopropylamino)-9H-purin-9-yl)-3ethynyl-3,4-dihydroxytetrahydrofuran-2 -yl)methoxy)-3-(4-(3-(3-chloropropyl)-ureido)phenyl)-2-(thiazol4-yl)propanoate (1.23 g) in THF (12 ml) was added NaH (290, 84 mg, 7.27 mmol, 60% in mineral oil). The mixture was stirred at 15°C for 5 hours, then quenched with H2O (6 ml) followed by the addition of NaOH (290.85 mg, 7.27 mmol). The mixture was stirred at 15°C for 16 hours and then at 40°C for 8 hours. Additional NaOH (600 mg) was added to the mixture and the mixture was stirred at 40°C for 4 hours, after which the mixture was quenched with water ( 20 ml). The resulting solution was extracted using EtOAc (15 ml). Vodn. the layer was acidified using 2 N aq. HCl (15 ml) to form a precipitate. The solid was collected by filtration and purified by preparative HPLC (column: YMCActus Triart C18 150-30 mm-5 µm; mobile phase: [water (0.225%FA)-ACN]; B%: 30%-50%, 10 min) to give a diastereomeric mixture (approximately 1:1) 2-(((2R,3S,4R,5R)-5-(2-chloro-6(isopropylamino)-9H-purin-9-yl)-3-ethynyl-3 ,4-dihydroxytetrahydrofuran-2-yl)methoxy)-3-(4-(2oxotetrahydropyrimidin-1(2H)-yl)phenyl)-2-(thiazol-4-yl)propanoic acid (245 mg) as a white solid .

1H NMR (400 MHz, CD3OD) δ ppm. 8.99 (d, J=1.6 Hz, 1H), 7.88-8.18 (m, 1H), 7.53-7.79 (m, 1H), 7.30 (d, J= 8.3 Hz, 1H), 7.03-7.13 (m, 2H), 6.95 (d, J=8.4 Hz, 1H), 5.83-6.03 (m, 1H), 4.63-4.74 (m, 1H), 4.33-4.43 (m, 1H), 4.09-4.29 (m, 1H), 3.72-4.01 (m, 3H ), 3.59-3.70 (m, 1H), 3.41-3.53 (m, 2H), 3.33-3.38 (m, 2H), 2.91-3.14 (m , 1H), 1.83-2.05 (m, 2H), 1.25-1.35 (m, 6H); LC/MS [M+H] = 697.4.

Example 177. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-chloro-4-(isopropylamino)-1H-pyrazolo[3,4d]pyrimidin-1 -yl)-3-ethynyl- 3,4-dihydroxytetrahydrofuran-2-yl)methoxy)-3-phenyl-2-(thiazol-4yl)propanoic acid

Example 1

Stage 1.

To a solution of 4,6-dichloro-1H-pyrazolo[3,4D]pyrimidine (620 mg, 1.11 mmol, 1 equiv.) and (3R,4R,5R)-5(((1-ethoxy-1-oxo -3-phenyl-2-(thiazol-4-yl)propan-2-yl)oxy)methyl)-4-ethynyltetrahydrofuran-2,3,4-triyltriacetate (230.35 mg, 1.22 mmol, 1.1 eq.) in MeCN (6.5 mL) under N2 at 0°C, add DBU (501 μL, 3.32 mmol, 3 eq.). The mixture was stirred at 0°C for 5 minutes, then added dropwise

- 141 046381

TMSOTf (900.93 µl, 4.99 mmol, 4.5 eq). The mixture was stirred at 0°C for 30 min and then at 65°C for 16 h before quenching with saturated aq. NaHCO ₃ solution (10 ml) and extracted using EtOAc (3x6 ml). The combined organic phase was dried over anhydrous Na ₂ SO ₄ , filtered and evaporated. The crude residue was purified by silica gel flash column chromatography (10-40% EtOA in petroleum ether) to give (2R,3R,4R,5R)-5-(4,6-dichloro-1Hpyrazolo[3,4d]pyrimidine- 1-yl)-2-((( 1-ethoxy-1-oxo-3-phenyl-2-(thiazol-4-yl)propan-2-yl)oxy)methyl)3-ethynyltetrahydrofuran-3,4-diyldiacetate (220 mg) as foam.

Stage 2.

K (2R,3R,4R,5R)-5-(4,6-dichloro-1H-pyrazolo[3,4^]pyrimidin-1-yl)-2-((( 1-ethoxy-1-oxo-3 -phenyl-2(thiazol-4-yl)propan-2-yl)oxy)methyl)-3-ethynyltetrahydrofuran-3,4-diyldiacetate (260 mg, 377.61 µmol, 1 eq.) in EtOH (2 ml) propan-2-amine (64.89 µl, 755.23 µmol, 2 equiv.) and DIEA (131.55 µl, 755.23 µmol, 2 equiv.) were added. The mixture was stirred at 15°C for 4 hours, then diluted with EtOAc (30 ml), washed with water (8 ml), brine (8 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated. The crude residue was purified by preparative TLC (EtOAc:petroleum ether=2:1) to give (2R,3R,4R,5R)-5-(6-chloro-4-(isonropylamino)-1H-pyrazolo[3,4- d]pyrimidin-1yl)-2-(((1-ethoxy-1-oxo-3-phenyl-2-(thiazol-4-yl)propan-2-yl)oxy)methyl)-3-ethynyltetrahydrofuran-3, 4diyldiacetate (70 mg) in the form of foam.

Stage 3.

To a solution of (2R,3R,4R,5R)-5-(6-chloro-4-(isonropylamino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2(((1-ethoxy- 1-oxo-3-phenyl-2-(thiazol-4-yl)propan-2-yl)oxy)methyl)-3-ethynyltetrahydrofuran-3,4-diyldiacetate (70 mg, 98.43 µmol, 1 eq.) LiOH-H ₂ O (4.13 mg) was added to THF (1 ml). The mixture was stirred at 50°C for 14 hours before evaporation to dryness. The crude residue was purified by preparative HPLC (column: YMC-Actus Triart C18 150-30 mm-5 µm; mobile phase: [water (0.225%FA)-ACN]; B%: 43% - 63%, 10 min) and dried by lyophilization to obtain a diastereomeric mixture (approximately 1:1) 2-(((2R,3S,4R,5R)-5-(6-chloro-4-(isopropylamino)-1H-pyrazolo[3,4-d]pyrimigine -1-yl)-3ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)-3-phenyl-2-(thiazol-4-yl)propanoic acid (15.7 mg) as a white solid.

1H NMR (400 MHz, CD3OD) δ ppm. 8.76-9.10 (m, 1H), 7.93-8.08 (m, 1H), 7.40-7.65 (m, 1H), 6.89-7.08 (m, 4H ), 6.80-6.88 (m, 1H), 6.11-6.20 (m, 1H), 5.14-5.28 (m, 1H), 4.38-4.50 (m , 1H), 4.29-4.37 (m, 1H), 3.934.11 (m, 1H), 3.78-3.86 (m, 1H), 3.47-3.63 (m, 2H ), 2.97-3.09 (m, 1H), 1.25-1.31 (m, 6H); LC/MS [M+H] = 598.7.

Example 178. Synthesis of 2-(((2R,3S,4R,5R)-5-(4-amino-6-chloro-1H-pyrazolo[3,4d]pyrimidin-1-yl)-3ethynyl-3,4- dihydroxytetrahydrofuran-2-yl)methoxy)-3-phenyl-2-(thiazol-4-yl)propanoic acid

Example 17S

Stage 1.

To a solution of (2R,3R,4R,5R)-5-(4,6-dichloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-(((1-ethoxy-1-oxo3 -phenyl-2-(thiazol-4-yl)propan-2-yl)oxy)methyl)-3-ethynyltetrahydrofuran-3,4-diyldiacetate (100 mg, 145.24 µmol, 1 eq.) in THF (1 ml ) NH4OH (199.76 µl, 1.45 mmol, 10 eq.) was added. The mixture was stirred at 15°C for 14 hours and then evaporated to dryness to obtain crude (2R,3R,4R,5R)-5-(4-amino-6-chloro-1H-pyrazolo-[3,4-d] pyrimidin-1-yl)-2-(((1-ethoxy-1-oxo-3-phenyl-2(thiazol-4-yl)propan-2-yl)oxy)methyl)-3-ethynyltetrahydrofuran-3,4 -diyl diacetate (120 mg) as a white solid.

Stage 2.

To a solution of crude (2R,3R,4R,5R)-5-(4-amino-6-chloro-1H-pyrazolo-[3,4-d]pyrimidin-1-yl)2-(((1-ethoxy- 1-oxo-3-phenyl-2-(thiazol-4-yl)propan-2-yl)oxy)methyl)-3-ethynyltetrahydrofuran-3,4-diyldiacetate (145.24 µmol, 1 eq.) in THF ( 4 ml) and H2O (2 ml), LiOH-H ₂ O (60.94 mg, 1.45 mmol, 10 eq.) was added. The mixture was stirred at 50°C for 16 hours before evaporation to dryness. The crude residue was purified by preparative HPLC (column: YMC-Actus Triart C18 150-30 mm-5 µm; mobile phase: [water (0.225%FA)-ACN]; B%: 28% - 48%, 10 min) and dried by lyophilization to obtain a diastereomeric mixture (approximately 1:1) 2-(((2R,3S,4R,5R)-5-(4-amino-6-chloro-1Hpyrazolo[3,4^]pyrimidin-1 -yl) -3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)-3-phenyl-2(thiazol-4-yl)propanoic acid (32.8 mg) as a white solid.

1H NMR (400 MHz, CD3OD) δ ppm. 8.87-9.01 (m, 1H), 7.91-8.06 (m, 1H), 7.47-7.67 (m, 1H), 7.01-7.06 (m, 2H ), 6.93-7.00 (m, 2H), 6.84-6.90 (m, 1H), 6.12-6.20 (m, 1H), 5.14-5.27 (m , 1H), 4.26-4.33 (m, 1H), 3.764.03 (m, 2H), 3.48-3.70 (m, 2H), 2.96-3.05 (m, 1H ); LC/MS [M+H] = 557.0.

- 142 046381

Example 179. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl) methoxy)-2-(thiazol-4-yl)acetic acid

Priz'er 1

Stage 1.

To a solution of (2R,3R,4R,5R)-5-((6-N,N'-bis-(tert-butoxycarbonyl)amino)-2-chloro-9H-purin-9-yl)-3ethynyl-2- (hydroxymethyl)tetrahydrofuran-3,4-diyldiacetate (2 g, 3.28 mmol) in toluene (10 ml) was added ethyl 2-diazo-2-(thiazol-4-yl) acetate (841 mg, 4.26 mmol) and Rh ₂ (OAc) ₄ (145 mg, 0.328 mmol) under argon. The resulting mixture was stirred at 70°C for 2 hours, after which it was left to cool to room temperature. Volatile organic compounds were removed under reduced pressure. The resulting crude product was silica gel column chromatography (0-40% EtOAc in hexane) to give (2R,3R,4R,5R)-5-(6-(bis-(tert-butoxycarbonyl)amino)-2-chloro- 9Hpurin-9-yl)-2-((2-ethoxy-2-oxo-1-(thiazol-4-yl)ethoxy)methyl)-3-ethynyltetrahydrofuran-3,4-diyldiacetate (1.78 g) as syrup.

Stage 2.

To a solution of (2R,3R,4R,5R)-5-(6-(bis-(Ίret-butoxycarbonyl)αmino)-2-chloro-9H-purin-9-yl)-2-((2ethoxy-2-oxo -1-(thiazol-4-yl)ethoxy)methyl)-3-ethynylte1rahydrofuran-3,4-diyldiacetate (310 mg) in DCM (3 ml) was added with TFA (2 ml) at 25°C. The mixture was stirred for 2 hours and then evaporated under reduced pressure to give crude (2R,3R,4R,5R)-5-(6-amino-2-chloro-9H-purin-9yl)-2-((2- ethoxy-2-oxo-1-(thiazol-4-yl)ethoxy)methyl)-3-ethynyltetrahydrofuran-3,4-diyldiacetate.

To a solution of crude (2R,3R,4R,5R)-5-(6-αmino-2-chloro-9H-purin-9-yl)-2-((2-ethoxy-2-oxo-1(thiazol-4 -yl)ethoxy)methyl)-3-ethynyltetrahydrofuran-3,4-diyldiacetate in THF (1 ml) and H ₂ O (1 ml) at 0°C, LiOH monohydrate (100 mg) was added. The resulting mixture was stirred at 25°C overnight, after which volatile organic compounds were removed under reduced pressure. The mixture was cooled to 0°C and acidified to pH ~6 using 1 N aq. HCl solution and evaporated under reduced pressure. The crude residue was purified by preparative reverse phase HPLC to give a diastereomeric mixture (approximately 1:1) 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purine-9 -yl)-3ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)-2-(thiazol-4-yl)acetic acid as a white solid.

^1H NMR (CD3OD, 300 MHz) δ 8.84-9.00 (m, 2H), 7.67-7.68 (m, 1H), 6.02-6.06 (m, 1H), 5 .28-5.32 (d, J=12.27 Hz, 1.5H), 5.14-5.16 (d, J=7.56 Hz, 0.5H), 4.24-4.28 (m, 1H), 3.69-4.09 (m, 2H), 3.16 (s, 0.5H), 2.95 (s, 0.5H); LC/MS [M+H] = 467.0.

Examples 180 and 181. Synthesis of (S)-2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3, 4dihydroxytetrahydrofuran-2-yl)methoxy)-3-phenyl-2-(thiazol-4-yl)propanoic acid and (R)-2(((2R,3S,4R,5R)-5-(6-amino-2 -chloro-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)-3-phenyl-2-(thia ^> ol-4-yl)yropanoic acid

Proceeding as described in Example 169 above, but replacing 3-(bromomethyl)thiophene and 2-chloro-Nisopropyl-9-purine-6-amine with benzyl bromide and 2-chloroadenine, a pair of diastereomeric products was obtained, the stereoconfiguration of which was arbitrary. Both products were purified by preparative HPLC and isolated as white solids.

(S)-2-(((2R,3S,4R,5R)-5-(6-Amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2- yl)methoxy)-3-phenyl-2-(thiazol-4-yl)propanoic acid 1H NMR (CD ₃ OD, 300 MHz) δ 8.95-8.96 (d, J=2.01 Hz, 1H) , 8.34 (s, 1H), 7.54-7.55 (d, J=2.01 Hz, 1H), 6.97-7.12 (m, 5H), 5.97-5.99 (d, J=6.99 Hz, 1H), 4.97-4.99 (d, J=7.08 Hz, 1H), 4.27-4.29 (t, J=4.23, 3 ,18 Hz, 1H), 3.88-3.99 (m, 2H), 3.62-3.79 (k, J=14.82, 39.24 Hz, 2H), 2.97 (s, 1H); LC/MS [M+H] = 557.0.

- 143 046381 (R)-2-(((2R,3S,4R,5R)-5-(6-Amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran -2-yl)methoxy)-3-phenyl-2-(thiazol-4-yl)propanoic acid 1H NMR (CD ₃ OD, 300 MHz) δ 8.95-8.96 (m, 1H), 7.99 (s, 1H), 7.70-7.71 (d, J=1.98 Hz, 1H), 7.05-7.25 (m, 5H), 5.92-5.94 (d, J =7.02 Hz, 1H), 4.85-4.87 (d, J=7.29 Hz, 1H), 4.20-4.22 (k, J=2.64 Hz, 1H), 3 .58-3.90 (m, 4H), 3.02 (s, 1H); LC/MS [M+H] = 557.0.

Example 182. Synthesis of 3-(2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2 -yl)methoxy)-2-carboxy-2-(thiazol-4-yl)ethyl)benzoic acid

Proceeding as described in Example 179 above, but replacing BnBr with methyl 3-(bromomethyl)benzoate, the title compound was obtained as a mixture of diastereomers (approximately 1:1) and isolated as a white solid.

1H NMR (CD3OD, 300 MHz) δ 8.99-9.01 (m, 1H), 8.33 (s, 0.5H), 8.14 (s, 0.5H), 7.66-7, 88 (m, 3H), 7.457.48 (d, J=7.29 Hz, 0.5H), 7.36-7.39 (d, J=7.86 Hz, 0.5H), 7.17 -7.22 (t, J=7.56 Hz, 0.5H), 7.00-7.05 (d, J=7.47 Hz, 0.5H), 5.99-6.01 (d , J=7.29 Hz, 0.5H), 5.93-5.95 (d, J=6.87 Hz, 0.5H), 5.00-5.03 (d, J=7.38 Hz, 0.5H), 4.904.95 (d, J=6.80 Hz, 0.5H), 4.23-4.31 (m, 1H), 3.80-4.01 (m, 2H) , 3.63-3.69 (m, 2H), 3.01 (s, 0.5H), 2.92 (s, 0.5H); LC/MS [M+H] = 601.0.

Example 183. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl) methoxy)-2-(thiazol-4-yl)-3-(3-(trifluoromethoxy)phenyl)propanoic acid

Proceeding as described in Example 179 above, but replacing BnBr with 1-(bromomethyl)-3(trifluoromethoxy)benzene, the title compound was obtained as a mixture of diastereomers (approximately 1:1) and isolated as a white solid.

1H NMR (CD3OD, 300 MHz) δ 8.97-9.00 (m, 1H), 8.41 (s, 0.5H), 8.26 (s, 0.5H), 7.68-7, 69 (d, J=1.92 Hz, 0.5H), 7.62-7.63 (d, J=1.83 Hz, 0.5H), 6.88-7.21 (m, 4H) , 6.00-6.02 (d, J=7.14 Hz, 0.5H), 5.94-5.96 (d, J=6.78 Hz, 0.5H), 5.04-5 .07 (d, J=7.44 Hz, 0.5H), 4.91-4.94 (d, J=6.87 Hz, 0.5H), 4.28-4.33 (m, 1H ), 3.62-3.96 (m, 4H), 2.98 (s, 0.5H), 2.96 (s, 0.5H); LC/MS [M+H] = 641.0.

Example 184. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl) methoxy)-2-(thiazol-4-yl)pent-4-ynic acid

Proceeding as described in Example 179 above, but replacing BnBr with propargyl bromide, the title compound was obtained as a mixture of diastereomers (approximately 1:1) and isolated as a white solid.

1H NMR (CD ₃ OD, 300 MHz) δ 8.95 (s, 1H), 8.79-8.83 (d, J=13.95 Hz, 1H), 7.73 (s, 1H), 6 .04-6.06 (d, J=7.05 Hz, 1H), 4.97-5.05 (dd, J=7.29, 17.91 Hz, 1H), 4.24-4.30 (m, 1H), 3.69-3.94 (m, 2H), 3.34-3.38 (m, 2H), 3.04 (s, 0.5H), 2.93 (s, 0 ,5H), 2.22-2.30 (dt, J=1.74, 19.62 Hz, 1H); LC/MS [M+H] = 505.0.

Examples 185 and 186. Synthesis of (S)-2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3, 4dihydroxytetrahydrofuran-2-yl)methoxy)-X-hydroxy-2-(thiazol-4-yl)acetamide (S)-2(((2R,3S,4R,5R)-5-(6-amino-2-chloro -9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)-H-hydroxy-2-(thiazol-4-yl)acetamide

(2R,3R,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-2-((2-ethoxy-2-oxo-1-(thiazol -4K solution yl )ethoxy)methyl)-3-ethynyltetrahydrofuran-3,4-diyldiacetate (2 g, 3.45 mmol) in MeOH (20 ml) adding

- 144 046381 50% NH2OH was added to H2O (30 ml). The mixture was stirred at 40°C for 1 hour and then evaporated under reduced pressure. The crude residue was purified by preparative reverse phase HPLC to provide a pair of diastereomeric title products, the stereoconfiguration of which was randomly assigned. Both products were isolated as white solids.

(S)-2-(((2R,3S,4R,5R)-5-(6-Amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2- yl)methoxy)-N-hydroxy-2-(thiazol-4-yl)acetamide: 1H NMR (CD ₃ OD, 300 MHz) δ 9.02-9.03 (d, J=1.77 Hz, 1H) , 8.45 (s, 1H), 7.68-7.69 (d, J=1.8 Hz, 1H), 5.98-6.01 (d, J=6.96 Hz, 1H), 4.92-4.95 (d, J=6.93 Hz, 1H), 4.29-4.32 (k, J=2.82, 2.28 Hz, 1H), 3.91-4, 10 (m, 2H), 3.18 (s, 1H); LC/MS [M+H] = 482.0.

(R)-2-(((2R,3S,4R,5R)-5-(6-Amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2- yl)methoxy)-N-hydroxy-2-(thiazol-4-yl)acetamide ¹ H NMR (CD3OD, 300 MHz) δ 8.97 (s, 1H), 8.46 (s, 1H), 7.68 (s, 1H), 5.97-6.00 (d, J=6.96 Hz, 1H), 4.88-4.90 (d, J=6.93 Hz, 1H), 4.33- 4.35 (m, 1H), 3.96-4.08 (m, 2H), 3.12 (s, 1H); LC/MS [M+H] = 482.0.

Examples 187 and 188. Synthesis of (S)-2-(((2R,3S,4R,5R)-5-(2-chloro-6-(methylamino)-9H-purin-9-yl)-3ethynyl-3, 4-dihydroxytetrahydrofuran-2-yl)methoxy)-3-phenyl-2-(thiazol-4-yl)propanoic acid and (R)-2-(((2R,3S,4R,5R)-5-(2- chloro-6-(methylαamino)-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)-3-phenyl-2-(thiazol-4-yl)propanoic acid

Proceeding as described in Examples 170 and 171 above, but replacing 2-chloro-N-isopropyl-9H-purine-6-amine with 2-chloro-N-methyl-9H-purine-6-amine, the title compound was obtained as diastereomer pairs (approximately 1:1) and isolated as white solids.

(S)-2-(((2R,3S,4R,5R)-5-(2-Chloro-6-(methylamino)-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran- 2-yl)methoxy)-3-phenyl-2-(thiazol-4-yl)propanoic acid: 1H NMR (CD3OD, 300 MHz) δ 8.99 (s, 1H), 7.97 (s, 1H), 7.69 (s, 1H), 7.08-7.22 (m, 5H), 5.93-5.95 (d, J=6.9 Hz, 1H), 4.96-4.98 ( d, J=6.0 Hz, 1H), 3.59-4.22 (m, 5H), 3.01-3.06 (m, 4H); LC/MS [M+H] = 571.0.

(R)-2-(((2R,3S,4R,5R)-5-(2-Chloro-6-(methylamino)-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran- 2-yl)methoxy)-3-phenyl-2-(thiazol-4-yl)propanoic acid: 1H NMR (CD3OD, 300 MHz) δ 8.978.98 (d, J=1.83 Hz, 1H), 8, 15 (s, 1H), 7.58-7.59 (d, J=1.77 Hz, 1H), 6.94-7.09 (m, 5H), 5.98-6.00 (d, J=7.17 Hz, 1H), 4.98-5.01 (d, J=7.26 Hz, 1H), 4.27-4.29 (t, J=3.48 Hz, 1H), 3.93 (m, 2H), 3.58-3.80 (k, J=14.46, 38.7 Hz, 2H), 3.07 (s, 3H), 2.94 (s, 1H) ; LC/MS [M+H] = 571.0.

Examples 189 and 190. Synthesis of (S)-2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3, 4dihydroxytetrahydrofuran-2-yl)methoxy)-2-cyano-3-phenylpropanoic acid and (R)-2(((2R,3S,4R,5R)-5-(6-αmino-2-chloro-9H-purine- 9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)-2-cyano-3-phenylpropanoic acid

Stage 1.

To a solution of (2R,3R,4R,5R)-5-(6-N,N'-(bis-(tert-butoxycarbonyl)amino)-2-chloro-9H-purin-9-yl)-3ethynyl-2- (hydroxymethyl)tetrahydrofuran-3,4-diyldiacetate (510 mg, 0.836 mmol) in toluene (5 ml) was added H ethyl 2-cyano-2-diazoacetate (134 mg, 0.961 mmol). The mixture was evaporated in vacuo. The mixture was placed in dry toluene (2 ml) followed by the addition of Rh ₂ (OAc) ₄ (8 mg, 17 µmol) under argon. The material was stirred and heated at 80°C for 30 min before adding additional ethyl 2-cyano-2-diazoacetate (254 mg, 1.82 mmol) for 60 min at 80°C. The reaction mixture was further heated at 80°C for 80 min, then cooled to room temperature and evaporated. The resulting crude product was subjected to combi-flash silica gel chromatography (5-65% EtOAc in hexanes) to yield the diastereomeric mixture (2R,3R,4R,5R)-5-(6-N,N'-(bis-(tert-butoxycarbonyl) amino)-2-chloro-9H-purin-9-yl)-2-((1-cyαno-2-ethoxy-2-oxoethoxy)methyl)-3-ethynyltetrahydrofuran-3,4-diyldiacetate (230 mg, yield 38 %) in the form of almost white foam.

- 145 046381

Stage 2.

((2R,3R,4R,5R)-5-(6-N,N'-(6uc-(mpeTbutoxycarbonyl)amino)-2-chloro-9H-purin-9-yl)-2 was loaded into an oven-dried flask -((1-cyano-2-ethoxy-2-oxoethoxy)methyl)-3-ethynyltetrahydrofuran-3,4-diyldiacetate (230 mg, 0.319 mmol) and combined with dry DMF (4 ml) To this mixture was added Cs ₂ CO ₃ (208 mg, 0.639 mmol) followed by the addition of benzyl bromide (109 mg, 0.639 mmol).The mixture was stirred at 25°C for 30 min, then diluted with cold saturated aqueous NH ₄ Cl (40 ml) and extracted using EtOAc ( 40 ml) The aqueous phases were extracted using EtOAc (2x40 ml). The combined organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and evaporated. The resulting crude product was obtained by combi-flash column chromatography with silica gel (10-70% EtOAc in hexane ) to obtain a diastereomeric mixture (2R,3R,4R,5R)-5(6-N,N'-(bis-(tert-butoxycarbonyl)amino)-2-chloro-9H-purin-9-yl)-2- (((2-cyano-1-ethoxy-1-oxo-3-phenylpropan-2-yl)oxy)methyl)-3-ethynyltetrahydrofuran-3,4-diyldiacetate as a pair of diastereomers (215 mg, 83% yield) in as an almost white solid.

Stage 3.

Solution of (2R,3R,4R,5R)-5-(6-N,N'-(bis-(tert-butoxycarbonyl)amino)-2-chloro-9H-purin-9-yl)-2-((( 2cyano-1-ethoxy-1-oxo-3-phenylpropan-2-yl)oxy)methyl)-3-ethynyltetrahydrofuran-3,4-diyldiacetate (215 mg, 0.265 mmol) in a solution of TFA (1 ml) in DCM (1 ml) was stirred for 2 hours, then evaporated under reduced pressure. The residue was azetropically distilled with DCM (8x8 ml) under reduced pressure. The residue was taken up in MeOH in H2O (2.2 mL, 5:1=v:v) followed by LiOH-H2O (77 mg, 1.86 mmol, 7 eq.) and THF (0.5 mL) . The mixture was stirred at ambient temperature for 40 minutes before evaporating to dryness. The resulting precipitate was dissolved in H2O (15 ml). The aqueous phase was extracted using EtOAc (2x10 ml). The aqueous phase was acidified to pH 2.5 using 1 N aq. HCl solution. The aqueous phase was extracted using EtOAc (3x50 ml). The combined organic layer was washed with brine (50 ml), dried over Na ₂ SO ₄ , filtered and evaporated. The crude residue was purified by preparative reverse phase HPLC to give the title compound as a pair of diastereomers: (S)-2(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H -purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)-2-cyano-3-phenylpropanoic acid (33.7 mg, 26% yield) and (R)-2- (((2R,3S,4R,5R)-5-(6-amino-2chloro-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)-2-cyano -3-phenylpropanoic acid (30 mg, 23% yield), the stereoconfiguration of which was arbitrarily assigned. Both compounds were isolated as almost white solids.

(S)-2-(((2R,3S,4R,5R)-5-(6-Amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2- yl)methoxy)-2-cyano-3-phenylpropanoic acid: ^1H NMR (CD3OD, 300 MHz) δ 8.32 (br. s, 1H), 7.20-7.36 (m, 5H), 6, 02 (d, J=7.00 Hz, 1H), 4.68 (d, J=7.02 Hz, 1H), 4.33-4.37 (m, 1H), 4.18 (dd, J =9.91, 3.96 Hz, 1H), 3.99 (dd, J=9.94, 2.19 Hz, 1H), 3.41 (br. s, 2H), 3.03 (s, 1H); LC/MS [M+H] = 499.1.

(R)-2-(((2R,3S,4R,5R)-5-(6-Amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2- yl)methoxy)-2-cyano-3-phenylpropanoic acid: 1H NMR (CD3OD, 300 MHz) δ 8.04 (br. s, 1H), 7.21-7.39 (m, 5H), 5.99 (d, J=6.90 Hz, 1H), 4.79 (d, J=6.93 Hz, 1H), 4.31-4.36 (m, 1H), 4.19 (dd, J= 10.07, 4.34 Hz, 1H), 4.12 (dd, J=10.10, 3.30 Hz, 1H), 3.38-3.44 (m, 2H), 3.09 (s , 1H); LC/MS [M+H] = 499.1.

Examples 191 and 192. Synthesis of (S)-2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3, 4dihydroxytetrahydrofuran-2-yl)methoxy)-3-phenyl-2-(1H-tetrazol-5-yl)propanoic acid and (R)-2(((2R,3S,4R,5R)-5-(6-amino -2-chloro-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)-3-phenyl-2-(1H-tetrazol-5-yl)propanoic acid

Stage 1.

To a solution of ethyl 1H-tetrazole-5-acetate (3 g, 19.21 mmol) in DMF (40 ml) under argon at 25°C was added 2-(trimethylsilyl)ethoxymethyl chloride (4.1 ml, 23.05 mmol ) and powdered potassium carbonate (5.31 g, 38.42 mmol). The reaction mixture was stirred overnight, then diluted with brine (70 ml) and EtOAc (70 ml). The aqueous phase was extracted using EtOAc (2x70 ml).

- 146 046381

The organic layer was washed with brine (70 ml) and water (70 ml), dried over Na ₂ SO ₄ and evaporated. The residue was purified by silica gel column chromatography (15-48% EtOAc in hexanes) to give ethyl 2-(2-((2-(trimethylsilyl)ethoxy)methyl)-2H-tetrazol-5-yl)acetate (2.379 g) in the form of a light yellow oil.

Stage 2.

DBU (1, 87 ml, 12.47 mmol) under argon. To this mixture was added 4-acetamidobenzenesulfonyl azide (2.395 g, 9.96 mmol) in 3 equal portions over 5 minutes. The reaction mixture was stirred for 3.5 hours, after which volatile organic compounds were removed under reduced pressure. The residue was purified by silica gel column chromatography (20% EtOAc in hexanes) to give ethyl 2-diazo-2-(2-((2-(trimethylsilyl)ethoxy)methyl)2H-tetrazol-5-yl)acetate (2.316 d) in the form of oil.

Stage 3.

To a solution of (2R,3R,4R,5R)-5-(6-(bis-(tert-butoxycarbonyl)amino)-2-chloro-9H-purin-9-yl)-3ethynyl-2-(hydroxymethyl)tetrahydrofuran- 3,4-diyldiacetate (2 g, 3.28 mmol) in toluene (8 ml) at 20°C under N2 was added Rh ₂ (OAc) ₄ (29 mg, 0.066 mmol, 0.066 eq.) and ethyl-2- diazo-2-(2-((2(trimethylsilyl)ethoxy)methyl)-2H-tetrazol-5-yl)acetate (1.08 g, 3.44 mmol, 1.05 eq.). The mixture was stirred at 75°C for 1 hour before adding additional ethyl 2-diazo-2-(2-((2(trimethylsilyl)ethoxy)methyl)-2H-tetrazol-5-yl)acetate (720 mg) for 80 min. The reaction mixture was cooled to ambient temperature and evaporated. The crude material was purified by combi-flash silica gel column chromatography (5-80% EtOAc in hexanes) to give the diastereomeric mixture (2R,3R,4R,5R)-5-(6-(bis-(tert-butoxycarbonyl)amino) -2-chloro-9H-purin-9-yl)-2((2-ethoxy-2-oxo-1-(2-((2-(trimethylsilyl)ethoxy)methyl)-2H-tetrazol-5-yl) ethoxy)methyl)-3-ethynyltetrahydrofuran-3,4-diyldiacetate (1.608 g) as gum.

Stage 4.

To a solution of (2R,3R,4R,5R)-5-(6-(bis-(tert-butoxycarbonyl)amino)-2-chloro-9H-purin-9-yl)-2-((2ethoxy-2-oxo -1-(2-((2-(trimethylsilyl)ethoxy)methyl)-2H-tetrazol-5-yl)ethoxy)methyl)-3-ethynyltetrahydrofuran-3,4-diyldiacetate (1.555 g, 1.739 mmol) in dry toluene (10 ml). The mixture was evaporated under reduced pressure. The residue was dissolved in anhydrous DMF (10 ml) and then benzyl bromide (1.189 g, 6.96 mmol) and dried Cs ₂ CO ₃ (1.133 g, 3.478 mmol) were added. The mixture was stirred at 25°C for 5.5 hours before diluting with saturated aq. NH ₄ Cl solution (60 ml). The aqueous phase was extracted using EtOAc (3x60 ml). The combined organic layer was washed with brine (60 ml), dried over Na ₂ SO ₄ , filtered and evaporated. The crude product was purified by silica gel flash column chromatography (5-65% EtOAc in hexanes) to give the diastereomeric mixture (2R,3R,4R,5R)-5-(6-(bis-(tert-butoxycarbonyl)amino)- 2-chloro-9H-purin-9-yl)-2-(((1-ethoxy1-oxo-3-phenyl-2-(2-((2-(trimethylsilyl)ethoxy)methyl)-2H-tetrazole-5 -yl)propan-2-yl)oxy)methyl)-3ethynyltetrahydrofuran-3,4-diyldiacetate (1.041 g) as gum.

Stage 5.

To a solution of (2R,3R,4R,5R)-5-(6-(bis-(tert-butoxycarbonyl)amino)-2-chloro-9H-purin-9-yl)-2-(((1ethoxy-1- oxo-3-phenyl-2-(2-((2-(trimethylsilyl)ethoxy)methyl)-2H-tetrazol-5-yl)propan-2-yl)oxy)methyl)-3-ethynyltetrahydrofuran-3,4- diyl diacetate (1.041 g, 1.057 mmol) in a mixture of MeOH and H2O (12 ml, 6:1=v:v) was added powdered LiOH-H ₂ O (349 mg, 8.5 mmol). The mixture was stirred at 23°C for 16 hours before evaporation to dryness. The residue was diluted with H ₂ O (40 ml), then extracted using EtOAc (40 ml). The aqueous phase was acidified to pH 2.5 using 1 N aq. HCl solution and extracted using EtOAc (3x40 ml). The combined organic layer was washed with brine (40 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated to form a diastereomeric mixture of 2-(((2R,3S,4R,5R)-5-(6-((tert-butoxycarbonyl )amino)-2-chloro-9H-purin-9-yl)-3ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)-3-phenyl-2-(2-((2-(trimethylsilyl)ethoxy) Methyl)2H-tetrazol-5-yl)propanoic acid (784 mg) as an oil.

Stage 6.

To a solution of 2-(((2R,3S,4R,5R)-5-(6-((tert-butoxycarbonyl)amino)-2-chloro-9H-purin-9-yl)-3-ethynyl3,4-dihydroxytetrahydrofuran -2-yl)methoxy)-3-phenyl-2-(2-((2-(trimethylsilyl)ethoxy)methyl)-2H-tetrazol-5-yl)propanoic acid (138 mg, 0.179 mmol) in DCM (0 .9 ml) TFA (0.9 ml) was added under argon at 0°C. The mixture was stirred at 0°C for 5 hours and then stirred at ambient temperature for 15 minutes, after which the volatile organic compounds were removed under reduced pressure. The residue was azetropically distilled with DCM (3x15 ml) under reduced pressure. The crude residue was purified by preparative reverse phase HPLC to provide the two title compounds as a pair of diastereomers: (S)-2-(((2R,3S,4R,5R)-5-(6-amino-2chloro-9H -purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)-3-phenyl-2-(1H-tetrazol5-yl)propanoic acid from the first fraction and (R)-2- (((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)-3- phenyl-2-(1H-tetrazol-5-yl)propane acid

- 147 046381 slots from the subsequent faction. Both compounds were isolated as almost white solids.

(S)-2-(((2R,3S,4R,5R)-5-(6-Amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4-digvdroxitetragvdrofuran-2- yl)methoxy)-3-phenyl-2-(III-tetrazol-5-yl)propanoic acid: 1H NMR (CD3OD, 300 MHz); δ 8.37 (s, 1H), 7.11-7.23 (m, 5H), 5.96 (d, J=6.57 Hz, 1H), 4.81 (d, J=6.57 Hz, 1H), 4.25-4.30 (m, 1H), 4.01 (dd, J=10.19, 2.29 Hz, 1H), 3.78 (d, J=13.90 Hz , 1H), 3.67 (d, J=13.90 Hz, 1H), 3.72-3.79 (m, 1H), 3.06 (s, 1H); LC/MS [M+H] = 542.2.

(R)-2-(((2R,3S,4R,5R)-5-(6-Amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2- yl)methoxy)-3-phenyl-2-(1H-tetrazol-5-yl)propanoic acid: ¹ H NMR (CD3OD, 300 MHz); δ 8.37 (s, 1H), 6.92-7.11 (m, 5H), 6.01 (d, J=7.11 Hz, 1H), 5.06 (d, J=7.11 Hz, 1H), 4.35-4.39 (m, 1H), 4.11 (dd, J=10.06, 2.52 Hz, 1H), 4.01 (dd, J=10.06, 5.49 Hz, 1H), 3.80 (d, J=14.75 Hz, 1H), 3.67 (d, J=14.75 Hz, 1H), 2.96 (s, 1H); LC/MS [M+H] = 542.2.

Examples 193 and 194. Synthesis of (S)-2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3, 4dihydroxytetrahydrofuran-2-yl)methoxy)-2-(2-phenylthiazol-4-yl)acetic acid and (R)-2(((2R,3S,4R,5R)-5-(6-amino-2-chloro -9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)-2-(2-phenylthiazol-4-yl)acetic acid

Proceeding as described in Example 179, but replacing ethyl 2-diazo-2-(thiazol-4-yl) acetate with ethyl (2-phenylthiazol-4-yl) diazoacetate, which was prepared according to the procedure described by Lefebvre, Quentin, et al. (Chemical Communications 2014, 50, 6617-6619), obtained the title compounds as a pair of diastereomers (approximately 1:1). The stereo configuration was set arbitrarily. Both compounds were isolated as almost white solids.

(S)-2-(((2R,3S,4R,5R)-5-(6-Amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2- yl)methoxy)-2-(2-phenylthiazol-4-yl)acetic acid: 1H NMR (CD3OD, 300 MHz); δ 9.03 (br.s, 1H), 7.91-7.97 (m, 2H), 7.60 (s, 1H), 7.39-7.45 (m, 3H), 6.06 (d, J=7.45 Hz, 1H), 5.28 (s, 1H), 4.92 (d, J=7.45 Hz, 1H), 4.29-4.33 (m, 1H) , 4.12 (dd, J=10.45, 2.51 Hz, 1H), 4.01 (dd, J=10.46, 2.56 Hz, 1H), 2.92 (s, 1H); LC/MS [M+H] = 543.1.

(R)-2-(((2R,3S,4R,5R)-5-(6-Amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2- yl)methoxy)-2-(2-phenylthiazol-4-yl)acetic acid: 1H NMR (CD3OD, 300 MHz); δ 9.15 (s, 1H), 7.90-7.96 (m, 2H), 7.61 (s, 1H), 7.38-7.44 (m, 3H), 6.09 (d , J=7.48 Hz, 1H), 5.23 (d, J=7.48 Hz, 1H), 5.30 (s, 1H), 4.26-4.29 (m, 1H), 3 .93 (dd, J=10.67, 2.18 Hz, 1H), 3.76 (dd, J=10.64, 2.48 Hz, 1H), 3.17 (s, 1H); LC/MS [M+H] = 543.2.

Example 195. Synthesis of 4-((((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl )methoxy)(carboxy)methyl)thiazole-2-carboxylic acid

NiBosja

Przyr 195

Stage 1.

Ethyl thioxamate (1.91 g, 14.36 mmol) and ethyl 4-bromoacetoacetate (3 g, 14.36 mmol) in dry toluene (27 ml) were charged into a microwave-safe vessel. The mixture was irradiated in a microwave reactor

- 148 046381 at 90°C for 1 hour. The reaction mixture was cooled to ambient and the solvent was decanted and then evaporated. The crude residue was purified by combi-flash silica gel chromatography (2-56% EtOAc in hexanes) to give ethyl 4-(2-ethoxy-2-oxoethyl)thiazole-2-carboxylate (890 mg, 26% yield) in in the form of thick oil.

Stage 2.

To a solution of ethyl 4-(2-ethoxy-2-oxoethyl)thiazole-2-carboxylate (890 mg, 3.66 mmol) in dry acetonitrile (12 ml) under argon was added DBU (0.82 ml, 5.49 mmol) and 4-acetamidobenzenesulfonyl azide (1.055 g, 4.39 mmol). The reaction mixture was stirred for 1.5 hours, after which it was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (20% EtOAc in hexanes) to provide ethyl 4-(1-diazo-2-ethoxy-2-oxoethyl)thiazole-2-carboxylate (894 mg, 90% yield) as a yellowish solid. .

Stage 3.

Proceeding as described in example 179, but replacing ethyl 2-diazo-2-(thiazol-4-yl) acetate with ethyl 4-(1diazo-2-ethoxy-2-oxoethyl)thiazole-2-carboxylate, we obtained the following: the title compound as a mixture of diastereomers (approximately 1:1) and isolated as an off-white solid.

Isomer 1: 1H NMR (CD3OD, 300 MHz): δ 8.93 (s, 1H), 7.91 (s, 1H), 6.06 (d, J=7.42 Hz, 1H), 5.39 (s, 1H), 5.13 (d, J=7.42 Hz, 1H), 4.24-4.31 (m, 1H), 4.03-4.09 (m, 1H), 3, 75-3.83 (m, 1H), 2.95 (s, 1H); LC/MS [M+H] = 511.1.

Isomer 2: 1H NMR (CD3OD, 300 MHz): δ 8.87 (s, 1H), 7.93 (s, 1H), 6.04 (d, J=7.42 Hz, 1H), 5.33 (s, 1H), 4.91 (d, J=7.45 Hz, 1H), 4.24-4.31 (m, 1H), 3.92-4.01 (m, 2H), 3, 19 (s, 1H); LC/MS [M+H] = 511.1.

Example 196. Synthesis of 4-(1-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2 -yl)methoxy)-1-carboxy-2-phenylethyl)thiazole-2-carboxylic acid

Proceeding as described in Example 179 above, but replacing (2R,3R,4R,5R)-5-(6-N,N'-(bis-(tert-butoxycarbonyl)amino)-2-chloro-9H-purine-9- yl)-3-ethynyl-2-(hydroxylmethyl)tetrahydrofuran-3,4-diyldiacetate on (2R,3R,4R,5R)-5-(6-N,N-(bis-(tert-butoxycarbonyl)amino)) -2-chloro-9H-purin-9-yl)-2-((2ethoxy-1-(2-(ethoxycarbonyl)thiazol-4-yl)-2-oxoethoxy)methyl)-3-ethynyltetrahydrofuran-3,4- diyl diacetate gave the title compound as a mixture of diastereomers (approximately 1:1) and isolated as an off-white solid.

Isomer 1: 1H NMR (CD ₃ OD, 300 MHz): δ 8.17 (br.s, 1H), 7.81 (s, 1H), 6.92-7.25 (m, 5H), 5. 95 (d, J=7.02 Hz, 1H), 4.89 (d, J=7.02 Hz, 1H), 4.29-4.34 (m, 1H), 3.59-4.05 (m, 4H), 3.01 (s, 1H); LC/MS [M+H] = 601.1.

Isomer 2: 1H NMR (CD ₃ OD, 300 MHz): δ 8.05 (br.s, 1H), 7.90 (s, 1H), 6.92-7.25 (m, 5H), 6. 00 (d, J=7.41 Hz, 1H), 5.04 (d, J=7.42 Hz, 1H), 4.21-4.26 (m, 1H), 3.59-4.05 (m, 4H), 3.06 (s, 1H); LC/MS [M+H] = 601.1.

Example 197. Synthesis of 2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl) methoxy)-2-(4-(2-aminopyridin-3-yl)benzyl)malonic acid

Proceeding as described in Example 22 above, but replacing (2-oxo-1,2-dihydropyridin-3-yl)boronic acid with 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane- 2-yl)pyridin-2-amine gave the title compound as a white solid.

1H NMR (CD ₃ OD, 300 MHz) δ 8.43 (s, 1H), 7.85-7.87 (dd, J=1.5, 6.42 Hz, 1H), 7.65-7, 68 (dd, J=1.53, 7.38 Hz, 1H), 7.37-7.40 (d, J=8.13 Hz, 2H), 7.09-7.12 (d, J= 8.07 Hz, 2H), 6.88-6.93 (t, J=6.9 Hz, 1H), 5.99-6.01 (d, J=6.72 Hz, 1H), 4, 77-4.79 (d, J=7.0, 1H), 4.37-4.40 (m, 1H), 3.98-4.12 (m, 2H), 3.34-3.42 (m, 2H), 3.09 (s, 1H); LC/MS [M+H] = 610.1.

- 149 046381

Examples 198 and 199. Synthesis of (S)-3-([1,1'-biphenyl]-4-yl)-2-(((2R,3S,4R,5R)-5-(6-αmino-2- chloro-9Hpurin-9-yl)-3,4-digvdroxy-3-methyltetrahydrofuran-2-yl)methoxy)-2-(thiazol-4-yl)propanoic acid and (R)-3-([1,1' -biphenyl]-4-yl)-2-(((2R,3S,4R,5R)-5-(6-αmino-2-chloro-9H-purin-9-yl)-3,4-dihydroxy-3methyltetrahydrofuran -2-yl)methoxy)-2-(thiazol-4-yl)propanoic acid

Proceeding as described in Example 1 above, but substituting (2R,3R,4R,5R)-5-(6-(bis-(tert-butoxycarbonyl)amino)-2-chloro-9H-purin-9-yl)-2 -(((tert-butyldiphenylsilyl)oxy)methyl)-3-ethynyltetrahydrofuran-3,4-diyldiacetate and diethyl-2-diazomalonate on (2R,3R,4R,5R)-5-(6-(N,N'- bis-(tertbutoxycarbonyl)αmino)-2-chloro-9H-purin-9-yl)-2-(hydroxylmethyl)-3-methyltetrahydrofuran-3,4-diyldiacetate and methyl 2-diazo-2-(thiazol-4-yl )acetate, a pair of diastereomeric title products (approximately 1:1) was obtained, the stereoconfiguration of which was arbitrarily assigned. Both products were purified by preparative HPLC and isolated as white solids.

(S)-3-([1,1'-Biphenyl]-4-yl)-2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purine- 9-yl)-3,4-dihydroxy-3methyltetrahydrofuran-2-yl)methoxy)-2-(thiazol-4-yl)propanoic acid: ¹ H NMR (CD ₃ OD, 300 MHz) δ 9.04 (s, 1H), 8.42 (s, 1H), 7.74 (s, 1H), 7.21-7.45 (m, 9H), 6.00-6.03 (d, J=8 Hz, 1H ), 4.67-4.70 (d, J=7 Hz, 1H), 4.13 (s, 1H), 3.88-3.85 (m, 2H), 3.62-3.66 ( d, J=14 Hz, 1H), 3.44-3.47 (d, J=11 Hz, 1H), 1.37 (s, 3H); LC/MS [M+H] = 623.2.

(R)-3-([1,1'-Biphenyl]-4-yl)-2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purine- 9-yl)-3,4-dihydroxy-3methyltetrahydrofuran-2-yl)methoxy)-2-(thiazol-4-yl)propanoic acid: ^1H NMR (CD3OD, 300 MHz) δ 9.04 (s, 1H) , 8.21 (s, 1H), 7.27-7.69 (m, 10H), 5.93-5.95 (d, J=7 Hz, 1H), 4.52-4.55 (d , J=8 Hz, 1H), 4.04 (s, 1H), 3.79-3.85 (m, 3H), 1.36 (s, 3H); LC/MS [M+H] = 623.2.

Example 200. Synthesis of 4'-(2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3,4-ethynyl-3dihydroxytetrahydrofuran- 2-yl)methoxy)-2-carboxy-2-(thiazol-4-yl)ethyl)-[1,1'-biphenyl]-2-carboxylic acid

179 above, but replacing (2R,3R,4R,5R)-5-((6-N,N'-(bis-(tertActing as described in the example butoxycarbonyl)amino)-2-chloro-9H-purine-9 -yl)-3-ethynyl-2-(hydroxylmethyl)tetrahydrofuran-3,4-diyldiacetate and BnBr on (2R,3R,4R,5R)-5-(6-N,N'-(bis-(tert-butoxycarbonyl )amino))-2-chloro-9H-purin-9yl)-2-((2-methoxy-2-oxo-1-(thiazol-4-yl)ethoxy)methyl)-3-ethynyltetrahydrofuran-3,4- diyl diacetate and 4(bromomethyl)-1,1'-biphenyl gave the title compound as a mixture of diastereomers (approximately 1:1) and isolated as an off-white solid.

LC/MS [M+H]=667.2.

- 150 046381

Example 201. Synthesis of 4'-(2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3,4-ethynyl-3dihydroxytetrahydrofuran- 2-yl)methoxy)-2-carboxy-2-(1H-tetrazol-5-yl)ethyl)-[1,1'-biphenyl]-2-carboxylic acid

Stage 1.

To a solution of ethyl 2-(III-tetrazol-5-yl)acetate (500 mg, 3.24 mmol) and trimethylsilyl)ethoxymethyl chloride (0.69 ml, 3.89 mmol) in anhydrous DMF (7 ml) under argon at Powdered potassium carbonate (896 mg, 6.48 mmol) was added at 25°C. The reaction mixture was stirred overnight, then diluted with H2O (30 ml) and extracted using EtOAc (3x30 ml). The combined organic layer was washed with brine (30 ml) and water (30 ml), dried over Na ₂ SO ₄ and evaporated. The residue was purified by silica gel column chromatography (8-58% EtOAc in hexanes) to give ethyl 2-(2-((2-(trimethylsilyl)ethoxy)methyl)-2H-tetrazol-5-yl)acetate (200 mg ) in the form of oil.

Stages 2-6.

Proceeding as described in Example 1 above, but replacing methyl 2-(thiazol-4-yl) acetate with ethyl 2-(2-((2(trimethylsilyl)ethoxy)methyl)-2H-tetrazol-5-yl) acetate, a pair of diastereomeric title products (approximately 1:1) was obtained, the stereoconfiguration of which was arbitrarily specified. Both products were purified by preparative HPLC and isolated as white solids.

^1H NMR (CD3OD, 300 MHz): Isomer 1: δ 8.50 (s, 1H), 7.73-7.79 (m, 2H), 7.31-7.56 (m, 3H), 7 .01-7.24 (m, 4H), 6.04 (d, J=7.87 Hz, 1H), 4.64 (d, J=7.88 Hz, 1H), 4.10-4, 14 (m, 1H), 3.46-4.00 (m, 4H), 1.35 (s, 3H); Isomer 2: δ 8.31 (s, 1H), 7.73-7.79 (m, 2H), 7.31-7.56 (m, 3H), 7.01-7.24 (m, 4H ), 5.99 (d, J=7.75 Hz, 1H), 4.48 (d, J=7.72 Hz, 1H), 4.19-4.23 (m, 1H), 3.46 -4.00 (m, 4H), 1.42 (s, 3H); LC/MS [M+H] = 652.2.

Example 202. Synthesis of (((((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2-yl) methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid

Stage 1.

To a solution of (2R,3R,4R,5R)-5-(6-(bis-(tert-butoxycarbonyl)amino)-2-chloro-9H-purin-9-yl)-3ethynyl-2-(hydroxymethyl)tetrahydrofuran- 3,4-diyldiacetate (329 mg, 0.539 mmol) in i-PrOH (1.6 ml), MeOH (1.1 ml) and H2O (0.8 ml) was added powdered LiOH (111 mg, 2.69 mmol) . The mixture was stirred for 30 min, after which the volatile organic compounds were removed under reduced pressure and the residue was diluted with H2O (12 ml). The pH of the aqueous layer was adjusted to ~3 using 1 N aq. HCl solution and extracted using EtOAc (3x12 ml). The combined organic layer was dried over MgSO ₄ , filtered and evaporated to give tert-butyl (2-chloro-9-((2R,3R,4S,5R)-4-ethynyl-3,4dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2 -yl)-9H-purin-6-yl)carbamate, which was directly used in the next step without further purification.

Stage 2.

A solution of tert-butyl (2-chloro-9-((2R,3R,4S,5R)-4-ethynyl-3,4-dihydroxy-5-(hydroxymethyl)tet

- 151 046381 ragidofuran-2-yl)-9H-purin-6-yl)carbamate (0.539 mmol) was combined with a mixture of DCM (1 ml) and TFA (0.5 ml). The reaction mixture was stirred for 3 hours, then it was evaporated. The residue was combined with DCM (10 ml) and evaporated again (5 cycles were repeated). The residue was further dried in a vacuum oven for 18 hours to obtain crude (2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-ethynyl-2(hydroxymethyl )tetrahydrofuran-3,4-diol as an almost white solid.

Stage 3.

Crude (2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin9-yl)-3-ethynyl-2-(hydroxymethyl)tetrahydrofuran-3,4- was loaded into an oven-dried flask. diol and dry trimethylphosphate (2.5 ml) under argon. The mixture was cooled to 0°C followed by the addition of a solution of methylenebis(phosphonic dichloride) (673 mg, 2.7 mmol) in dry trimethylphosphate (1.1 ml) dropwise over 10 min. The reaction mixture was stirred at 0°C for 3 hours, after which a solution of triethylammonium carbonate (1 M, 1.9 ml) was added dropwise. The mixture was stirred for 15 minutes at 0°C and then stirred for 2 hours at ambient temperature. The crude mixture was purified by preparative reverse phase HPLC to obtain the crude product. This crude product was further purified twice by reverse phase HPLC to obtain the desired (((((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3 -ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid (34 mg) as a light brown solid.

1H NMR (CD3OD, 300 MHz): δ 8.75 (br.s, 1H), 6.07 (br.s, 1H), 4.86 (br.s, 1H), 4.31-4.61 ( m, 3H), 3.20 (s, 1H), 2.54 (br., 1H); LC/MS [M+H] = 484.0.

Example 203. Synthesis of (((((2R,3S,4R,5R)-5-(6-(benzylαamino)-2-chloro-9H-purin-9-yl)-3-ethynyl-3,4dihydroxytetrahydrofuran-2- yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid

Stage 1.

In a nitrogen atmosphere, a solution of (3aR,5R,6R,6aR)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-6-ethynyl2,2-dimethyltetrahydrofuro[2,3D][1,3]dioxol-6-ol (14.48 g, 32 mmol) (prepared using the methods described in Hulpia, F. et al. Bioorg. Med. Chem. Lett. 2016, 26, 1970-1972) in acetic acid (130 ml) cooled to 14 -17°C and treated with acetic anhydride (32.01 ml, 341 mmol, 10.7 eq.) and concentrated sulfuric acid (576 μl, 10.8 mmol, 0.34 eq.). After stirring for 2.5 hours, the mixture was diluted with ethyl acetate (200 ml each) and washed with water. The aqueous phase was extracted with ethyl acetate (25 ml) and the combined organic solution was washed with sodium bicarbonate (aq, saturated, 200 ml), dried over Na ₂ SO ₄ , filtered and evaporated. The residual oil was purified by silica gel flash column chromatography (0-3% ethyl acetate in dichloromethane) to give (3R,4R,5R)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-4-ethynyltetrahydrofuran-2. 3,4-triyltriacetate as a mixture of anomers and isolated as a white solid in good yield (9.5 g, 55%).

Stage 2.

Under nitrogen, 2,6-dichloroadenine (2.91 g, 15.4 mmol, 1.01 eq.) and N,O-6uc(trimethylsilyl)acetamide (4.87 mL, 19.6 mmol, 1.29 eq. .) in anhydrous acetonitrile (90 ml) was stirred at room temperature. A solution of (2R,3R,4R,5R)-2,4-bis-(acetyloxy)-5{[(tert-butyldiphenylsilyl)oxy]methyl}-4-ethynyloxolan-3-yl acetate (8.2 g, 15 .22 mmol) in anhydrous acetonitrile (10 ml) followed by dropwise addition of trimethylsilyl trifluoromethanesulfonate

- 152 046381 (3.67 ml, 20.3 mmol, 1.33 eq.). The reaction mixture was heated to 50°C for 18 h, then cooled to room temperature. Saturated aqueous sodium bicarbonate (10 ml) was added and the mixture was stirred for ten minutes. The resulting mixture was extracted with ethyl acetate (3x100 ml), and the combined organic layer was dried (Na ₂ SO ₄ ), filtered and evaporated. The residue was purified by silica gel flash column chromatography (0-30% ethyl acetate in dichloromethane) to give (2R,3R,4R,5R)-2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-(2, 6-Dichloro-9H-purin-9-yl)-3-ethynyltetrahydrofuran-3,4-diyldiacetate as a white solid (8.2 g, 81%).

Stage 3.

Solution of (2R,3R,4R,5R)-2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-(2,6-dichloro-9H-purin-9-yl)-3ethynyltetrahydrofuran-3,4-diyldiacetate (1.6 g, 2.4 mmol) in anhydrous THF (25 ml) was cooled to 0°C and treated with acetic acid (0.192 ml, 3.36 mmol) and tetrabutylammonium fluoride in THF (1 N, 3.36 ml, 3.36 mmol). After addition was complete, the reaction mixture was warmed to room temperature and stirring continued for 3 hours. The reaction mixture was evaporated. The reaction mixture was purified by flash column chromatography (0-50% ethyl acetate in hexanes) to give (2R,3R,4R,5R)-5-(2,6-dichloro-9H-purin-9-yl)-3- ethynyl 2-(hydroxymethyl)tetrahydrofuran-3,4diyldiacetate (0.88 g, 86%) as a white foam.

Stage 4.

(2R,3R,4R,5R)-5-(2,6-dichloro-9H-purin-9-yl)-3-ethynyl-2-(hydroxymethyl)tetrahydrofuran3,4-diyldiacetate solution (100 mg, 0.233 mmol) in trimethylphosphate (4 ml) was cooled to 0°C and treated with a second solution of methylenebis-(phosphonodichloride) (116 mg, 0.467 mmol, 2 eq.) in trimethylphosphate (4 ml). After addition was complete, stirring was continued for 2 hours, then the cooling bath was removed and stirring was continued for another 18 hours. Ammonium bicarbonate (0.7 M aqueous TEAB, pH 8.5) was added slowly with vigorous stirring until no more gas is released. After quenching, NaHC.'O ₃ (saturated, aq.; 5 ml) was added and the mixture was stirred for 1 hour at room temperature. The reaction mixture was washed with dichloromethane, acidified with 2 N HCl to pH ~ 1 and extracted with ethyl acetate (10x50 ml). The combined organic layer was dried over sodium sulfate, filtered and evaporated in vacuo. The residual oil was azeotroped with toluene (3x10 ml) to give (((((2R,3R,4R,5R)-3,4-diacetoxy-5-(2,6-dichloro-9H-purin-9yl)-3 -ethynyltetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid as an off-white solid, which was used in the next step without further purification.

Stage 5.

Solution (((((2R,3R,4R,5R)-3,4-diacetoxy-5-(2,6-dichloro-9H-purin-9-yl)-3-ethynyltetrahydrofuran2-yl)methoxy)(hydroxy) Phosphoryl)methyl)phosphonic acid from step 4 (~90 mg) was dissolved in anhydrous dioxane (8 ml), cooled to 0°C, then treated with diisopropylethylamine (0.085 ml, 0.513 mmol, 2.2 eq.) and benzylamine (0.036 ml , 0.33 mmol, 1.4 eq.). After addition was complete, the reaction mixture was stirred at room temperature for 18 hours and evaporated to give (((((2R,3R,4R,5R)-3,4-diacetoxy-5-(6-(benzylamino)-2-chloro- 9H-purin-9-yl)-3-ethynyltetrahydrofuran2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid. The crude product was directly used in subsequent hydrolysis without further purification.

The crude product from step 5 was dissolved in 1:1 MeOH/THF (2 ml) and treated with LiOH (84 mg, 3.5 mmol, 15 eq.) in water (1 ml). After addition was complete, the reaction mixture was stirred at room temperature for 18 h, then acidified to pH ~ 1 using 2 N HCl and evaporated. The resulting reaction mixture was diluted 1:1 with acetonitrile in water with 0.1% TFA (4 mL) and purified by reverse phase HPLC to give (((((2R,3S,4R,5R)-5-(6-(benzylamino) -2-chloro-9H-purin-9-yl)-3ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid as a white solid (9.2 mg, 7% ) after lyophilization.

1H NMR (D2O) δ 8.63 (s, 1H), 7.41 (m, 5H), 6.07 (d, J=6.9 Hz, 1H), 4.96 (d, J=7, 0 Hz, 1H), 4.46 (s, 1H), 4.37 (d, J=12.0 Hz, 1H), 4.25 (d, 1=11.5Hz, 1H), 3.21 ( s, 1H), 2.38 (t, J=20.0 Hz, 2H). HPLC: Rt=17.2 min, 97.9%. ESI-MS for C ₂₁ H ₂₄ ClN ₅ O ₉ P ₂ calculated 587.07, found 586.8 (M-); ESI-MS for C ₁₂ H ₉ ClN ₅ calculated 258.05, found 258.4 (M-ribose fragment).

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Example 204. Synthesis of ((((2R,3S,4R,5R)-5-(6-(6eH3HTaMUHo)-2-xnop-9H-nypuH-9-un)-3^TUHun-3,4dihydroxytetrahydrofuran-2-yl )methoxy)methyl)phosphonic acid

Stage 1.

A flame dried round bottom flask was charged with diethyl hydroxymethylphosphonate (780 mg, 4.64 mmol) and triethylamine (0.838 mL, 6.031 mmol, 1.3 eq) in anhydrous dichloromethane (20 mL), cooled to -78°C and treated dropwise with trifluoromethane -sulfonic anhydride (0.847 ml, 5.10 mmol, 1.3 eq.). The reaction mixture was stirred for 10 minutes while the reaction mixture warmed to 0°C. After 30 minutes, the reaction mixture was poured into ether (pre-cooled to 0°C) and the crystalline precipitate was filtered. The filtrate was then washed successively with water (1x100 ml), 1 M HCl (1x100 ml) and saturated aqueous sodium chloride solution (1x125 ml). The organic layer was dried (MgSO ₄ ), filtered and evaporated to give crude (diethoxyphosphoryl)methyl trifluoromethanesulfonate as a yellow oil, dissolved in anhydrous THF and this solution was directly used in the next step without further purification.

Stage 2.

Solution of (2R,3R,4R,5R)-5-(2,6-dichloro-9H-pyrin-9-yl)-3-ethynyl-2-(hydroxylmethyl)tetrahydrofuran-3,4-diyldiacetate (350 mg, 0.815 mmol) and (diethoxyphosphoryl)methyltrifluoromethanesulfonate (294 mg, 0.978 mmol, 1.2 eq.) in THF (20 ml) were cooled to -78°C and treated dropwise with LiHMDS (1M in THF; 0.980 ml, 0.978 mmol, 1. 2 eq.). After stirring for 1.5 hours, the reaction was quenched with solid NH ₄ Cl, diluted with water and extracted with ethyl acetate. The organic layer was dried (MgSO ₄ ), filtered and evaporated. The mixture was purified by flash column chromatography (0-100% ethyl acetate in hexanes) to give (2R,3R,4R,5R)-5-(2,6-dichloro-9H-purin-9-yl)-2-( ((diethoxyphosphoryl)methoxy)methyl)-3-ethynyltetrahydrofuran-3,4-diyldiacetate (140 mg, 30%) as a pale yellow oil.

Stage 3.

Solution of (2R,3R,4R,5R)-5-(2,6-dichloro-9H-pyrin-9-yl)-2-(((diethoxyphosphoryl)methoxy)methyl)-3ethynyltetrahydrofuran-3,4-diyldiacetate (85 mg, 0.147 mmol) and diisopropylethylamine (40 μl, 0.235 mmol, 1.6 eq.) in anhydrous dioxane (8 ml) were cooled to 0°C, treated with benzylamine (19 μl, 0.176 mmol, 1.2 eq.). After addition was complete, the reaction mixture was warmed to room temperature and stirred for 18 hours. The mixture was diluted with water and extracted with ethyl acetate. The organic layer was dried (Na ₂ SO ₄ ), filtered and evaporated to give crude (2R,3R,4R,5R)-5-(6-(benzylamino)-2-chloro-9H-purin-9-yl)-2- (((diethoxyphosphoryl)methoxy)methyl)-3-ethynyltetrahydrofuran-3,4-diyldiacetate (93 mg, 96%) as a white solid, which was directly used in the next step without further purification.

Stage 4.

The crude product from the previous step was dissolved in anhydrous acetonitrile (10 ml) and treated dropwise with bromotrimethylsilane (0.24 ml, 1.8 mmol, 12 eq.). After addition was complete, the solution was stirred at room temperature for 22 hours and quenched with water (5 ml). After stirring for an additional 2-3 minutes, the solution was extracted with ethyl acetate (4x100 ml). The organic layer was dried (Na ₂ SO ₄ ), filtered and evaporated to give ((((2R,3S,4R,5R)-5-(6-(benzylamino)-2chloro-9H-nyrin-9-yl)-3- ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)methyl)phosphonic acid as an almost white solid. This crude solid was dissolved in absolute EtOH at 0°C and treated with KOEt (51 mg, 0.61 mmol, 4 eq.) in one portion. The reaction mixture was stirred at room temperature for 20 minutes, then it was acidified with AcOH (0.52 ml, 0.91 mmol, 6 eq.) and stirred for an additional 10 minutes. The crude product was purified by reverse phase HPLC and lyophilized to give ((((2R,3S,4R,5R)-5-(6-(benzylamino)-2-chloro-9H-purin-9yl)-3-ethynyl -3,4-dihydroxytetrahydrofuran-2-yl)methoxy)methyl)phosphonic acid (6 mg, 7%) as a white solid.

¹ H NMR (d2O) δ 8.41 (s, 1H), 7.19 (m, 5H), 5.84 (d, J=7.2 Hz, 1H), 4.82 (d, J=7 ,1 Hz, 1H), 4.21 (m, 1H), 4.19 (m, 2H), 3.79 (d, J=3.8 Hz, 2H), 3.57 (m, 2H), 2.98 (s, 1H). HPLC: Rt=7.19 min, 97.5%. ESI-MS for C ₂₀ H ₂₁ ClN ₅ O ₇ P calculated 509.09, found 509 (M+); ESI-MS for C11H8QN6 calculated 258.05, found 259 (M-ribose fragment).

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Example 205. Synthesis of (((((2R,3S,4R,5R)-5-(6-(benzylamino)-2-chloro-9H-purin-9-yl)-3,4-dihydroxy-3dihydroxytetrahydrofuran-2- yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid

Dov bde

4. aq. LiOH, THF, SHON

Example 205

Stage 1.

In a nitrogen atmosphere, an ice-cooled solution of (3aR,5R,6aS)-5-(((tertbutyldiphenylsilyl)oxy)methyl)-2,2-dimethyldihydrofuro[2,3-d][1,3]dioxol-6(5H)- it (6 g, 14.1 mmol) in anhydrous THF (100 ml) was treated dropwise with 3M methylmagnesium chloride in THF (5.9 ml, 1.4 eq.). After addition was complete, the cooling bath was removed and stirring was continued for 1 hour. The mixture was again cooled to 0°C and quenched with saturated aqueous ammonium chloride (10 ml), diluted with ethyl acetate (100 ml) and washed with water (80 ml). The aqueous layer was re-extracted with ethyl acetate (1x50 ml) and the combined organic layer was dried over sodium sulfate, filtered and evaporated. The residue was purified by silica gel flash column chromatography (0-30% ethyl acetate in hexane) to give (3aR,5R,6R,6aR)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-2,2,6trimethyltetrahydrofuro [2,3D][1,3]dioxol-6-ol as a light viscous oil (4.2 g, 67%).

Stage 2.

In a nitrogen atmosphere, a solution of (3aR,5R,6aS)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-2,2dimethyltetrahydrofuro[2,3-d][1,3]dioxol-6(5H)-one ( 2.25 g, 5.08 mmol) in dichloromethane (35 ml) and water (3.5 ml) were cooled to 0°C and treated with trifluoroacetic acid (15 ml). After 2.5 hours, saturated aqueous NaHCO ₃ was added until the pH of the solution reached ~8 and the mixture was extracted with dichloromethane (2x150 ml). The combined organic layer was dried over sodium sulfate, filtered and evaporated. The crude oil was azeotroped with toluene (3x5 ml), diluted with dichloromethane (45 ml), treated with pyridine (12 ml), acetic anhydride (4.77 ml, 50.83 mmol) and catalytic 4-DMAP (142 mg, 1. 17 mmol). After stirring for 18 hours, the reaction mixture was diluted with ethyl acetate (200 ml each) and washed successively with saturated aqueous NH4Cl (3x100 ml), 0.5 N HCl (2x100 ml) and saturated aqueous sodium chloride (1x120 ml). The combined organic layer was dried over sodium sulfate, filtered and evaporated. The residual oil was purified by silica gel flash column chromatography (0-30% ethyl acetate in hexane) to give (3R,4R,5R)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-4-methyltetrahydrofuran-2. 3,4triyl triacetate (2.1 g, 78%) as a colorless solid.

Stages 3-6.

Proceeding as described in Example 203 above, but replacing (3aR,5R,6R,6aR)-5-(((m-retbutyldiphenylsilyl)oxy)methyl)-6-ethynyl-2,2-dimethyltetrahydrofuro[2,3D][1, 3]-dioxol-6-ol on (3aR,5R,6aS)-5-(((mret-butyldiphenylsilyl)oxy)methyl)-2,2-dimethyldihydrofuro[2,3-d][1,3] dioxol6(5H)-one, the title compound (8 mg, 3%) was obtained as a white solid.

1H NMR (d2O:DMSOD _& 6:1) 8.46 (br. s, 1H), 7.23 (m, 5H), 5.84 (d, J=6.9 Hz, 1H), 4.45 (d, J=7.0 Hz, 1H), 4.12 (s, 1H), 3.95 (m, 3H), 3.54 (m, 1H), 2.18 (br. s, 2H) , 1.31 (s, 3H). ESI-MS for CH _; 4Cl\,OP _; calculated 563.1, found 562.1 (M-).

Example 206 Assay 1: CD73 enzyme inhibition in vitro.

To measure soluble CD73 enzyme activity, recombinant CD73 was obtained from R&D Systems, Cat. No. 5795-EN-010. Serial dilutions of the test compounds were incubated with recombinant CD73 and AMP in reaction buffer (25 mM Tris-HCl pH 7.5, 5 mM MgCl ₂ , 50 mM NaCl, 0.25 mM DTT, 0.005% Triton X-100). The final reaction volume was 25 μL, and the final concentrations of recombinant CD73 and AMP were 0.5 nM and 50 μM, respectively. Reactions were allowed to continue for 30 min at room temperature before adding 100 μl of malachite green (Cell Signaling Technology, Cat. No. 12776). After 5 min at room temperature, the optical density at 630 nm was determined using a microplate spectrophotometer. Inorganic phosphate concentration was determined using a phosphate standard curve.

Quantitation Assay 2: In Vitro CD73 Enzyme Inhibition.

To measure soluble CD73 enzyme activity, recombinant CD73 was obtained from R&D Systems, Cat. No. 5795-EN-010. Serial dilutions of analyzed compounds incubated

- 155 046381 were mixed with recombinant CD73 and AMP in reaction buffer (25 mM Tris-HCl pH 7.5, 5 mM MgCl ₂ , 50 mM NaCl, 0.25 mM DTT, 0.005% Triton X-100). The final reaction volume was 25 μL, and the final concentrations of recombinant CD73 and AMP were 0.05 nM and 50 μM, respectively. Reactions were allowed to continue for 1 hour at 37°C before adding 100 μl of malachite green (Cell Signaling Technology, Cat. No. 12776). After 5 min at room temperature, the optical density at 630 nm was determined using a microplate spectrophotometer. Inorganic phosphate concentration was determined using a phosphate standard curve.

IC ₅₀ data for both analyzes are given in table. 2. BUT means not defined.

table 2

Example No. Compound Analysis 1 CD73 IC50 (nM) Analysis 2 CD73 IC50 (nM) 1 o nh ₂ H 4 ^0H ν^Λ _μ Yes NO J O-d o A ήΛι (V >2 ......... —4·---- but ' Ън BUT 1.7

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2 o NH ₂ o _h Uon n^L, HO/ O-do O Un HO 147 3 0 »2 0 Uon N^X %.........../ / | ^N Λ yy^^xy HO OH HO 1 4 0 NH ₂ О У OH |sr X yy < Il ^{N Л} ^С! ^UuHn^ Oh HO 0.5 5 o mn ₂ o Uona y X yy < ^z Ύ, ®Лхо7 ^Л Л| ci-^/XYY Yu HO 4 6 0 0 <Y ^H 7% HO JO“-w _o NY ° ns? Ън 52 HO 7 O NH ₂ O _X У OH N-^X, % / ,// у ^X N io ibAolA/a О ХУ N 4'< % Htf ΌΗ HO 8 8 0 NH ₂ o U-on hk X b<< D 1 but/ _o n^ _n X _c| У ХУ ^u HO OH HO 2

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9 o nh ₂ O y_Q|_| W Ο ΐ Д/ °Лх°у ^N^CI 0 f J ^H0< bН nm^ BUT 0.17 10 o ΝΗ ₂ O V-0H hk/L >7 HO / Ovo/^N^CI o £ I -/ ^{but bn} But 0.26 eleven o nh ₂ O y_ _{he N} 1 N < ^z J 1 ™ ^Ν %=1 °. O AA l—/ BUT OH Z^'N tt -o But 0.06 12 O NH ₂ o U-on g*i bG but /o-y ₀ n^ _n ^4 _c| з 0 ^0 V/ but' He HN 1 But 0.1 13 o nh ₂ o y~on n^-A y.............7 ”V°y Ho'i / -ξ no' Ън o But 0.3 14 $0^0 сА^-о 1 ft °'’ 1I L ° But 0.6

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15 About NHj O U-ON s 4 ъГ <20 but' Ън BUT 20 16 O MH ₂ O U-OH U uG ^HO /°-^oy^N^ ^c i no' bn But 24 17 O ΝΗ ₂ 0 У ОН n4x ьГ <Ду ^Н ° ^N ^ ^CI no' Ън But 347 18 U,S o T 0 У ОН N-^Ух n fl! Ϊ уоу °“А ОyN'^N^CI £ 3 ^еЛ / but' Ън But 0.3 19 o nh ₂ 0 U-ON 1\kUx b< Sh no u o-z _o Ν\ ^Λ α 0 C/ I -Z c G< Uch LJ pi Un ~^y^N 1 But 0.2 20 o nh ₂ yU ⁿ Uy-ν,ο P _M A _C1 o y J ^=9-4__/ Chu^^ but' Ън y^-N a But 0.2 21 O ΝΗ ₂ 0 У-ОН N-^Ux уЧ с 1ι J^y °»уу ^A ^ci ⁰ 1J ^*Lu Uuno''on H0-4N 1 \r~U But 0.3

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22 0 ΝΗ ₂ o Χ—on n Jx A......../ f | NsGu b-τι Q _N hf^CI O L-ch b~~7 BUT OH o BUT 0.3 23 BUT νη ₂ o '>=O y A e li ϊ BUT 1 O— _ Ν·% .ι-ίΑ, _F \7 n^ci рД-f/A / but' he 5 BUT 24 ϋ)— Z j3° ^{x xO} 41 L g θ o' ¹ \/...... A At Α~^2Ζ m o 1.8 But 25 but νη ₂ 0 \=О Αχ Ут\ С ιίι but Ad ₀ nA _n A _c| // \ / ¹ H V·*·** ? but he 7 But 26 o nh ₂ Ο _λ Aon Ν^Α _μ bl < ^ζ 20 ^Н0 °Д0 SO but He 58 But 27 but νη ₂ QV=O |sj Χχ............/ HO Ολ,οΑ^Ν^ΟΙ nA but He HNxO 18 But

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28 O 0^,0 ill? η o'L^ ° [ O I O 3 HO 29 but _h nh ₂ /.............\ s 1ι ^χ HO O-woyqA _N A _c| €Α^Α ^{ο he} 2 HO thirty l but NH 0 ™О |\| ^Н0 Ц°^0у ^М ^1<^С1 4 # -% h Υ-Υ but he HO 5 31 BUT ^X 'A o Y=o >7<< ^ζ Υ 1 ^H °J °~y°y ^{N Ν} οι A/ but he HO 2 32 imkh-^^ON BUT ΗΝ °Х Ао N-^D., y........./ with ϋ ^N Γ\θ^Υ°7^ ^x s ^z but He HO 3 33 HO ^NH 0 V=o ^ύ^^ί\ι Л7\ f J ) ^HO I °Vv ^N n ^A ci O ₌ D / ^x s ^z but Oh HO 2

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34 ykG o he ^Μ γ HO-^\^O Ν ° “\-°У \\ zj ~~t·· 3 .......t HO 0H But 2 35 jOH 0 ^OH n \* ^H _H0 AA ₀ ⁰ - Ж ο N ^N^CI O m-< HO OH But 4 36 l7 ⁿ 0 OH HN У ^H0 V° Μ ⁰ “”Л,0 M ¹ 3 //'2ζζ>»4—1 Η0 &Η But 4 37 but ν-χ ζ-^ΟΗ 0 he II L ГкА ηο-'ΜΜ^ο (Υ ^Ν ^ν° γ ^Ν but bn But 6 38 ,0. UKI o ^{he n} 7 1J L· ькМх no^ЧМ^о f | ^{N Ζ} but bn But 3 39 z\^0^ o he II 1 hk Jx HO'-'VMND ^/ γ Ν _/ Μ'°Αχ°γ ^Ν ' ^Λ ' ^Ν ^ι but he But 3

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40 OH O OH ^NG ?^CHZ JJ A A NO-YES=O <R | ^N /=¾^ °~^y°y ^>NN ^ ^CI but bn BUT 3 41 o ο-χΧ^^· X o J O Χΐ'Ό X II g ,1, I °Ko ill zn O \ D But 0.6 42 <yJ ^H 0 he And L IX As, but // y ^N /ААу «Άι _________f H0 He But 1 43 0 OH ΗΝ^^-ΟΗ jj Γ hL Jx °Λ/ _Ο γΝ lAci ~——A—/ ' BUT He But 2 44 D/On o Hhr o _x Don n^A _m but/ °~\ _x .O ₄ J ^M '^'N ⁱⁱ ^CI BUT He But 62

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45 0HN^ o. VoH IN_X, K/\ c jFji NO ο-χ O^^hT^CI o but he But 1 46 ⁿ ^x> o N o, Voh ν^Α _μ v/ ( ^ζ Η ι HO/O^^Q^I n^qi Ηΰ' Ън But 1 47 <^ ^0H O Ν Kon n^-L, ν/ K n '^=*K--Ζ BUT' He But 2 48 o 4/7 o /....... \ / ' X .о- ° J x ^^7 'Ό 1.0 1/1 ¹ сг'о 7^ But 0.5 49 0 /— NH.2 yj“° HO уО—« q F ₃ c^p4 ^: BUT He 273 BUT 50 o NH ₂ v/ ^0H Н0~ He 3 But

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51 ΝΗ·2 Ν < ^Ζ Υ 1 UL he o-l ' ^{ΌΗ 7} u-OH J[ He Cl 3 But 52 nh ₂ N^X // J ^N J ^N Cl OH O-D ' ^OH \\ L—U VOH iff OH / -/ q HI MeO ^u 4 But 53 /X L^ ^and ί z/ ² g J O \ oz t MP O \> o I o But 0.3 54 Ο NH2 o Voh hu JL Wf < ^z Ίι ? _o F/ ^o DL/ ^A mA:| HO i\ ......................../ Htf Ън But 0.2 55 o.^/° ^H O^/O /=N X yyyx /*У-- M Ή ^ζ ηο he Ύ Cl 35 BUT 56 ο νη·2 Ο V-OH Kk Αχ ' W^ci ⁷⁷ no' Ън BUT >200

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57 o nh ₂ o V-on y to Sy <& f ^{Ν s|} but he But 103 58 But SkC ./U Uk BUT / O - ixi'^'^q । But he But 1.6 59 Ph CO-jH μ \ t g—~HCH Li HO ₂ Uo-2f/ ^N yY ^N ^ ^Ph ---- 2 ·, NN HO OH ] CI 1 But 60 Ph CO ₂ H _N kA N k no ₂ s Y ^N \Af Ύ 1 ₌ .....dD............./ 1 IL o no he I CI 3 But 61 ΗΟ ₂ (?^Ο^\^ ^Ο ^ ^Ν уХу ^N ^ ^J T 3 Νχχ,Ν A BUT OH Ύ CI 1.5 But 62 Ph—\CO ₂ H ^_ _{N n} but ₂ s ^o'^s^s* ^N Y^Sf S ⁴ but he k c ci Chk But 2 63 Ph—\CO ₂ H |_| ^Н ° ^2С ^А2Ч°У ^М y^ ⁿ V — - Νί^χΝ but he ] [ η ci kk But 1 64 Ph co ₂ n _n HO ₂ C^O^Y^°k* ^N yАу ^N k^ ^x O =^dd and -.L NO OH ] CI But 3

- 166 046381

65 _Ph ^C0 ₂ H Γ\η ₀ Η HOgC^O^ ⁴ / T^ ^N y^f --* - s N^/ ^N HO OH ] Cl HO 1 66 Ph [ CO ₂ H ^r=N ] ho2c^o^ ⁴ y°\^ ⁿ %Λγ ^Ν -* ; 2 N ^.N but He ] Cl HO 0.3 67 Ph 1 CO ₂ H _H ( I ,-*< 0 N NOchS O l Γ* ,τ i ² ______ 1 1! F --° ' : _ |\| N HO OH ( Cl HO 0.8 68 Ph I CO ₂ H y—— |S| |_| ί Π ^HOaC ^°i^ ^o T ^N v^ ^N '^^ z ₂ N-4/ ^N HO OH ] Cl HO 0.8 69 Ph Lco ₂ h _h HOgC^o^^ ⁰ ^ ^N γ^γ ^N ' (2) ....................... ^: У f—i HO OH IA // ci HO 3 70 Ph I CO2H /τζζΝ |-| HO ₂ C^O'^ ⁴ f°y ^,NN .................G,;; \ / 1 jjj but he ''~| ^Z Cl HO 1 71 HO ^X NH o _x )=O N-^L, b<< J Ϊ ^but 1 °Ύ°ν ^ΝΛΝ % /==( ......... D J but' Oh HO 6

- 167 046381

72 Ph k/ ^C ° ^2H _n /=1 n ^NN VA -/¾% but he U (\ \ ci Kj/ But 3 73 Ph ί 0Ο ₂ Η /=N μ ΗΟ ₂ Ο^Ο^^°Ύ ^N y^y ^N - £ Νί-χ-Ν BUT OH \ CI BUT 1 74 Ph 1уо ₂ n |m Ho ₂ c^O'^y°y ^N уМ''-р n6 bn ^Νΐ γ Cl BUT 2 75 Ph I co ₂ H _{( A} Ηθ ₂ ο^ο^γ °y ^N y^y ^N — - - N ,N HO OH ] Cl But 0.6 76 Ph I CONH ,—ki ., kN^,CO ₂ H but ₂ с о у / i if but bн ^Νΐ γ Cl But 6 77 Ph I COoH J— И u HQ ₂ c^o^ ^Q y ^N y^y ^N but bн ^N y Cl But 2 78 Ph ί 0Ο ₂ Η /-=14 |_| but ₂ с^о'^<°у ^N y^y ^N -- 14 x but he 1 Cl But 2 79 Ph~=co ₂ H _N yy 3h. χ-%. _ζ θ\ κι V N. / HO ₂ CO^Y ν* ^Ν 4^Υζ —- _w \ / 1 1' - - 3 ·, ΝίχχΝ HO OH 1 Cl But 4

- 168 046381

80 Ph—v CO ₂ H y3=N H0 ₂ C^0^7°y* ^N aA ^*4...............( N -.A but he A Cl HO 2 81 Ph ICO ₂ H _N | kA χθχ. κι L Ns. HO ₂ C^O^7 y* ^N Y(f \\ 2 2 N /N HO OH Ύ Cl HO 0.4 82 Ph I co ₂ h । Η0 ₂ 0^0^7 ⁰ ν· ^Ν γ4γ- ^Ν У ...... 2 1- |\| , I ' but he ..... A Cl HO 2 83 Ph ί CO ₂ H ^=Ν μ HO ₂ C^O^/ ^O >* ^N γ^γ ^Ν \~7 - 1 ·, |\| N BUT OH\Cl HO 1 84 Ph l θθ2^Ι ^_|sj |_| i | but ₂ с^оу^°у ^ы уУ^ ^m ^ ^/ ^ ^У _ . ·, Νί^/Ν but he is J Cl HO 3 85 F Ph l CO ₂ H /-=Ν |_| ί HO ₂ C^O^7 ^O 7^ ⁿ . ., _NNHOOH \Cl HO 2 86 Cl ^Ph /¼ l\ CO ₂ H .—n |_| । ηAs. ζθ\ n ks HO ₂ C'O'A^ T^Vlf - ''. Ki - - but he is A Cl HO 1

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87 Ph ^^.Cl LcO ₂ H _H fj HO ₂ C'^O ^x ^y'°y- ^N yAf ^N ............1 KN but it is in Cl HO 0.6 88 Ph—l QOjH /= Ν 1 ~i HO ₂ C'^O ^x ^ ⁴ /°'y ^N yA' ^N -1 N-/N HO OH Y Cl HO 1.2 89 Ph— _x CO,H । x 0 n VN HO ₂ C 0^- ₁ / —' rL N HO OH Cl HO 1.1 90 Ph—\CO ₂ H y\ H0 ₂ C^0'^ ⁴ V°'y ^N xAy ^N —-:...................1 NN but He U Cl 0.9 HO 91 Ph—\ CO ₂ H /=N _H HO ₂ C ^X O^°y ^N Αγ ^Ν Ά) =4b_J _/ 1 V1 L.—J but Oh \ Cl 0.3 HO 92 Ph^ CO ₂ H /=N _n Λ kIu ₂ C^O'2y°>' ^N yAy ^M '^^ · ·, hl -X. x N but he Y Cl 0.8 HO 93 Ph CO ₂ H _ _{N u} HO ₂ <Ао-УУ y,N γ HO OH ^N y Cl 1.7 HO 94 ph—, CO ₂ H ^—hi |_| \ / ^H ° ^2C ^°Ai^ ^N y^ ⁿ but He U Cl HO 5

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95 Ph— CO ₂ H _{H H} o ₂ c ^X o^Y°y ^N γ^γ^ --i N<;x ^N but he Y Cl HO 7 96 Ph ^: —\ COjH ^r=N HO ₂ C^O ^X ^V ^O V· ^N γΚγ ^Ν -.........i Niy ^N but he Y Cl HO 3 97 Ph—. CO ₂ H _H ΗΟ ₂ Ο'^Ο^γ°'γ ^Ν γ^γ ^Ν -^OH =^7—i N^N HO OH I Cl HO 2 98 Ph—, CO ₂ H ^—hl hi HO ₂ C^O^^°^ ^N γ^γ ^Μ *γ^ΟΗ — ^; y N ^>x N but he Y Cl HO 2 99 Ph I CO ₂ H ^__hi Η0 ₂ 0^0^^°γ ^Ν γ^γ ^Ν ^OH — : i HO OH .....T Cl HO 6 100 Ph I UU2r1 rrzzN Г\ .,ni । — : _ N . N HO OH Y Cl HO 2 101 Ph „ OH \CO ₂ H Г ) -.,/ Η0 ₂ (Τθ'^ ⁴ γ ⁰ γ ^Ν γ^γ ^Ν - - but he Y Cl HO 1 102 ^p \co ₂ H YyJ'H HO ₂ C^O^Y^°Y ^N ^Λγ nJ — ^— 2 . N--..N but he is Y Cl HO 1

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103 Ph HO ₂ C^'O'^y°y ^N γ^γ· ^N I ---x Nx; _? ,- ⁿ OH HO OH ] Cl HO 3 104 Ph I COgH ^=n kk ⁿ ° ₂ с^сху ^о у ^N yhr ^N v Ηό bн ^Νΐ γ Cl HO 2 105 Ph CO ₂ H 1 ho ₂ c^o ^x *\'°>- ^{n N} X? HO OH ]Cl HO 2 106 Ph _ V _n / l H /D θν'ο'Χ y ^N Jv ^N -X / v J, /NH -, N HO OH T Cl HO 296 107 Ph 1 COoH _m and g and nV P \ J k 2 ⁰ y ^N |\| Ts Ya—111.D |^| but bn Ύ Cl HO 506 108 Ph (CO ₂ HH £J Eto/Mo''·' ⁴ / y ^N 'V>r' ^N '^| ^/ \............./ 1 y, Cl — - ' N^/N but he is 1 Cl HO 3 109 Ph i CO ₂ H y=N |-| y H BOaC-^O^^y ⁰ ^ ^NN but bn ^Νΐ γ Cl HO 284

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BY X-O \co ₂ n _n - ; - Ν—but he\CI But 3 111 x^o [1 ,N _F \CO ₂ N n G | ho ₂ 4o ^z V°y ⁿ v/f ^N =4—ί N -.A HO OH ] Cl But 2,666 112 l/ ξ^0Ο ₂ Η /-ζχΝ |-| jj y ho ₂ <Xo^'V°N^ ^ν \ ₍ Χ ^/Νχ/ ^ — - ίχΝ BUT OH 7 Cl But 1 357 113 o V ^N V° HO ₂ C^O ^x ^°^ ^N yK^ ^NH2 HO OH Ύ Cl 331 BUT 114 CZn-^jO GT-^bl |-j X ζΟχ m to hk but ₂ c^LU^X 7^ ^N V|f \ / .........g _Ν4χ Ν HO OH 1 Cl 177 BUT 115 CX-OH ^Η2Ν Λ°χ°ζ· ^Ν γΛ ^Ν Λ but' He Cl 135 BUT 116 O^NH.2 ^Η2Ν Λ°Χ°/ ^Ν 4γ ^Ν Χ) o ^=Л—{ Ν'-. L ' BUT OH Ύ CI 8,049 BUT

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117 !—-Η |_| ^H °2 ^C ^°^( ^Q y ^Ν уЦ ^Ν but 0Η ] CI HO 58 118 9θ ² ^ ^=Ν |_) ί/^Ι but ₂ with ^{Ν Ν} — ' ₇ Μ but όΗ 1 CI HO 48 119 0 OVe-Ph _к1 /'''''Ο ΗΟ ₂ Ο^ο^ν°^ ^Ν y^f ^N ЗУ''— ¹ ----' J j μ ...... - - Ny.. ·^HOOH\Cl HO 4,612 120 OyOH Ub OH\ Cl 4,628 HO 121 О NH ₂ 0 3— OEt N Ух yy 3ypN Н+,ί ° -y>y/γ ^N + 3 \ θ but he HO 1,711 122 O NH ₂ o _x Voh n^A„ b< O ϊ HO J o-d QN^ _N <y _f .NH ₂ ζ \ -^S. 7 © y/ but he HO 40 123 O.r ^OH uon θ λ=Ν MyN ly ^N H ₂ 3? .......h N^x^l Zyu he is at Cl 71 HO 124 CO ₂ Et _N Уу yy-yV-Jy-^ 0 N ^H0 &h У Cl 27 HO

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125 o 0. %OH χ °XX ^N ^ ^N ^ ^CF3 ntf He HO 235 126 o ^Z <V- ^Z 4^ ,,Ο 0 J γ r 1 X ° ιΑ Ο^Ο [Γ ¹ ''ΰ) HO 9 127 ο νη ₂ Ο ΆθΗ ьк А χ e XI HO Jo^ _o Ν-Λχο but' he HO 170 128 0 ΝΗ ₂ 0 —OH %_/ ¢(1 ' ^Ν fj.- _N A, _C| s ^*4—I but' Oh HO 6 129 pikJL SO /—-N |_| HO ₂ C^O'^^ ^Q '^ ^N 4^^' ^N Xj? HO OH ICl 1 580 HO 130 o HN^ °ь4 ^0Н X hoJ oy ₀ A4 XX^ but Ън HO 0.36

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131 Ο ί i h ^OH K/ m| Ί DLdd KfAci o | \nO bn HN^J^ But >100 132 O O O '/—ON Ν y_/ with Ίι ? ⁿ ™ / ^D ^O _v ^nA nA3I o | y Km'^^ ⁷ HO bn HN But But 133 O \ o O /—OH K/ f NH but J _o nK γΚ y y o o U no^ he ^Hl ^jj But 7 134 o o 0 y—OH W C NH but / o—» _o nK Ζ=Κ γ U o o IJ HO' OH ^ΗΝ κΖ But 16 135 o νη ₂ Q у_0|-| y.—-U but J o^L ₀ νΚ z==K y U o q I y Km ⁷ ^ BUT He ^Hh yjj But 91

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136 θ ' NH O V-OH J AX f V ⁿ HO J η N— ^J . J -^Y f Cl o MU JYm HO OH ^Hh LA But 0.24 137 Ο HN' 0 V-OH hk Hell *\ πι yuA-y νΑ4ι Q A /y KA ^- ^ but he But 0.31 138 O y—ON NJ U~/\ γΖ _Ν HO J O—» _η Ν-Λ U ΑΖ ν ^υ γ νΑ _γ , o 4 L = «j*4A ΚιΑ^ but he ΗΟ_ζ^Ν' Ts A/ But 0.15 139 O An q U—ON Ad 4 Ί/ ^Ν А/ ν°χ/ А o IJ Аы ⁷ ^ but he ^ηο ·^τα But 0.13 140 Q Άη V> ^0H 2mm G D \ N HO / O—» _η N-Λ J =D Y / ^N CI o \j\ '*==<L / ''C-ki N(5 OH HN 1 V. But 0.29

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141 Ax NH 0 y OH NJ Ύ N HO _o nA I ' / ^N Cl ο \\ \ / K _N HO OH HN But 0.23 142 o A ^H у—/ сУГ sOZ °A° nA _n A _c| U. Κι ~~~ _- ι X-_/ ns? Oh But 5 143 _K®I n'l J ° X χ I L ° Ο'' But 13 144 0, NH,> o Don y J nsЪ V ^J ' NO / o-d ₀ ы _N ' _C | 1k xD I— IIX 1 |_|^у But 1 145 o nh ₂ O * OH N - _; y ' N y ^/o d.o YES ' ” 1 yu he But 108 146 (D° %Du° iiji Q o \ / //~* Ά* ¹ XL A I /“-”2. ke Q But 0.41

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147 o hiX О V-OH JL, %.........../ / ^N \ ,0 f ,//, °« О Уч HO OH HN ft But 0.58 148 o HiX 0 Uon N X. %..........XC Ί| ^N ™>'Xγ ^Ν ^Λ p IJ —X ( ΚιΧ ⁷ but But 0.08 149 Ο ΝΗτ C) '/—QI IN . ¹ -- _f ____' 7ι N HoХd _o .Хм ^J ' _CJ o ' ₌ -Δ BUT OH But 0.11 150 ΟχΥ> I cry ⁰ X χ σ' %Χ ₂ ^z y^~S Q But 0.16 151 1 I Ο ΝΗ Ο Ύ Oil Ν. ί X' ϊ J but /Od _ο 'U _α o \\ —-a / _Χν ^{ΗΟ ΟΗ} But 0.25

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152 0 NH ₂ <) '?—ΟΙ 1 N ^λ d__ ΟΓ ' N ^h 7 ^7o A- ^o / ⁿ ' ^nJ ' ^c ' o \ / “Ky no” he But 0.05 153 0 HN A q he nA 'Η---V I A A ^/0 ~d o , ^N 'nA _ci c Ά \ / C J y -} ——g / but he ΗΟ^^Ν But 0.48 154 o _h νη ₂ O VoH N Ax b( Ш no/ o-l o n^n^ci 0 C χχ ~~—**4— ua* ⁷ but ί )' _χ Ν_/ But 2 155 o in _g Q y—QH ,A b << J 1 ''' y.O y ^N IT'ci o AA ~ch l _HN ^{but on} but ϊι ) N—sZ But 2 156 □Me МрО— ^к ''—> о ^X=J Nl 1 О VoH N _ А ^Н у О-Д □ ^N- _N '- - _С | o -—-A ' % _Ν but in But 0.14

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157 o nh ₂ o Voh W < J Ί O' \ r- / nd he ^HN ^J) But 0.37 158 O NHj o У-ОН N_X, M 31 Ϊ ^H0 ν Ύ NH --—,Δ--/ ^N HO' t)H But 8 159 o. ,o ''S'' CJ k / ο γ ⁷ М==у о %-он ы А Μ <Τΐ BUT ^; J O— ¹ · l ΝΆμΑ-, υΎ ” ci but he But 476 160 o HN· O 7—OH N XL χΎ f μ ^Ν but/Huo N'4/ci A ..................... \ / o y) y — ^{and |} J ns? 'He But 0.31 161 o νη ₂ 0 --OH hk J * J j HO / 0 o N- ^U _N ⁷ w^\ / ⁿ « but he But 561 162 O NH2 O 7—OH N XL ^but / >Aci Y U o | J ——shL—( Ch/~~ not him Ό But 0.42

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163 0 Ο o Don m Jx y/ < ll ^N 1 / ) ( but 'on But >100 164 o nh ₂ 0 V-OH NA w <Αι i ЕУ? O-do N L _N A _C| about | J =3—/ A-s nd Oh ^Hf v3 But 261 165 О NHj О / OH Nx Αχ % f < ^z Ai 7 Eto^ Ά N^^ci □ A =3........J An no' He ^HN v3 But 45 166 O NH ₂ %— OH N Ax η-a < jfi □ C 3 =3-/ nd Oh HN \ kA But 145 167 O NH ₂ Koh n^Ax NL / J 1 7 D/N -Cl o 'A -3 / Ar^ but Oh HN \ o But 139 168 1 11.,,OH O HN o X—on NA Cl °D°y ^mAMLs| nA An But 14

- 182 046381

169 0 ΗΝ·^\ <5-<х Ъ~ОН Un < Ji /У °γ/Αι ^s nb Ън But 162 170 ο HN'^\ _с ^х Ά-on ν JL Ι Π\ \ JI l/M n ci HO Ън But 8 171 o HN^X cx Voh n^A. _v1 O'-Λ < lij \ ~ ^N ' ^ci H(f Ън But 96 172 0 HN^V, with Ά-ΟΗ nx? У /i N ζ7 ^ο γ/Α, (j =>4 / but' Ън But 6 173 o 1 IN v-7 s-' L ^{0N n} An L f' h 1 -N / O-d _o N' n Cl P =X HO oi 1 But 112 174 ο UN ^-- S%3” ^OH /V^N A / t\ L NJO~XQN'''Μ'^-'.ζ,ι 0 3 / p ₃ ssL^ ......... ^.171ΐ * But 17

- 183 046381

175 0 NH ₂ Voh N ' _M γ Г__/ η N 7/7°-a о, ⁿ N '' Cl о Δ ' hO he But 1 176 / ° $o w Г 11 i X ,o 1 ο ρΓ S/SD ABOUT But 5 177 ~~S /? Ж'^ ⁱⁿ у 21 \ -*~- у θ,-Α^χ) 11 Cj^O But BUT 178 o nh ₂ ' OH ' - o w / s/ g·'· '< κι I '—7* N 11 । ' °~D O * ^N ^' N ^J 'CI ~~~—« V 1 1 yu he But BUT 179 0 NH ₂ e^l. V~OH N XL / f fl ^{Ή N θΛ} ί ⁰ / ^H|<: ^ ^C1 BUT he 320 But 180 About Nib C--X%—OH N GN' O '1 N /o-d O^.T'hrHi (Г =Г- Ϊ 9 But

- 184 046381

181 0 Nbfe QtC 1Χλ N y O-dp N^ _n -A _c| 1D =A— 21 HO 182 O NH ₂ C'-Ov A—one Y %_У у у ⁴ N О· У ° ^υ γ%/ HO OH 24 HO 183 Ο NH2 _C ^X У-ОН s^y/ f II ί Ο—* η Ν'^Ζ.,Ζχ 1 and—» θ N οι FsCO'^A hcT OH 56 HO 184 qn»2 C—-OH hk < ^z J'J ^N °A oJ^A^ci II - HO OH 102 HO 185 O ₄ OH NH _{2 C} ^X V-NH N Λ g y_ y J| ' N AA 0-^0^ _N ^J _Cl HO OH 11748 HO 186 o OH NH ₂ U”MN N U t>z ^z 1 i ^N °~Λ Ο NN 41 —_ _r \ / HO OH 8833 HO 187 uD ² ^ Ά·., lu i o o · ' A _z ^ _z o 10 HO

- 185 046381

188 Ο HN- <ΎΓ XL/ Ol See ^>4............1 but he 134 HO 189 O MN ₂ Ν-χ. %—OH Μ < ^ζ 20 /=/ “ΛΑ ^Ν ' ^Λα Hcf Ън 1851 HO 190 0NH; N - /—OH N _ l· J ? ____ ° ^N ci HO oi 1 9316 HO 191 N'NH o ^nh 2 NI q 1 _N --\ ^ ^N -y4 °~\°7^^ ^W ^ ^CI but bn 7 HO 192 .ANH o NH ₂ NI « 1 25 HO 193 cL V z^w _g iiyA Άθ Go O' ^if T\ // J/r T 0 263 HO

- 186 046381

194 /~У s-γ L_ N MH ₂ O I HO °y ..O y^N^ 'CI 336 BUT 195 S-У ' L,N NH ₂ /yAi HO °Vy ^N ^N^CI ho' Oh 236 But 196 11°y-z iU J /G -l o Q. ' _>* C '- -X m d 82 But 197 0 NH ₂ o _h Lon N_l, FZ J ? psj ⁰ \°Z ^N 'n'-'CI HjN A --/ Λχ ^ζ but Oh try BUT 0.73 198 ABOUT G o o 2268 BUT

- 187 046381

199 0 NHz c--n. OH L У/ < ^ζ ΐίι у°-^у ^mА ьА ^с| hH nd he 5757 BUT 200 About NH; e-M X ΟΝ Ν' । y-/ '7' Ν Hud ° / ^N ' ^{JI νΛ} ° O o XX HO OH ΗΟ'^^ηΧ]] L II 6091 BUT 201 o i_^ ^z X _z ^Z V ^z mJ3 I 3-Τ'·ό C g t - X. .o ⁰ hkhne ΣΖ 'Z ^v --';—' \ ί ~__/ ~ζ - Ζ _χ θ 204 BUT 202 I o CL / I y O s® 1 AM ΙΙί O ''Ό G Cf.....V z Q 148 BUT 203 o ηνΎυ no-r ^/ _p m X G\ /G H NO ( ^z fl NO ^ ^N<> OI but bn 4.2 BUT

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Example 207 Activation of tumor-targeted immune response by CD73 inhibitors.

C57BL/6 mice were implanted subcutaneously with EG7 cells. Compound 9 (50 mg/kg) or vehicle was administered orally twice daily starting on the first day after implantation (N=10 per group). Tumors were excised on day 14 and analyzed by flow cytometry. Compound 9 increases the % of CD8+ cells in CD45+ cells as demonstrated in FIG. 1A (* means p < 0.05). C57BL/6 mice were implanted subcutaneously with EG7 cells. Anti-CD8 antibody was administered intraperitoneally. on days 1, 0, 5 and 10. Compound 9 (50 mg/kg) or vehicle was administered orally twice daily starting on day 1. FIG. 1B demonstrates that CD8+ T cell depletion reduces efficacy (**** indicates p < 0.0001 compared to Compound 9+anti-CD8). Compound 9 alone demonstrated greater tumor volume reduction than the combination of Compound 9 with anti-CD8 antibody.

Example 208: Reversal of AMP-mediated CD8+ T cell suppression using CD73 inhibitors.

Human CD8+ T cells were labeled with CellTrace CFSE and then preincubated with adenosine deaminase inhibitor and compound 9 or vehicle for 20 min. To assess T cell proliferation and CD25 expression, 20 μM AMP was added. To assess cytokine production, 10 μM AMP was added. T cells were activated by a-CD3, a-CD28 and hIL2. After 4 days, proliferation and CD25 expression were assessed by flow cytometry, and cytokine levels in the supernatant were measured by ELISA. EC ₅₀ values were determined using a four-parameter dose-response curve equation. Fig. 2A shows EC ₅₀ =11.6 nM for CD8+ T cell proliferation. Fig. 2B shows EC ₅₀ =9.6 nM for CD8+ T cell activation. Fig. 2C shows EC ₅₀ =4.5 nM for IFNy production. Fig. 2D shows EC ₅₀ =5.6 nM for Granzyme B production.

Example 209 Selectivity of CD73 inhibitors

The compounds of this invention are selective for CD73 and do not exhibit proliferative effects. Using compound 9, cell surface CD39 activity was assessed using K562 cells expressing human CD39 and human kinase-Glo. The activity of recombinant human ENTPD2 and ENTPD3 was assessed using a malachite green quantitation assay. The enzymes CD39, ENTPD2 and ENTPD3 each showed an IC ₅₀ value of >10,000 M. Compound 9 was screened on the Eurofins Safety Screen Panel and the Eurofins Express Diversity Kinase Profile Panel. In the safety panel, 1/87 targets were inhibited by >50% at the 10 μM dose of compound 9. PDE3 enzyme was inhibited by 59% In the kinase panel, none of the 45 targets were inhibited by more than 50%.

In addition, compound 9 did not exhibit antiproliferative effects against the three cell lines. The viability of EG7 and A375 cells treated with 100 μM compound 9 was measured using CellTiter-Glo after 3 days. Human CD8+ T cell proliferation was measured by flow cytometry after 4 days of treatment with 100 μM compound 9 using the CellTrace CFSE Cell Proliferation Kit. Fig. 3A shows comparable % survival of EG7 cells, a mouse T-cell lymphoma cell line. Fig. 3B shows comparable % survival of A375 cells, a human melanoma cell line. Fig. 3C shows comparable % of human CD8+ T cells separated.

Example 210 Inhibition of CD73.

The activity of compound 9 was assessed against recombinant CD73 and CD73 expressing SK-MEL-28 cells using a malachite green quantitation assay. Inhibition of CD73 in plasma was measured using LC/MS to assess the conversion of ¹⁵ N5-AMP d ¹⁵ N5-ADO. Fig. 4A demonstrates nanomolar inhibition of CD73 cells from both humans and mice. Fig. 4B shows EC ₅₀ =0.17 nM for recombinant human CD73 cells.

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Fig. 4C shows EC5 ₀ =0.38 nM for human plasma CD73 cells. Inhibition of CD73 in plasma was measured using LC/MS to assess the conversion of ¹⁵ N5-AMP d ¹⁵ N5-ADO. Fig. 4D shows EC50=0.21 nM for human CD73 cell surfaces.

Example 211: Pharmacodynamics of an oral dose of a CD73 inhibitor.

A single dose of Compound 9 (50 mg/kg) was administered orally to mice, and plasma was collected at the indicated time points. Levels of compound 9 were measured using LC/MS. The IC ₅₀ value in mouse plasma was 1 nM, as demonstrated in FIG. 5A. Plasma was collected from mice 2 h post-dose and supplemented with ¹⁵ N5-AMP and TNAP inhibitor. Levels of ¹⁵ N5-ADO were measured using LC/MS. Fig. 5B shows 92% inhibition of mouse plasma CD73 cells.

Example 212: Efficacy of oral monotherapy with CD73 inhibitors.

The compounds of this invention exhibit potent antitumor effects, as evidenced by a reduction in tumor volume in a mouse model. In one model, EG7 cells were implanted subcutaneously into C57BL/6 mice. Compound 9 or vehicle was administered orally twice daily starting on the first day after implantation (N=10 per group). Fig. 6A demonstrates further reduction in tumor volume with increasing doses of Compound 9. In another model, EG7 cells were implanted subcutaneously into C57BL/6 mice. Compound 9 was administered orally twice daily (100 mg/kg) starting on the first day after implantation (N=10 per group). Vehicle was administered orally twice daily from the first day after implantation (N=20) until the fifth day after implantation, after which mice were randomized according to tumor volume into two groups. Compound 9 (100 mg/kg) or vehicle was administered orally twice daily starting on the sixth day after implantation, N=10 per group. Fig. 13A shows a decrease in tumor volume when compound 9 was administered to mice with confirmed tumors. Fig. 13B-D show separate repeats of this measurement for each dosage. In fig. 13B represents the carrier. In fig. 13C shows dosing of compound 9 started on day 1. FIG. 13D shows dosing of compound 9 started on day 6. In another model, CT26 cells were implanted subcutaneously into Balb/c mice. Compound 9 at a dose of 100 mg/kg or vehicle was administered orally twice daily starting on the first day after implantation (N=10 per group). Fig. 6B shows a decrease in tumor volume compared to vehicle. **** indicates p < 0.0001 compared to vehicle; NS indicates non-significance (two-way ANOVA).

Example 213: Efficacy of a CD73 inhibitor in combination with immuno-oncology and chemotherapeutic agents.

For each experiment, C57BL/6 mice were implanted subcutaneously with EG7 cells. Antu-PD-LI antibody (5 mg/kg) was administered intravenously on days 3, 5, 7, 9, 11, 13. Compound 9 (100 mg/kg) or vehicle was administered orally twice daily, starting on the first days after implantation. Fig. 7A demonstrates tumor volume reduction with monotherapy and combination therapy. ** means p < 0.01; **** means p < 0.0001 (two-way ANOVA). Fig. 7B-7E show separate repeats of this measurement for each dosage. In fig. 7B shows the carrier, FIG. 7C shows an anti-PD-L1 antibody, FIG. 7D shows connection 9, and FIG. 7E shows compound 9+anti-PD-L1.

Oxaliplatin was administered intramuscularly. at a dose of 6 mg/kg on study days 7 and 14. Compound 9 (100 mg/kg) or vehicle was administered orally twice daily starting on the first day after implantation. Fig. 8A demonstrates tumor volume reduction with monotherapy and combination therapy. **** means p < 0.0001 (two-way ANOVA). Fig. 8B-8E show separate repeats of this measurement for each dosage. Fig. 8B shows the carrier, FIG. 8C shows oxaliplatin, FIG. 8D shows connection 9, and FIG. 8E shows compound 9+oxaliplatin.

Doxorubicin was administered intravenously. at a dose of 2.5 mg/kg on study days 7 and 14. Compound 9 (50 mg/kg) or vehicle was administered orally twice daily starting on the first day after implantation. Fig. 9A demonstrates tumor volume reduction with monotherapy and combination therapy. * means p < 0.05; *** means p < 0.001 (two-way ANOVA). Fig. 9B-9E show separate repeats of this measurement for each dosage. In fig. 9B shows the carrier, FIG. 9C shows doxorubicin, FIG. 9D shows connection 9, and FIG. 9E shows compound 9+doxorubicin.

Docetaxel was administered intramuscularly. at a dose of 5 mg/kg on study days 5, 12 and 19. Compound 9 (100 mg/kg) or vehicle was administered orally twice daily starting on the first day after implantation. Fig. 12A demonstrates tumor volume reduction with monotherapy and combination therapy. * means p < 0.05; **** means p < 0.0001 (two-way ANOVA). Fig. 12B-1OE show separate repeats of this measurement for each dosage. In fig. 12B shows the carrier, FIG. 12C shows docetaxel, FIG. 12D shows connection 9, and FIG. 12E shows compound 9+docetaxel.

Example 214 Efficacy of a CD73 inhibitor in multiple tumors.

The serum was purchased from Discovery Life Sciences. Serum from patients with head and neck squamous cell carcinoma (HNSCC), ovarian cancer, and triple negative breast cancer

- 190 046381 pom and esophageal cancer were incubated with a serial dilution of compound 9 in the presence of a TNAP inhibitor. The conversion of ¹⁵ N5-AMP to ¹⁵ N5-ADO was measured by LC/MS. Fig. 10A demonstrates sub-nanomolar inhibition of HNSCC in serum. Fig. 10B demonstrates sub-nanomolar inhibition of ovarian cancer in serum. Fig. 10C demonstrates sub-nanomolar inhibition of TNBC in serum. Fig. 10D demonstrates sub-nanomolar inhibition of esophageal cancer in serum.

Example 215 CD73 expression in multiple human tumors.

Fig. 11 shows normalized levels of CD73 mRNA expression in tumor and normal tissues. CD73 (NT5E) expression levels were obtained from the TCGA (tumor tissues) or GTEX (normal tissues) databases using the UCSC Xena platform and analyzed using an unpaired t test. CD73 expression, measured by Log2 (normalized count +1), was higher compared with vehicle for clear cell tumors of the pancreas, esophagus, stomach, head and neck, colon, lung, and kidney.

Incorporation by reference.

All publications and patents mentioned herein are hereby incorporated herein by reference to the same extent that each individual publication or patent is specifically and individually indicated for inclusion by reference. In the event of a conflict, this application, including any definitions herein, will control.

Equivalents.

Although specific embodiments of the subject matter have been discussed, the above description is illustrative and not limiting. Many variations of the invention will become apparent to those skilled in the art upon consideration of this specification and the following claims. The entire scope of the invention is to be determined by reference to the claims, together with their full scope of equivalents and description, together with such variations.

Claims (72)

1. Compound of formula (I) R ³ where Y is Het represents itself or its pharmaceutically acceptable salts, where Z represents CH or N; R ^a is selected from H, halogen, C1.6 alkyl, thienyl, -NR7R ⁸ , C5.1 ₀ aryl and 5-10 membered heteroaryl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur; R ^b is selected from halogen, C ₁ . ₆ alkyl, _halogenC1 . ₆ alkyl, hydroxyC ₁ . ₆ alkyl, C^alkylthio, amido, carbonyl and 5-10 membered heteroaryl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur; - 191 046381 R ⁷ is selected from H, hydroxy, C _1-6 alkyl, C _5-10 arylC _1-6 alkyl, 5-10 membered heteroarylC _1-6 alkyl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur , C ₃ - ₆ cycloalkyl and 3-10 membered heterocyclyl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur; And R ⁸ represents H or C ₁ - ₆ alkyl or R ⁷ and R ⁸ together with the nitrogen atom to which they are attached form a 4-7 membered heterocyclyl ring containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur; R ^1a represents H or hydroxy; R ^1b represents H or hydroxy; R ^2a represents hydroxy or C ₁ - ₆ alkyl; R ^2b represents C ₂ - ₆ alkynyl; R ^{2 3} represents H; R ⁴ is selected from H, CN, 5-10 membered heteroaryl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur, -C(O)OR ⁹ , -C(O)NR ¹¹ R ¹² and - S(O) ₂ R ¹⁰ ; R ⁵ selected from H, C _1-6 alkyl, 3-10 membered heterocyclylC1- ₆ alkyl containing from one to four heteroatoms _{independently} selected from nitrogen, oxygen and sulfur, C _5-10 arylC1- ₆ alkyl, 5-10 membered heteroarylC _1-6 alkyl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur; R ⁶ is selected from -C(O)OR ⁹ , -C(O)NR ¹⁶ R ¹⁷ and -P(O)(OR11)(OR ¹² ); R ⁹ represents H, C ₁ - ₆ alkyl or 3-10 membered heterocyclyl containing from one to four nitrogen atoms; R ¹⁰ is C _1-6 alkyl or C ₅ - ₁₀ aryl and each of R ¹¹ and R ¹² is H or C _1-6 alkyl or R ¹¹ and R ¹² together with the carbon atom to which they are attached form a 5-7 membered heterocyclyl containing from one to four nitrogen atoms; each R ¹⁶ and R ¹⁷ is independently selected from H, hydroxy or C _1-6 alkyl; provided that the specified connection does not represent 2 o nh ₂ o Yoh n 1 Ъ< and no/o^ _o « ^L Х _С | O'he 2. Connection according to claim 1, characterized in that a) or b): a) if each of R ⁴ and R ⁶ is -C(O)OH and R ⁵ is benzyl substituted on the phenyl ring by a 3-10 membered heterocyclyl or 5-10 membered heteroaryl substituent containing from one to four heteroatoms , independently selected from nitrogen, oxygen and sulfur, then said heterocyclyl or heteroaryl substituent is selected from pyrrolidinyl, piperazinonyl, piperidonyl, tetrahydropyrimidonyl, pyridonyl and pyridyl, each of which is unsubstituted or substituted with C _1-6 alkyl, C _1-6 alkoxy, hydroxyC _{1 -6} alkyl or C ₁ - ₆ alkoxyC ₁ - ₆ alkyl; And b) if R ⁴ is -C(O)OH or tetrazolyl, R ⁶ is -C(O)OH, and R ⁵ is benzyl substituted on the phenyl ring by a second phenyl ring, then either a benzylphenyl ring or a second phenyl ring the ring is substituted -C(O)OR ⁹ where R ⁹ represents H or C1- ₆ alkyl. - 192 046381 3 o nh ₂ O Khon xx < ^z У ΐ Н°7 °Y,Oy ^N ' ^A N^CI but''on 3. A compound according to claim 1 or 2, having the following structure: 4 about in ₂ About Hong NX У/ °Л/°у Αι Ън 4. A compound according to claim 3, characterized in that the compound of formula (I) has structure (IA) 5 o MN ₂ O Hon N A nG <17 Α°γο ^L L| ci-XY \ / L\ \ __/ but'' He 5. A compound according to any one of claims 1 to 4, characterized in that R ^1a is in the β configuration. 6 O O ° X ^he /уХ /7\ \ ^ΝΗ but /o^ ₀ Νγ / ⁵ ^ \ 7 O no' He - 209 046381 - 210 046381 6. A compound according to claim 5, characterized in that the compound of formula (I) has structure (IB) 7. The connection according to any of the previous paragraphs, characterized in that R ^2a is in the α configuration. 8. A compound according to claim 7, characterized in that the compound of formula (I) has the structure (IC) 9. A compound according to any one of claims 1 to 6, characterized in that R ^2a is in the β configuration. 10. A compound according to claim 9, characterized in that the compound of formula (I) has the structure (ID) 11. A compound according to claim 3, characterized in that the compound of formula (I) has the structure (IE) - 193 046381 12. The connection according to any one of claims 1-11, characterized in that R ⁵ represents C ₅ .1 ₀ arylC1. ₆ alkyl or 5-10 membered heteroarylC _1-6 alkyl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur, with a para substituent on the aryl or heteroaryl ring selected from 3-10 membered heterocyclyl containing from one up to four heteroatoms independently selected from nitrogen, oxygen and sulfur, 5-10 membered heteroaryl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur and C ₅ - ₁₀ aryl. 13. The compound according to claim 1, characterized in that each C ₁ _ ₆ alkyl, C ₅ _ ₁₀ arylC ₁ _ ₆ alkyl and 5-10-membered heteroarylC ₁ _ ₆ alkyl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur, in R ⁵ is unsubstituted or substituted with one or more substituents selected from halogen, C _1-6 alkyl, C ₁ _ ₆ alkoxy, carbonyl, amino, amido, C ₃ _ ₆ cycloalkyl, 3-10 membered a heterocyclyl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur, and a 510-membered heteroaryl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur. 14 o 9 νλ ^ΟΗ 911 ^ν ο ' but 'OH 14. The compound according to claim 13, characterized in that the substituents in C ₁ _ ₆ alkyl, C ₅ _ ₁₀ arylC ₁ _ ₆ alkyl and 510-membered heteroarylC _1-6 alkyl containing from one to four heteroatoms independently selected from nitrogen , oxygen and sulfur, in R ⁵ selected from halogen, halogenC ₁ - ₆ alkyl, C ₁ - ₆ alkoxy, amino, carbonyl, C ₅ - ₁₀ aryl, 3-10-membered heterocyclyl containing from one to four heteroatoms, independently selected of nitrogen, oxygen and sulfur and 5-10 membered heteroaryl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur. 15 o nh ₂ O Uon N U Uu ¢191 ⁿ ° \ °991/ ^{J n} ' ^ci but''''on 15. A compound according to any one of claims 1 to 11, characterized in that R ⁵ is benzyl substituted on the phenyl ring, preferably in the para position, by a 3-10 membered heterocyclyl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur, or a 5-10 membered heteroaryl substituent containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur, preferably the phenyl ring substituent being selected from substituted piperidonyl, piperazinonyl, tetrahydropyrimidonyl, pyridonyl and pyridyl, and Optionally, piperidonyl, _{tetrahydropyrimidonyl} , pyridonyl or pyridyl is substituted with one or more of _{C1_6} alkyl, _hydroxyC1-6 alkyl or C| _-6 alkoxyC| _-6 alkyl. 16 o NH2 o Uon N U Uy < ^z ly but ^C| but'' 16. A compound according to any one of claims 1 to 12, characterized in that R ⁵ is benzyl substituted on the phenyl ring, preferably at position 4, - 194 046381 17 о nh ₂ о УOn gкУ ъГ ¢199 ^but 999Χ ^{Ν <} ' ^αι but' Ън 17. Connection according to any one of paragraphs. 1-11, characterized in that represents - 195 046381 - 196 046381 - 197 046381 - 198 046381 18 y$ ⁰ g O Uon M^U _m UU UI but/ °^7 ” O -^997 but''on 18. Connection according to any one of paragraphs. 1-17, characterized in that Het is selected from - 199 046381 19 o NH ₂ O Uon Uch ¢11 but/ 0 o N^tAci o 99 ^9-7 U/ but''on 19. Connection according to any one of paragraphs. 1-17, characterized in that Het is 20 O MN ₂ O _x Uon Ν.^Λ _ΚΙ Н < ^z J ΐ Х/°Л/0 n^n^ci o ο У/^ but''on /у 9 - 211 046381 21 o nh ₂ 0 con N - χ-ζ. Η < ^Z J ϊ °ΛΑ, ^νΛ Αι о £j HO 'OH H0-k~ ^N ft 21. The compound according to claim 19 or 20, characterized in that R ⁷ is selected from H, C ₁ . ₆ αlkyl, C5. ₁₀ arylS ₁ . ₆ alkyl, 5-10 membered _heteroarylC1 . ₆ alkyl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur, C _3-6 cycloalkyl and 3-10 membered heterocyclyl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur. 22 o nh ₂ o con nJ. W < Г HOJ Oy _o kj _c| o ^z £j k/''/ but' Oh A 22. Compound of formula (II) (P) or pharmaceutically acceptable salts thereof, where Het represents where Z represents CH or N; R ^a is selected from H, halogen, C1-6αalkyl, thiophenyl, ^-NR7R8 , C5-10aryl and 5-10 membered heteroaryl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur; R ^b is selected from halogen, C1 _-6 alkyl, haloC1 _-6 alkyl, hydroxyC1 _-6 alkyl, C1 _-6 alkylthio, amido, carbonyl and 5-10 membered heteroaryl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur; R ⁷ is selected from H, hydroxy, C _1-6 alkyl, C _5-10 arylC _1-6 alkyl, 5-10 membered heteroarylC _1-6 alkyl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur , C _3-6 cycloalkyl and 3-10 membered heterocyclyl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur; And R ⁸ represents H or C _1-6 alkyl or R ⁷ and R ⁸ together with the nitrogen atom to which they are attached form a 4-7 membered heterocyclyl ring containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur; R ^1a represents H or hydroxy; R ^1b represents H or hydroxy; R ^2a represents hydroxy or C _1-6 alkyl; R ^2b represents C _2-6 alkynyl; - 200 046381 R ³ represents H; R ⁴ is selected from 5-10 membered heteroaryl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur, -C(O)OR ⁹ , -C(O)NR ¹¹ R ¹² or -S(O) ₂ R ¹⁰ ; R ⁵ is selected from H, C ₁ . ₆ αlkyl, 3-10 membered _{heterocyclylC1} . ₆ alkyl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur, C ₅ . ₁₀ arylS ₁ . ₆ alkyl, 5-10 membered _heteroarylC1 . ₆ alkyl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur; R ⁶ is selected from -C(O)OR ⁹ , -C(O)NR ¹¹ R ¹² and -P(O)(OR ¹¹ )(OR ¹² ); R ⁹ represents H, C ₁ . ₆ αlkyl or 3-10 membered heterocyclyl containing from one to four nitrogen atoms; R ¹⁰ represents C ₁ . ₆ alkyl or _C5 . ₁₀ aryl and each of R ¹¹ and R ¹² represents H or C ₁ . ₆ alkyl or R ¹¹ and R ¹² together with the carbon atom to which they are attached form a 5-7-membered 3-10-membered heterocyclyl containing from one to four nitrogen atoms; provided that a) and b) or a) and c); a) the connection is not b) if R ⁴ and R ⁶ are each -C(O)OH and R ⁵ is benzyl substituted on the phenyl ring by a 3 to 10 membered heterocyclyl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur, or a 5-10 membered heteroaryl substituent containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur, then the phenyl ring substituent is selected from piperidonyl, tetrahydropyrimidonyl, pyridonyl and pyridyl, each of which is unsubstituted or substituted with C ₁ . ₆ αlkyl, C ₁ . ₆ alkoxy, hydroxyC ₁ . ₆ alkyl or C ₁ . _{6 alkoxyC1} . ₆ alkyl; And c) if R ⁴ is -C(O)OH or tetrazolyl, R ⁶ is -C(O)OH, and R ⁵ is benzyl substituted on the phenyl ring by a second phenyl ring, then either a benzylphenyl ring or a second phenyl ring the ring is substituted -C(O)OR ⁹ where R ⁹ represents H or C ₁ . ₆ alkyl, or a compound selected from the group consisting of 23 o J; o K ¹ ° O 1/ ¹ u-jy ⁰ 23. A compound according to any one of claims 1 to 22, characterized in that R ^1a and R ^2a each represent hydroxy. 24 HO nh ₂ o. S° n^A _ki >a ( ^Z T ^Н °У °y,oJ ^A N ⁴ tl si# =-? ( HO OH 24. A compound according to any one of claims 1 to 22, characterized in that R ^1a represents hydroxy and R ^1b represents H. 25 HO NH ₂ °AA ^{0 N} -<A.. b<< ^z J 1 cm ^{n ci} but Oh 25. A compound according to any one of claims 1 to 24, characterized in that R ^2a represents Me and R ^2b represents ethynyl. 26 o ⁵ “-rt ° J o '° XJ if ο γ o ^;: 'o X 26. A compound according to any one of claims 1 to 24, characterized in that R ^2a represents hydroxy and R ^2b represents ethynyl. 27 HO NH ₂ o _h Ao Nx J,.. M <'J 1 ^but °уоу! ^m ' ^h mk| n£ ho' Oh HN^S - 212 046381 27. A compound according to any one of claims 1 to 24, characterized in that R ^2b represents propynyl, NN butynyl, ' ^or ' which is substituted with benzyl. 28 O V A J ° s o ° O O'S }--4— Γ·ύ ri 28. A compound according to any one of claims 22 to 27, characterized in that the compound of formula (II) has the following structure: - 201 046381 29 HO NH ₂ 0. A ^{0 N} / . $O < ^z Ji ^H0 °Λ ^ζί / ^{N cl} < /Airi ^H0 ° ^H 29. A compound according to any one of claims 22 to 28, characterized in that R ^1a is in the α configuration. 30 CAA ² ΊΟ ,o 11 ° r χ ° o Q 30. A compound according to claim 29, characterized in that the compound of formula (II) has the structure (IIAa) R ^fl R ^J (IIAa) 31 but ^X O Cl Ao >7<< J 1 z\ °^' ^,0 / ^NCI AA no' bn 31. The compound according to any one of claims 22-30, characterized in that R ^1a is in the β-configuration. 32 _UK1 /\O ^H HO HN a A ^{0 n} X>m _u Xa ^(/ - 2oj ^H /\ °X( ^o y ^N '^<N ^ ^c ' s but'' Oh 32. A compound according to claim 31, characterized in that the compound of formula (II) has structure (IIB) R ⁺ R ³ (PVA) 33 HO ^NH o _h Ao n^A.. O < ^z J 1 O “yjOi HO' OH - 213 046381 33. The compound according to any one of claims 22-32, characterized in that R ^2a is in the α configuration. 34 _ΗΚ χΧθΗ 0 OH ^H j HoX-yX (yN l..- ⁰ ·/χχ ¹ O but he 34. A compound according to claim 33, characterized in that the compound of formula (II) has the structure (IICa) R ⁴ R ³ (PSa) 35 ^he o he ⁿ y v ^H noХд-Хо Х°у О but he 35. The compound according to any one of claims 22-32, characterized in that R ^2a is in the β configuration. 36 he O OH d\NO' -уХо <Х]| ^N /^=У ⁰ X/°'/ ^ ^N< ^ ^CI o but he 36. A compound according to claim 35, characterized in that the compound of formula (II) has the structure (IIDa) R ⁴ R ³ (IIDa) 37 but ν-\ /\^.ON about he U JI 1 Νχ,Λ HOХХ ,4- Ji 'N ^у°у°у X, ⁷ but' He 37. A compound according to claim 28, characterized in that the compound of formula (II) has the structure (IIEa) R ⁴ R ¹ (PEa) 38 0 OH HN ^/ ^' JI L Nx/4 НО^ууХ pf|'N У' ⁰ У ^Ν<Ιχ<:ι О but He 38. A compound according to any one of claims 1 to 37, characterized in that R ⁴ is -C(O)OR ⁹ , -C(O)NR ¹¹ R ¹² or -S(O)2R ¹⁰ . 39 \ /\Х\ о he II 1 ho-XXo / У ^Ν /=у ^о у°у ^|Л Х ^{| 7} but He - 214 046381 39. The compound according to claim 38, characterized in that R ⁴ represents -C(O)OR ⁹ and R ⁹ represents H or Y^alkyl. 40 OH O OH ^ΗΓ ίΆ3 11 L ιχ As xA°luuAA, ⁷ no' He 40. The compound according to claim 38, characterized in that R ⁴ is -C(O)NR ¹¹ R ¹² . 41 he 0 he U II I ιχ As NOADo ,Λ· Τ| N O ,/ ^N 'N^'ci O but he 41. The compound according to claim 38, characterized in that R ⁴ represents -S(O)2R ¹⁰ and R ¹⁰ represents Y^alkyl or C5. ₁₀ aryl. 42 <yJ ^H 0 OH y HO^-yAo y ^N K|'N xDDD ΑΧι ^Z no' bn 42. A compound according to any one of claims 22-41, characterized in that R ⁵ is selected from H, Y^alkyl, C ₅ . ₁₀ arylS ₁ . ₆ alkyl and 5-10 membered heteroarylCl. ₆ alkyl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur. - 202 046381 43 d O OH NN '. OH 11 A IX A, NOADo (/ D N DudA ^X BUT He 43. The compound according to claim 42, characterized in that each C _1-6 alkyl, C _5-10 arylC _1-6 alkyl and 5-10-membered heteroarylC _1-6 alkyl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur, in R ⁵ is unsubstituted or substituted with one or more substituents selected from halogen, C _1-6 alkyl, C _1-6 alkoxy, carbonyl, amino, amido, C _3-6 cycloalkyl, 3-10 membered heterocyclyl, containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur and 5-10-membered heteroaryl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur. 44 Aon o N|D O Don N__D A < ^z Dd U°D ⁰ “Ax°y ^N ^ ^N t:i O no he - 215 046381 44. The compound according to claim 43, characterized in that the substituents in C _1-6 alkyl, C _5-10 arylC _1-6 alkyl and 510-membered heteroarylC _1-6 alkyl containing from one to four heteroatoms independently selected from nitrogen , oxygen and sulfur, in R ⁵ selected from halogen, halogenC _1-6 alkyl, C _1-6 alkoxy, carbonyl, C ₅₁₀ aryl, 3-10 membered heterocyclyl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur and 5-10 membered heteroaryl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur. 45 U 0 HN o Uon n^A _k , w U ο ^U NO He 45. The compound according to claim 43, characterized in that R ⁵ represents a C _5-10 arylC _1-6 alkyl substituted on the C _5-10 aryl ring, preferably in the para position, with a 5-7 membered heterocyclyl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur, or a 5-7 membered heteroaryl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur. 46 U O N o Uon n^A _k1 U U d 1 Sy °^VOy ^N ^N^CI O ^U NO' He 46. A compound according to any one of claims 22 to 45, characterized in that R ⁵ is selected from H, methyl or ethyl. 47 \d ^he o 14 o, Uon W < ^z J1 Н°у °^ν°γ ^Ν ^ ^Ν<< ΌΙ but He 47. The compound according to claim 42, characterized in that R ⁵ is selected from benzyl, -CH ₂ -pyridyl, -CH 2 -pyridazinyl, -C^-oxazolyl, -CH ₂ -thiophenyl, -C^-furanyl, -CH ₂ -thiazolyl and -C^-benzothiazolyl. 48 Λ> ^0Η o N-^ ^J o _x Uon n^A _k1 Y < ^z d ϊ ^U θ^ν-Οχ/^Ν^ΟΙ BUT He 48. The compound according to claim 42, characterized in that R ⁵ represents benzyl, substituted in the phenyl ring with a 3-10-membered heterocyclyl or 5-10-membered heteroaryl substituent containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur, wherein preferably the phenyl ring substituent is selected from substituted piperidonyl, tetrahydropyrimidonyl, pyridonyl and pyridyl, and optionally piperidonyl, tetrahydropyrimidonyl, pyridonyl or pyridyl is substituted by one or more of C _1-6 alkyl, hydroxyC _1-6 alkyl or C _{1- 6} alkoxyC _1-6 alkyl. 49 o ΝΗ ₂ V” -'Л F _3C yy °у°/ ^{N Cl} HO' He 49. A compound according to claim 48, characterized in that R ⁵ is benzyl substituted on the phenyl ring (preferably at position 4) 50 o nh ₂ УУ ^Н ^дА _F3 cJyуу ^мЛс| But he - 216 046381 - 217 046381 - 218 046381 - 219 046381 - 220 046381 - 221 046381 88 Ph— _χ ΟΟ ₂ Η n fp JX хч, _Ζ Ο ₄ m ί N-D but ₂ s op; Y^ ^N Yif —- n< _:/ n but he ] Cl 89 Ph—\ CO ₂ HN 1 X /°x N to Νχ but ₂ with ο'^Ρ / Y ;f — - l·. Νχ.- ^Ν BUT OH 7 Cl 90 Ph CO ₂ H _N ΗΟ ₂ Ο^Ό-^··°'^ ^Ν dLu ^N - : -, Νχ,Ν BUT he 7 Cl 91 Ph— CO ₂ H _/Γ= Ν Η ho ₂ c^o^2)^ ^N yHf ^Ν χ2) Ηό 0Η ^Ν χ CI 92 Ph с ₂ Н Ν Η Λ urh Д 'Х'^/^''.^NX HO ₂ C 0 / rif -—: Νχ-Ν but he 7 01 93 ^Ph с° ₂ n /=Ν Η Η0 ₂ Γ^ο-^ρ°/· ^Ν ·χ _[ Ρ^ ^Ν ρ —', Νχ ^Ν BUT OH ] Cl 94 Ph—\00 ₂ Η H \/ HO ₂ c^'O'^Y°r^ ^N γ^γ ^Ν —~ 7 Νχ, ^Ν HO OH ] Cl 95 Ph—. C0 ₂ H _H Η0,Λρ°ν ^Ν Χ ^Ν -p°H --“ ¹ X Νχ,· ^Ν HO OH 1 Cl - 222 046381 96 ph—, CO ₂ H D -~ i Nx/ ^N HO OH A Cl 97 _Ph CO ₂ H,^ _NH HO ₂ CV* ^NN —~ ' N _a .. ⁿ HO OH D Cl 98 Ph—\CO ₂ H ^_ _NH HO ₂ s4D°D ^4γ ^Ν *γ^ΟΗ BUT He ^N V Cl 99 Ph N7 lC0 ₂ H D H0 ₂ c ^Z 'O''*X-°v ⁿ y4y ^N ^OH --—; · N. ₄ ,.N HO OH A Cl 100 Ph lco ₂ H ,^ _N GDhon Н0 ₂ С'Х'Л · ' A^N-У --T : N _a .,N ho he 7 Cl 101 ^Ph ₍ rn μ X\ ^0H \CO ₂ H _N ry,,/ HO ₂ c ^> ^O'^<' ^O y ^N y^Y ^N --, N _a ,N HO OH ] Cl 102 ^P V co ₂ H . noLu^uh —- Νλ,/Ν but he is to Cl 103 Ph =4 ί Ν Ν ^he but he Υ CI - 223 046381 104 Ph -ΧΟΛ ί L· Ν ^ОН HO OH Ύ Cl 105 Ph X ^C0 2 ^H 1 but ₂ s- ^X 'O^M γ4γ ^Ν -- NO but oh A Cl 106 Ph (c° ₂ ^H N1 _H A ,.. nh =M ( Jj In HO OH Ύ Cl 107 Ph IC° ₂ H _N H f 1 ,.ΝΗ —Д ί Jj N HO OH Ύ Cl 108 Ph Lco ₂ H „ Π EtO ₂ C ⁷ ·ΐ' ^νί ^ ^Ν '/)γ'- 'O; l a but he 7 Cl 109 Ph XX... I CO ₂ HNH f 4 ESogC^O^* ⁴ / -' Nx ^N HO OH ] Cl 110 Oo Lz ⁿ 'ί CO ₂ H n |_| η o ₂ ⁿⁿ 'g'—> Ν.χΝ HO OH 1 Cl - 224 046381 - 225 046381 118 C°2 ^H /=N H fX HO ₂ C O VV* rif HO OH 7 CI 119 °APh but ₂ s Ά>·Ά° V ^Ν \Χν ^Ν —Λ Γ \u —- ν ₄ , ^ν BUT he 7 Cl 120 0.,.OH 1 /M ⁿ but he Ύ CI 121 o yn ₂ m ^0E * /Ά UvYM but' he 122 0 МН ₂ о U ^OH Н < J Ϊ but7 °^о Ν^Αγ ^ΝΗ2 О ⁰ but' He 123 оА ⁿ Y ^0H Ο^/'Ό y=N Аф° А\ΛγΝΗ2 ПЧО ОН А Cl 124 9°2 ^Et /=NA? ^H0 > ^N v Uv ^N hoY ⁰ \ Г Yn θ -jggA ί Ν--..ΔΙ HO OH ] Cl - 226 046381 125 0 0 U OH N-sxZ. yy ^N ^ ^CF3 Oh ~ but 'he 126 0 NH ₂ o. Uon n^A , H < ^Z A 1 h°A ^ο Λχ°ν ^{Ν N} ^ ^C| sQn ^Du but Oh 127 o nh ₂ o Uon rr A, H < J 1 no/ ₀ 0 ^U_7 o no 'on 128 o nh ₂ O 'A OH 'U,., W < J 1 U/ ^o D..o..y m^c1 ys —/ but 'on 129 PIvJA f ^-0 g, /=Ч ^Н but γί^2^-' Д jy \ ? - ί Ny.- ^N HO OH A Cl 130 O HN' O Won Ns. Ah Ay ¢10 n£y θΑ/Ογ^ΝΑ:! o £ y ₌ D [ but bn ΛΝ 1 - 227 046381 - 228 046381 - 229 046381 141 θ ^NH 0 OH NJ ku x γχ ^HO ΛλαΜνΑ μ ' Υ У ci О Н Κι but &н ^ng уу 142 o nh ₂ o Hon N_A _m M < ^z D H s°/ °Ύ°УХ ί Μ'no' OH 143 0 νη ₂ О Khon Ν X ku Ш "V °Λθ/ ^{nAci Ν} but Ън 144 0 νη ₂ o Hon ν^Χ _μ Μ < ^ζ JJ but/°-уо nX _n X _C | Oh ^but '' ^he 145 O NH ₂ o Hong gm X ku < ^z l ? but/ o-do ,Χ'νΧ'Ή Oh but' He 146 o MN ₂ o Hong gm X yy kdk jX / °Х ^o y 'Хс1 o Уу =Х t but' He /О-х^у к - 230 046381 - 231 046381 - 232 046381 - 233 046381 - 234 046381 169 0 HN'^% Q-h. Len Ν-._ χΧ ζ1Λ°Λχ°γ ^Ν ^ ^Ν ^ι ^s HO OH 170 o HN^\ qa VoH IskxX ГЧ < ^z Д i У / °^..0 H 'N ^z: <c| ^' ^:ΐ HO' OH 171 o HN'\ W < J 1 yX/ ^o y<y ^A N--ci but Ън 172 ο ^ΗΝ Λ7 ca Voh Ν._Χ. V Wk < ii 7 ^ΧΛ°Λ^°γ ^Ν ^ ^Ν ^ι ^ ^: 'but' OH 173 X ° J I XX <A< ¹ xo 174 “Π O O tsu ΗγΎ ⁰ L ° T χΖγ Q / - 235 046381 - 236 046381 181 o nh ₂ /-OH M Mx Μ·/ <' 1ϊ У/ ^Ο ^ν°γ ^Ν ^Ν^θΙ ^ ^: ' ^:ί BUT OH 182 Ο ΝΗ ₂ ΚθΗ N. C Cm < ^ζ he ^Ν J °^y,Oy ^N ^N' ^A 'CI ₀ j3' - BUT OH OH 183 O ΝΗ ₂ MM <' TU ^N ( ⁰ ^\Ογ ^Ν '^'Ν' ^Λ '0Ι F ₃ CO ' ^:: - BUT OH 184 o NH ₂ S^, Ch° ^{N M} -Dm Sm < ^ζ Ml ^Ν \ ^Ο ^\,Οχ/ ^Ν ^ ^Ν ' ^Λ ' ⁰ ' ¹¹ Mch but he 185 0 he НН ₂ о^х Knh n _v M /м <' Ml ^Ν °^γθγ· ^Ν ^Ν^ΟΙ but he 186 O OH ΝΗ ₂ S^X FnH N_A _m 0C <' 11 ^{N Ο} ^γθγ. ^Ν ^Ν^ΟΙ HO OH 187 and mN T \—/ .? MM, III o rC M ^z D - 237 046381 188 nY° O-'M 2 o ζ W ¹ o 189 0 ΝΗ ₂ Ύ OH S. Д Ш °^y°y ^NN CI ^' ^::ί but Ън 190 0 ΝΗ ₂ Mon Λ Μ _/= / ⁰ y°y ^{N Ν α} no' Ън 191 _μ Ύνη ο νη ₂ ν I Λ ., Ν=^ν Λ-ΟΗ Ν^Μ-, ^Ν Α X II ^Ν but Ън 192 ..Ύνη 0 ΝΗ ₂ Ν I \\ _t , ί νΑ^Τοη _<ζ Ν-Λ _ν ³ ^γθγ- ^Ν ^Ν^α nХ Ън 193 ω ^s w Ο 5 J UM θ L '1 ^χ \ ιι ^χ ' Ήγ ο ο τ - 238 046381 - 239 046381 or a pharmaceutically acceptable salt thereof. 50. A compound according to any of the previous paragraphs, characterized in that R ⁶ represents C(O)OR ⁹ and R ⁹ represents H or C _1-6 alkyl. 51. A compound according to any one of claims 1 to 49, characterized in that R ⁶ is -C(O)NR ¹⁶ R ¹⁷ . - 203 046381 52. The compound according to any one of claims 1 to 50, characterized in that R ⁴ and R ⁶ each represent -C(O)OH. 53. The connection according to any one of claims 22-37, characterized in that represents - 204 046381 - 205 046381 - 206 046381 54. The compound according to any one of claims 22-53, characterized in that the Het substituents are selected from halogen, C ₁ . ₆ alkyl, _halogenC1 . ₆ alkyl, amino and 5-8 membered heterocyclyl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur. 55. The compound according to any one of claims 22-53, characterized in that Het is 56. The connection according to any one of paragraphs. 1-53, characterized in that Het is where Z represents CH or N; R ^a is selected from H, halogen, C1-6αalkyl, thiophenyl, ^-NR7R8 , C5-10aryl and 5-10 membered heteroaryl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur; R ^b is selected from halogen, C _1-6 αlkyl, halogenC _1-6 alkyl, amido, carbonyl, amido and 5-10 membered heteroaryl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur; R ⁷ is selected from H, C _1-6 alkyl, C _5-10 arylC _1-6 alkyl, 5-10 membered heteroarylC _1-6 alkyl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur, C _3-6 cycloalkyl and 3-10 membered heterocyclyl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur; A R ⁸ represents H or _C1-6 alkyl or R ⁷ and R ⁸ together with the nitrogen atom to which they are attached form a 4-7 membered heterocyclyl ring containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur. 57. The compound according to claim 56, characterized in that Z represents CH. - 207 046381 58. The compound according to claim 56, characterized in that Z is N. 59. The compound according to any one of claims 56-58, characterized in that R ^a represents -NR7R ⁸ . 60. A compound according to any one of claims 56 to 59, characterized in that R ^b is selected from Cl, -CF3, carbonyl and -CONH2. 61. The compound according to any one of claims 56-60, characterized in that Het is 62. A compound according to any one of claims 56-61, characterized in that R ⁷ represents O^alkyl or C _3-6 cycloalkyl, with C1. ₆ alkyl or C ₃ . ₆ cycloalkyl is unsubstituted or substituted with one or more substituents selected from hydroxy, C1. ₆ alkoxy, C ₅ . ₁₀ aryl, amino and C ₃ . ₆ cycloalkyl. 63. A compound according to any one of claims 56-61, characterized in that R ⁷ represents C _5-10 arylC _1-6 alkyl or 5-10 membered heteroarylC1. ₆ alkyl containing from one to four heteroatoms independently selected from nitrogen, oxygen and sulfur, wherein the C _5-10 arylC _1-6 alkyl or 5-10 membered heteroarylC _1-6 alkyl is unsubstituted or substituted with halogen or C _1-6 alkyl. 64. The compound according to any one of claims 56-63, characterized in that R ⁸ is selected from H, methyl and ethyl. 65. A compound according to any one of claims 56-61, characterized in that R ⁷ and R ⁸ , together with the nitrogen atom to which they are attached, form a 3-10-membered heterocyclyl ring containing from one to four heteroatoms, independently selected from nitrogen, oxygen and sulfur, selected from azetidinyl, morpholino, pyrrolidinyl and azepanil. - 208 046381 66. Connection selected from the following: Example No. Compound 67. A pharmaceutical composition containing a compound or a pharmaceutically acceptable salt thereof according to any one of paragraphs. 1 -66 and one or more pharmaceutically acceptable excipients. 68. A method of inhibiting CD73 in a cell, comprising contacting the cell with a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 66. 69. A method of treating a disease or disorder selected from cancer, cerebral and cardiac ischemic diseases, fibrosis, immune and inflammatory disorders, inflammatory bowel motility disorder, neurological, neurodegenerative and central nervous system diseases, depression, Parkinson's disease and sleep disorders, comprising administering a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 66. 70. The method according to claim 69, characterized in that the cancer is selected from bladder cancer, bone cancer, brain cancer, breast cancer, heart cancer, cervical cancer, colon cancer, rectal cancer, esophageal cancer, fibrosarcoma, stomach cancer, gastrointestinal cancer, head and neck cancer, Kaposi's sarcoma, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, myeloma, ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, cancer testicular germ cells, thymoma and thymic carcinoma. - 240 046381 71. The method of claim 69 or 70, wherein the cancer is selected from breast cancer, brain cancer, colon cancer, fibrosarcoma, kidney cancer, lung cancer, melanoma, ovarian cancer and prostate cancer. 72. Method according to any one of claims 69 to 71, characterized in that the cancer is breast cancer.